taq dna polymerase Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Qiagen taq dna polymerase
    Sequences of contaminating bacterial <t>DNA</t> in three <t>Taq</t> polymerases. Sequence alignment of three contaminants from different commercial Taq polymerases showing the presence of different strains of the same Pseudomonas species. Roche FastStart, Platinum HiFi Platinum Taq polymerases contain similar strains of a Pseudomonas species with a single base difference in the region covered by the 16S350 PCR assay.
    Taq Dna Polymerase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 4735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/Qiagen
    Average 99 stars, based on 4735 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher taq dna polymerase
    Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed <t>Taq</t> <t>DNA</t> polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26450 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 26450 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    99
    Roche faststart taq dna polymerase
    Reduction of PCR Inhibition by Increased Amounts of <t>Taq</t> Polymerase. qPCR with HMW and template <t>DNA</t> from FFPE tissue using different amounts of Taq polymerase. PCR-amplification of increasing amounts of genomic HMW template DNA (2.5-3,840 ng) using (A) 2 U Taq , (B) 4 U Taq and PCR-amplification of template DNA from FFPE tissue using (C) 2 U Taq , (D) 4 U Taq . Shown are the mean values (± standard deviations) from triplicate measurements.
    Faststart Taq Dna Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 2342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/faststart taq dna polymerase/product/Roche
    Average 99 stars, based on 2342 article reviews
    Price from $9.99 to $1999.99
    faststart taq dna polymerase - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs taq dna polymerase supertaq
    <t>DNA</t> modification and bypass during bisulfite treatment. ( A ) Scheme of the chemical reaction of cytidine: Treatment with bisulfite generates the non-aromatic, non-planar 5,6-dihydrouridine-6-sulfonate (dhU6S), which decomposes to uracil upon treatment with base (and heat). ( B ) Primer extension activity of different polymerases <t>(Taq,</t> 5D4, 3A10, E10, TgoT) on template T1 either unmodified (C), bisulfite-treated and desulfonated (Reagent 1, 80°C, 20 min) (converting dC to dU) (D) or bisulfite-treated (Reagent 1) but not desulphonated (converting dC to dhU6S) (S). Polymerases 5D4 and 3A10 are able to generate full-length (+20) products even from the non-desulphonated template (S). ( C ) Time-course comparison of primer extension activity of Taq and 5D4 on T1 either unmodified (C) or bisulfite-treated with (D) or without desulphonation (S). (P: primer).
    Taq Dna Polymerase Supertaq, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase supertaq/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase supertaq - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    97
    New England Biolabs taq dna polymerase
    CE analysis of processing synthetic <t>DNA</t> by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized <t>Taq</t> DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.
    Taq Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 6203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/New England Biolabs
    Average 97 stars, based on 6203 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2019-12
    97/100 stars
      Buy from Supplier

    99
    TaKaRa la taq polymerase
    PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the <t>Taq</t> DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; <t>Takara-bio).</t> PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.
    La Taq Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 3423 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/la taq polymerase/product/TaKaRa
    Average 99 stars, based on 3423 article reviews
    Price from $9.99 to $1999.99
    la taq polymerase - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher platinium taq dna polymerase
    MNN2 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 23 colonies for each condition. Dark grey: Petri dishes cultures, light grey: liquid cultures. B ) Average of the 3 assays presented in A (standard deviation=19.55). C ) PCR product visualized on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications have been carried out with the <t>Platinium</t> <t>Taq.</t>
    Platinium Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinium taq dna polymerase/product/Thermo Fisher
    Average 94 stars, based on 119 article reviews
    Price from $9.99 to $1999.99
    platinium taq dna polymerase - by Bioz Stars, 2019-12
    94/100 stars
      Buy from Supplier

    78
    Affibody taq polymerase binding
    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z <t>EGFR:2377</t> -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z <t>Taq:3638</t> -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
    Taq Polymerase Binding, supplied by Affibody, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase binding/product/Affibody
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    taq polymerase binding - by Bioz Stars, 2019-12
    78/100 stars
      Buy from Supplier

    Image Search Results


    Sequences of contaminating bacterial DNA in three Taq polymerases. Sequence alignment of three contaminants from different commercial Taq polymerases showing the presence of different strains of the same Pseudomonas species. Roche FastStart, Platinum HiFi Platinum Taq polymerases contain similar strains of a Pseudomonas species with a single base difference in the region covered by the 16S350 PCR assay.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Sequences of contaminating bacterial DNA in three Taq polymerases. Sequence alignment of three contaminants from different commercial Taq polymerases showing the presence of different strains of the same Pseudomonas species. Roche FastStart, Platinum HiFi Platinum Taq polymerases contain similar strains of a Pseudomonas species with a single base difference in the region covered by the 16S350 PCR assay.

    Article Snippet: Qiagen Taq DNA polymerase contained a mixture of contaminants that did not give reliable sequencing data from reaction to reaction. shows the alignment of the broad host range primers for bacterial 16S rDNA used in this study.

    Techniques: Sequencing, Polymerase Chain Reaction

    Approximating the copy number of 16S rDNA in commercial Taq polymerases. Six DNA polymerases were used with primers for 16S rDNA on 7 serial 10-fold dilutions of E. coli genomic DNA (10 ng to 10 fg) with no added DNA in the 8 th sample. The least squares fit equation for each dilution series was used to assign a value to the signal from the 8 th sample, which contains Taq-associated DNA only. The efficiency of the reaction was determined from the slope of the linear fit plotting the base10 log of the DNA concentration vs. the threshold cycle. A slope of −3.322 indicates an average doubling rate of “2,” which is approximately 100% efficiency (2̂3.322∼10). The rDNA values assigned are for “E. coli equivalents.”

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Approximating the copy number of 16S rDNA in commercial Taq polymerases. Six DNA polymerases were used with primers for 16S rDNA on 7 serial 10-fold dilutions of E. coli genomic DNA (10 ng to 10 fg) with no added DNA in the 8 th sample. The least squares fit equation for each dilution series was used to assign a value to the signal from the 8 th sample, which contains Taq-associated DNA only. The efficiency of the reaction was determined from the slope of the linear fit plotting the base10 log of the DNA concentration vs. the threshold cycle. A slope of −3.322 indicates an average doubling rate of “2,” which is approximately 100% efficiency (2̂3.322∼10). The rDNA values assigned are for “E. coli equivalents.”

    Article Snippet: Qiagen Taq DNA polymerase contained a mixture of contaminants that did not give reliable sequencing data from reaction to reaction. shows the alignment of the broad host range primers for bacterial 16S rDNA used in this study.

    Techniques: Concentration Assay

    Detection of Pseudomonas fluorescens at high and low Taq polymerase concentrations. Bacterial detection with 0.5 Units (A) or 0.05 Units (B) Qiagen Taq DNA polymerase with 16S350B assay on samples containing 10 3 , 10 2 , 10 1 and zero Pseudomonas fluorescens bacteria. A composite of A and B is shown in C.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Detection of Pseudomonas fluorescens at high and low Taq polymerase concentrations. Bacterial detection with 0.5 Units (A) or 0.05 Units (B) Qiagen Taq DNA polymerase with 16S350B assay on samples containing 10 3 , 10 2 , 10 1 and zero Pseudomonas fluorescens bacteria. A composite of A and B is shown in C.

    Article Snippet: Qiagen Taq DNA polymerase contained a mixture of contaminants that did not give reliable sequencing data from reaction to reaction. shows the alignment of the broad host range primers for bacterial 16S rDNA used in this study.

    Techniques:

    Detection of bacterial DNA in six Taq polymerases. Four dilutions of six DNA polymerase were tested with primers for 16S rDNA in the presence of 100 pg (∼10̂5 16S rDNA) E. coli genomic DNA (circled) or H 2 O.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Detection of bacterial DNA in six Taq polymerases. Four dilutions of six DNA polymerase were tested with primers for 16S rDNA in the presence of 100 pg (∼10̂5 16S rDNA) E. coli genomic DNA (circled) or H 2 O.

    Article Snippet: Qiagen Taq DNA polymerase contained a mixture of contaminants that did not give reliable sequencing data from reaction to reaction. shows the alignment of the broad host range primers for bacterial 16S rDNA used in this study.

    Techniques:

    Detection of the beta-lactamase gene in commercial Taq polymerase. Four dilutions of Amplitaq DNA polymerase were tested with primers for the beta-lactamase gene in the presence of 10 3 pUC19 plasmids (labeled 1000 pUC19 genomes) or H 2 O.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Detection of the beta-lactamase gene in commercial Taq polymerase. Four dilutions of Amplitaq DNA polymerase were tested with primers for the beta-lactamase gene in the presence of 10 3 pUC19 plasmids (labeled 1000 pUC19 genomes) or H 2 O.

    Article Snippet: Qiagen Taq DNA polymerase contained a mixture of contaminants that did not give reliable sequencing data from reaction to reaction. shows the alignment of the broad host range primers for bacterial 16S rDNA used in this study.

    Techniques: Labeling

    Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Polymerase Chain Reaction

    One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Incubation

    Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Standard Deviation, Agarose Gel Electrophoresis

    Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.

    Article Snippet: M-MLV reverse transcriptase (25 U) (Invitrogen) and Taq DNA polymerase (2.5 U) (Invitrogen) were used in all quantitative real-time experiments.

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Journal: Nucleic Acids Research

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    doi: 10.1093/nar/gkn575

    Figure Lengend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Article Snippet: Thermus aquaticus (Taq ) DNA polymerase (recombinant) and Platinum® Taq DNA Polymerases were purchased from Invitrogen (Carlsbad, CA, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    Comparison of the performance of OXP-modified primers to other Hot Start DNA polymerases. ( A ) Agarose gel analysis of the PCR products resulting from the 35 thermal cycles of amplification of five copies of a 365-bp fragment from the HIV-1 tat gene using 0.5 μM unmodified, single OXP-modified and double OXP-modified primers. Reactions containing unmodified primers were amplified by Taq DNA polymerase, Platinum® Taq DNA Polymerase, AmpliTaq Gold® DNA Polymerase, HotStart-IT™ Taq DNA Polymerase and DyNazyme™ II Hot Start DNA Polymerase. Reactions containing single and double OXP-modified primers were amplified by Taq DNA polymerase. ( B ) Graphical representation of PCR amplicon yield. The results from triplicate experiments were averaged and are normalized to the yield of reactions containing single OXP-modified primers plus Taq DNA polymerase. Error bars represent the SD. (*), indicates Hot Start DNA polymerases.

    Journal: Nucleic Acids Research

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    doi: 10.1093/nar/gkn575

    Figure Lengend Snippet: Comparison of the performance of OXP-modified primers to other Hot Start DNA polymerases. ( A ) Agarose gel analysis of the PCR products resulting from the 35 thermal cycles of amplification of five copies of a 365-bp fragment from the HIV-1 tat gene using 0.5 μM unmodified, single OXP-modified and double OXP-modified primers. Reactions containing unmodified primers were amplified by Taq DNA polymerase, Platinum® Taq DNA Polymerase, AmpliTaq Gold® DNA Polymerase, HotStart-IT™ Taq DNA Polymerase and DyNazyme™ II Hot Start DNA Polymerase. Reactions containing single and double OXP-modified primers were amplified by Taq DNA polymerase. ( B ) Graphical representation of PCR amplicon yield. The results from triplicate experiments were averaged and are normalized to the yield of reactions containing single OXP-modified primers plus Taq DNA polymerase. Error bars represent the SD. (*), indicates Hot Start DNA polymerases.

    Article Snippet: Thermus aquaticus (Taq ) DNA polymerase (recombinant) and Platinum® Taq DNA Polymerases were purchased from Invitrogen (Carlsbad, CA, USA).

    Techniques: Modification, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

    Dependence of pre-PCR heating of PTE on primer dimer accumulation. Agarose gel analysis of primer dimer accumulation with preheated single OXP-modified primers in nontemplate system. Mixture of both primers was preheated at 95°C in 1× PCR buffer (pH 8.4 at 25°C) for increasing amounts of time. Samples were cooled on ice water, the Taq DNA polymerase was added followed by PCR amplification. PCR cycle parameters: 95°C (2 min); 30 cycles of [95°C (40 s); 56°C (30 s); and 72°C (2 min)]; 72°C (7 min). Primer dimer amplicon, indicated on the gel image, ran as a 50–80 bp DNA fragment (left lane: 50-bp ladder).

    Journal: Nucleic Acids Research

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    doi: 10.1093/nar/gkn575

    Figure Lengend Snippet: Dependence of pre-PCR heating of PTE on primer dimer accumulation. Agarose gel analysis of primer dimer accumulation with preheated single OXP-modified primers in nontemplate system. Mixture of both primers was preheated at 95°C in 1× PCR buffer (pH 8.4 at 25°C) for increasing amounts of time. Samples were cooled on ice water, the Taq DNA polymerase was added followed by PCR amplification. PCR cycle parameters: 95°C (2 min); 30 cycles of [95°C (40 s); 56°C (30 s); and 72°C (2 min)]; 72°C (7 min). Primer dimer amplicon, indicated on the gel image, ran as a 50–80 bp DNA fragment (left lane: 50-bp ladder).

    Article Snippet: Thermus aquaticus (Taq ) DNA polymerase (recombinant) and Platinum® Taq DNA Polymerases were purchased from Invitrogen (Carlsbad, CA, USA).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Modification, Amplification

    Evaluation of thermolabile primers in one-step RT–PCR. For each gene of interest ( PBGD , ABCA-1 and β-actin ), the PCR primers were unmodified, single OXP-modified or double OXP-modified primers. RT utilized a polydT 18 primer. Reactions contained Taq DNA polymerase and M-MLV reverse transcriptase.

    Journal: Nucleic Acids Research

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    doi: 10.1093/nar/gkn575

    Figure Lengend Snippet: Evaluation of thermolabile primers in one-step RT–PCR. For each gene of interest ( PBGD , ABCA-1 and β-actin ), the PCR primers were unmodified, single OXP-modified or double OXP-modified primers. RT utilized a polydT 18 primer. Reactions contained Taq DNA polymerase and M-MLV reverse transcriptase.

    Article Snippet: Thermus aquaticus (Taq ) DNA polymerase (recombinant) and Platinum® Taq DNA Polymerases were purchased from Invitrogen (Carlsbad, CA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Modification

    Reduction of PCR Inhibition by Increased Amounts of Taq Polymerase. qPCR with HMW and template DNA from FFPE tissue using different amounts of Taq polymerase. PCR-amplification of increasing amounts of genomic HMW template DNA (2.5-3,840 ng) using (A) 2 U Taq , (B) 4 U Taq and PCR-amplification of template DNA from FFPE tissue using (C) 2 U Taq , (D) 4 U Taq . Shown are the mean values (± standard deviations) from triplicate measurements.

    Journal: PLoS ONE

    Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

    doi: 10.1371/journal.pone.0077771

    Figure Lengend Snippet: Reduction of PCR Inhibition by Increased Amounts of Taq Polymerase. qPCR with HMW and template DNA from FFPE tissue using different amounts of Taq polymerase. PCR-amplification of increasing amounts of genomic HMW template DNA (2.5-3,840 ng) using (A) 2 U Taq , (B) 4 U Taq and PCR-amplification of template DNA from FFPE tissue using (C) 2 U Taq , (D) 4 U Taq . Shown are the mean values (± standard deviations) from triplicate measurements.

    Article Snippet: Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 .

    Techniques: Polymerase Chain Reaction, Inhibition, Real-time Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded, Amplification

    Inhibitory Effect of Template DNA from FFPE Tissues on PCR Performance. qPCR with 1 µg of genomic HMW template DNA and increasing amounts (60-1,440 ng) of spiked genomic template DNA from FFPE tissue Genomic DNA from FFPE tissue was treated beforehand with active DNase I (+) and heat-inactivated DNase I (-), respectively. qPCR was performed using a 150-bp fragment and 1 U Taq polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.

    Journal: PLoS ONE

    Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

    doi: 10.1371/journal.pone.0077771

    Figure Lengend Snippet: Inhibitory Effect of Template DNA from FFPE Tissues on PCR Performance. qPCR with 1 µg of genomic HMW template DNA and increasing amounts (60-1,440 ng) of spiked genomic template DNA from FFPE tissue Genomic DNA from FFPE tissue was treated beforehand with active DNase I (+) and heat-inactivated DNase I (-), respectively. qPCR was performed using a 150-bp fragment and 1 U Taq polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.

    Article Snippet: Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 .

    Techniques: Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Successful PCR Amplification of Larger Fragments by Overcoming PCR Inhibition. PCR-amplified DNA fragments of different sizes within the PITX2 gene locus using template DNA from FFPE tissue. The PCR was carried out using 1 µg (upper and middle panel) and 5 ng (lower panel) template DNA in the presence of 1 U and 4 U Taq DNA polymerase, respectively.

    Journal: PLoS ONE

    Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

    doi: 10.1371/journal.pone.0077771

    Figure Lengend Snippet: Successful PCR Amplification of Larger Fragments by Overcoming PCR Inhibition. PCR-amplified DNA fragments of different sizes within the PITX2 gene locus using template DNA from FFPE tissue. The PCR was carried out using 1 µg (upper and middle panel) and 5 ng (lower panel) template DNA in the presence of 1 U and 4 U Taq DNA polymerase, respectively.

    Article Snippet: Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 .

    Techniques: Polymerase Chain Reaction, Amplification, Inhibition, Formalin-fixed Paraffin-Embedded

    Degradation of DNA from FFPE Tissues and its Effect on PCR Amplification of Amplicons with Different Sizes. Analysis of DNA integrity of genomic and bisulfite-converted DNA from unfixed and FFPE tissues by means of (A) agarose gel electrophoresis and (B) end-point PCR using PCR fragments of different sizes within the PITX2 gene locus. (C) qPCR results applying increasing amounts (2.5–3,840 ng) of genomic template DNA from unfixed and (D) from FFPE tissue. Shown are the mean values (± standard deviations) from triplicate measurements. Each PCR was performed with 1 U Taq polymerase. DNA from unfixed specimens is considered high molecular weight (HMW) DNA.

    Journal: PLoS ONE

    Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

    doi: 10.1371/journal.pone.0077771

    Figure Lengend Snippet: Degradation of DNA from FFPE Tissues and its Effect on PCR Amplification of Amplicons with Different Sizes. Analysis of DNA integrity of genomic and bisulfite-converted DNA from unfixed and FFPE tissues by means of (A) agarose gel electrophoresis and (B) end-point PCR using PCR fragments of different sizes within the PITX2 gene locus. (C) qPCR results applying increasing amounts (2.5–3,840 ng) of genomic template DNA from unfixed and (D) from FFPE tissue. Shown are the mean values (± standard deviations) from triplicate measurements. Each PCR was performed with 1 U Taq polymerase. DNA from unfixed specimens is considered high molecular weight (HMW) DNA.

    Article Snippet: Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 .

    Techniques: Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Real-time Polymerase Chain Reaction, Molecular Weight

    Transferability of the Findings to a qPCR Targeting an Alternative Genomic Locus. qPCR applying a 200-bp PCR fragment within the ACTB gene locus. Amplification with (A) HMW DNA from unfixed tissue and (B) DNA from FFPE tissue (2.5 ng to 3,840 ng) in the presence of 1 U and 4 U Taq DNA polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.

    Journal: PLoS ONE

    Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

    doi: 10.1371/journal.pone.0077771

    Figure Lengend Snippet: Transferability of the Findings to a qPCR Targeting an Alternative Genomic Locus. qPCR applying a 200-bp PCR fragment within the ACTB gene locus. Amplification with (A) HMW DNA from unfixed tissue and (B) DNA from FFPE tissue (2.5 ng to 3,840 ng) in the presence of 1 U and 4 U Taq DNA polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.

    Article Snippet: Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 .

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Formalin-fixed Paraffin-Embedded

    PCR Inhibition by Bisulfite-Converted Template DNA from FFPE Tissues. (A) qPCR–amplification of different amounts (10–3,840 ng) of bisulfite-converted template DNA from FFPE tissue using a 129-bp PCR fragment within the ACTB gene locus. 1 U and 4 U Taq polymerase were used for qPCR. Shown are the mean values (± standard deviations) from triplicate measurements. (B) PCR amplification of the specific PCR product was confirmed by agarose gel electrophoresis using 1 U (upper panel) and 4 U (lower panel) Taq polymerase.

    Journal: PLoS ONE

    Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

    doi: 10.1371/journal.pone.0077771

    Figure Lengend Snippet: PCR Inhibition by Bisulfite-Converted Template DNA from FFPE Tissues. (A) qPCR–amplification of different amounts (10–3,840 ng) of bisulfite-converted template DNA from FFPE tissue using a 129-bp PCR fragment within the ACTB gene locus. 1 U and 4 U Taq polymerase were used for qPCR. Shown are the mean values (± standard deviations) from triplicate measurements. (B) PCR amplification of the specific PCR product was confirmed by agarose gel electrophoresis using 1 U (upper panel) and 4 U (lower panel) Taq polymerase.

    Article Snippet: Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 .

    Techniques: Polymerase Chain Reaction, Inhibition, Formalin-fixed Paraffin-Embedded, Real-time Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis

    PCR Inhibition by Template DNA from FFPE Tissues with Regard to PCR Conditions (dNTP Concentration and Thermal Cycling Profile). Shown are qPCR results applying a 200-bp fragment within the PITX2 gene locus using template DNA from FFPE tissue (80-1,920 ng) in the presence of 1 U Taq polymerase. (A) dNTP concentrations and (B) annealing and elongation times were varied. Shown are the mean values (± standard deviations) from triplicate measurements.

    Journal: PLoS ONE

    Article Title: Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

    doi: 10.1371/journal.pone.0077771

    Figure Lengend Snippet: PCR Inhibition by Template DNA from FFPE Tissues with Regard to PCR Conditions (dNTP Concentration and Thermal Cycling Profile). Shown are qPCR results applying a 200-bp fragment within the PITX2 gene locus using template DNA from FFPE tissue (80-1,920 ng) in the presence of 1 U Taq polymerase. (A) dNTP concentrations and (B) annealing and elongation times were varied. Shown are the mean values (± standard deviations) from triplicate measurements.

    Article Snippet: Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer , 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2 .

    Techniques: Polymerase Chain Reaction, Inhibition, Formalin-fixed Paraffin-Embedded, Concentration Assay, Real-time Polymerase Chain Reaction

    DNA modification and bypass during bisulfite treatment. ( A ) Scheme of the chemical reaction of cytidine: Treatment with bisulfite generates the non-aromatic, non-planar 5,6-dihydrouridine-6-sulfonate (dhU6S), which decomposes to uracil upon treatment with base (and heat). ( B ) Primer extension activity of different polymerases (Taq, 5D4, 3A10, E10, TgoT) on template T1 either unmodified (C), bisulfite-treated and desulfonated (Reagent 1, 80°C, 20 min) (converting dC to dU) (D) or bisulfite-treated (Reagent 1) but not desulphonated (converting dC to dhU6S) (S). Polymerases 5D4 and 3A10 are able to generate full-length (+20) products even from the non-desulphonated template (S). ( C ) Time-course comparison of primer extension activity of Taq and 5D4 on T1 either unmodified (C) or bisulfite-treated with (D) or without desulphonation (S). (P: primer).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: DNA modification and bypass during bisulfite treatment. ( A ) Scheme of the chemical reaction of cytidine: Treatment with bisulfite generates the non-aromatic, non-planar 5,6-dihydrouridine-6-sulfonate (dhU6S), which decomposes to uracil upon treatment with base (and heat). ( B ) Primer extension activity of different polymerases (Taq, 5D4, 3A10, E10, TgoT) on template T1 either unmodified (C), bisulfite-treated and desulfonated (Reagent 1, 80°C, 20 min) (converting dC to dU) (D) or bisulfite-treated (Reagent 1) but not desulphonated (converting dC to dhU6S) (S). Polymerases 5D4 and 3A10 are able to generate full-length (+20) products even from the non-desulphonated template (S). ( C ) Time-course comparison of primer extension activity of Taq and 5D4 on T1 either unmodified (C) or bisulfite-treated with (D) or without desulphonation (S). (P: primer).

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Modification, Activity Assay

    Amplification of 24 individual human genomic loci. Comparison of PCR performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic DNA. Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: Amplification of 24 individual human genomic loci. Comparison of PCR performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic DNA. Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Amplification, Polymerase Chain Reaction

    PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Gas Chromatography, Molecular Weight

    Degree of methylation of individual CpG sites. Promoter regions of four genes ( A — prkcdbp , B — dab2ip , C — ptgs2 , D — ezh2) were amplified with either Taq ot 5D4/Taq blends, using bisulfide-treated genomic DNA from normal cells or LNCapP cells as a template and subjected to deep sequencing. Cyan—methylated CpGs, orange—unmethylated CpGs. Individual CpGs are numbered starting from the 5′ end of the amplicon.

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: Degree of methylation of individual CpG sites. Promoter regions of four genes ( A — prkcdbp , B — dab2ip , C — ptgs2 , D — ezh2) were amplified with either Taq ot 5D4/Taq blends, using bisulfide-treated genomic DNA from normal cells or LNCapP cells as a template and subjected to deep sequencing. Cyan—methylated CpGs, orange—unmethylated CpGs. Individual CpGs are numbered starting from the 5′ end of the amplicon.

    Article Snippet: Taq DNA polymerase (SuperTaq) was obtained from HT Biotechnology (Cambridge) and Q5 polymerase from New England Biolabs.

    Techniques: Methylation, Amplification, Sequencing

    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Labeling, Gas Chromatography, Incubation, Negative Control

    Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Gas Chromatography, Polymerase Chain Reaction, Sequencing, Generated, Produced, Purification, Incubation

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Gas Chromatography, Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation, Activated Clotting Time Assay

    PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Article Snippet: The same results were obtained by the other polymerase systems: LA Taq polymerase (Takara-bio), Tth polymerase (Applied-boisystems); Expand High Fidelity, Expand High FidelityPLUS and Expand Long Template polymerase (Roche-diagnostics); TITANIUM Taq polymerase (Becton-Dickinson-Clontech); and Taq polymerase (Promega).

    Techniques: Polymerase Chain Reaction, Genomic Sequencing, Sequencing, Staining

    PCR with primers carrying mismatched bases. PCR was performed at two human genomic sites with primers (20 bp), one of which (forward primer) carried one, two or three mismatched bases in the middle of the primer, in the absence (left) or presence (right) of Tth RecA protein and ATP using the Taq DNA polymerase ( rTaq DNA polymerase plus ‘hot start’ antibody). ( a ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 66 562 and nt 66 581 in GenBank accession no AC006454 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set a-1); lanes 2 and 6, PCR products using primers (primer set a-2 with one mismatched base at nt 66 566, T to C); lanes 3 and 7, PCR products using primers (primer set a-3 with two mismatched base at nt 66 566 and nt 66 571, both T to C); and lanes 4 and 8, PCR products using primers (primer set a-4 with three mismatched base at nt 66 566 and nt 66 571, T to C and nt 66 576, G to C). The oligonucleotide sequences used for the forward primers (mismatched bases are underlined) are as follows: primer set a-1, 5′-CATGGCACCTGCTCTGAGAC-3′; primer set a-2, 5′-CATGGCACC C GCTCTGAGAC-3′; primer set a-3, 5′-CATGGCACC C GCTC C GAGAC-3′; and primer set a-4, 5′-CATG C CACC C GCTC C GAGAC-3′. ( b ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 38 501 and nt 38 520 in GenBank accession no. AC0937734 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set b-1); lanes 2 and 6, PCR products using primers (primer set b-2 with one mismatched base at nt 38 505, G to A); lanes 3 and 7, PCR products using primers (primer set b-3 with two mismatched base at nt 38 505 and nt 38 510, both G to A); and lanes 4 and 8, PCR products using primers (primer set b-4 with three mismatched base at nt 38 505, nt 38 510 and nt 38 515, all G to A). The oligonucleotide sequences used for the forward primers are as follows: primer set b-1, 5′-ATCTGTGTGGTTCGGCTCTG-3′; primer set b-2, 5′-ATCTGTGTG A TTCGGCTCTG-3′; primer set b-3, 5′-ATCTGTGTG A TTCG A CTCTG-3′; and primer set b-4, 5′-ATCT A TGTG A TTCG A CTCTG-3′. ( c ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 63 957 and nt 63 976 in GenBank accession no. AC004975 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set c-1); lanes 2 and 6, PCR products using primers (primer set c-2 with one mismatched base at nt 63 961, A to T); lanes 3 and 7, PCR products using primers (primer set c-3 with two mismatched base at nt 63 961 and nt 63 966, A to T and C to T); and lanes 4 and 8, PCR products using primers (primer set c-4 with three mismatched base at nt 63 961, nt 63 966 and nt 63 971, A to T, C to T and G to T). The oligonucleotide sequences used for the forward primers are as follows: primer set c-1, 5′-GCAGGCACCAAGAACTACTG-3′; primer set c-2, 5′-GCAGGCACC T AGAACTACTG-3′; primer set c-3, 5′-GCAGGCACC T AGAA T TACTG-3′; and primer set c-4, 5′-GCAG T CACC T AGAA T TACTG-3′. The sequences for the backward primers are 5′-TCACCTCCCAGCCTGGCCCA-3′ for ( a ), 5′-AGGGAGATGTTCTCATAAAT-3′ and 5′-CTGTAAGTGGCAGACATTAC-3′ for ( b ). Nucleotide numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: PCR with primers carrying mismatched bases. PCR was performed at two human genomic sites with primers (20 bp), one of which (forward primer) carried one, two or three mismatched bases in the middle of the primer, in the absence (left) or presence (right) of Tth RecA protein and ATP using the Taq DNA polymerase ( rTaq DNA polymerase plus ‘hot start’ antibody). ( a ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 66 562 and nt 66 581 in GenBank accession no AC006454 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set a-1); lanes 2 and 6, PCR products using primers (primer set a-2 with one mismatched base at nt 66 566, T to C); lanes 3 and 7, PCR products using primers (primer set a-3 with two mismatched base at nt 66 566 and nt 66 571, both T to C); and lanes 4 and 8, PCR products using primers (primer set a-4 with three mismatched base at nt 66 566 and nt 66 571, T to C and nt 66 576, G to C). The oligonucleotide sequences used for the forward primers (mismatched bases are underlined) are as follows: primer set a-1, 5′-CATGGCACCTGCTCTGAGAC-3′; primer set a-2, 5′-CATGGCACC C GCTCTGAGAC-3′; primer set a-3, 5′-CATGGCACC C GCTC C GAGAC-3′; and primer set a-4, 5′-CATG C CACC C GCTC C GAGAC-3′. ( b ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 38 501 and nt 38 520 in GenBank accession no. AC0937734 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set b-1); lanes 2 and 6, PCR products using primers (primer set b-2 with one mismatched base at nt 38 505, G to A); lanes 3 and 7, PCR products using primers (primer set b-3 with two mismatched base at nt 38 505 and nt 38 510, both G to A); and lanes 4 and 8, PCR products using primers (primer set b-4 with three mismatched base at nt 38 505, nt 38 510 and nt 38 515, all G to A). The oligonucleotide sequences used for the forward primers are as follows: primer set b-1, 5′-ATCTGTGTGGTTCGGCTCTG-3′; primer set b-2, 5′-ATCTGTGTG A TTCGGCTCTG-3′; primer set b-3, 5′-ATCTGTGTG A TTCG A CTCTG-3′; and primer set b-4, 5′-ATCT A TGTG A TTCG A CTCTG-3′. ( c ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 63 957 and nt 63 976 in GenBank accession no. AC004975 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set c-1); lanes 2 and 6, PCR products using primers (primer set c-2 with one mismatched base at nt 63 961, A to T); lanes 3 and 7, PCR products using primers (primer set c-3 with two mismatched base at nt 63 961 and nt 63 966, A to T and C to T); and lanes 4 and 8, PCR products using primers (primer set c-4 with three mismatched base at nt 63 961, nt 63 966 and nt 63 971, A to T, C to T and G to T). The oligonucleotide sequences used for the forward primers are as follows: primer set c-1, 5′-GCAGGCACCAAGAACTACTG-3′; primer set c-2, 5′-GCAGGCACC T AGAACTACTG-3′; primer set c-3, 5′-GCAGGCACC T AGAA T TACTG-3′; and primer set c-4, 5′-GCAG T CACC T AGAA T TACTG-3′. The sequences for the backward primers are 5′-TCACCTCCCAGCCTGGCCCA-3′ for ( a ), 5′-AGGGAGATGTTCTCATAAAT-3′ and 5′-CTGTAAGTGGCAGACATTAC-3′ for ( b ). Nucleotide numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Article Snippet: The same results were obtained by the other polymerase systems: LA Taq polymerase (Takara-bio), Tth polymerase (Applied-boisystems); Expand High Fidelity, Expand High FidelityPLUS and Expand Long Template polymerase (Roche-diagnostics); TITANIUM Taq polymerase (Becton-Dickinson-Clontech); and Taq polymerase (Promega).

    Techniques: Polymerase Chain Reaction

    Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).

    Article Snippet: The same results were obtained by the other polymerase systems: LA Taq polymerase (Takara-bio), Tth polymerase (Applied-boisystems); Expand High Fidelity, Expand High FidelityPLUS and Expand Long Template polymerase (Roche-diagnostics); TITANIUM Taq polymerase (Becton-Dickinson-Clontech); and Taq polymerase (Promega).

    Techniques: Polymerase Chain Reaction, Staining, Molecular Weight

    MNN2 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 23 colonies for each condition. Dark grey: Petri dishes cultures, light grey: liquid cultures. B ) Average of the 3 assays presented in A (standard deviation=19.55). C ) PCR product visualized on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications have been carried out with the Platinium Taq.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: MNN2 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 23 colonies for each condition. Dark grey: Petri dishes cultures, light grey: liquid cultures. B ) Average of the 3 assays presented in A (standard deviation=19.55). C ) PCR product visualized on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications have been carried out with the Platinium Taq.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification, Standard Deviation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Efficiency of MNN5 gene amplification from wild type and modified strains. A ) Percentage of colonies picked from Petri dishes that amplified MNN5. The experiment was carried out on 47 colonies for each strain. Dark grey: BY4742; light grey: YiMMOgène. Amplification was carried out using the DreamTaq or the Platinium Taq. B ). PCR product visualisation, after amplification with the Platinium Taq, on a 1% agarose gel stained with SYBR safe. Left panel (1): BY4742, right panel (2): YiMMOgène, ML: Molecular ladder.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: Efficiency of MNN5 gene amplification from wild type and modified strains. A ) Percentage of colonies picked from Petri dishes that amplified MNN5. The experiment was carried out on 47 colonies for each strain. Dark grey: BY4742; light grey: YiMMOgène. Amplification was carried out using the DreamTaq or the Platinium Taq. B ). PCR product visualisation, after amplification with the Platinium Taq, on a 1% agarose gel stained with SYBR safe. Left panel (1): BY4742, right panel (2): YiMMOgène, ML: Molecular ladder.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Efficiency of MNN5 gene amplification from liquid culture of modified strain. A ) Percentage of MNN5 amplification from 47 colonies picked on Petri dishes and 47 colonies picked from liquid cultures in micro-plates. B ) Growth curve in micro-plate (YPD medium), average of two colonies of YiMMOgène. The OD 600 was measured in a micro-plate reader. ♦: OD 600 after 6 hour culture (early exponential phase). ●: OD 600 after 17 hour culture (late exponential phase). ▄: OD 600 after 24 hour culture (early stationary phase). C ) Percentage amplification of MNN5 from 47 YiMMOgène after different culture times in micro-plate. All amplifications have been carried out with the Platinium Taq.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: Efficiency of MNN5 gene amplification from liquid culture of modified strain. A ) Percentage of MNN5 amplification from 47 colonies picked on Petri dishes and 47 colonies picked from liquid cultures in micro-plates. B ) Growth curve in micro-plate (YPD medium), average of two colonies of YiMMOgène. The OD 600 was measured in a micro-plate reader. ♦: OD 600 after 6 hour culture (early exponential phase). ●: OD 600 after 17 hour culture (late exponential phase). ▄: OD 600 after 24 hour culture (early stationary phase). C ) Percentage amplification of MNN5 from 47 YiMMOgène after different culture times in micro-plate. All amplifications have been carried out with the Platinium Taq.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification

    MNN5 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 47 colonies for each condition. Dark grey: Petri dishes cultures, light grey: micro-plate cultures. B ) Average of the 3 assays presented in A (standard deviation =22.07). C) PCR product visualised on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications were carried out using Platinium Taq.

    Journal: BMC Research Notes

    Article Title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    doi: 10.1186/1756-0500-6-201

    Figure Lengend Snippet: MNN5 gene amplification efficiency on modified strain colonies picked from Petri dishes or micro-plate cultures. A ) 3 independent assays carried out on 47 colonies for each condition. Dark grey: Petri dishes cultures, light grey: micro-plate cultures. B ) Average of the 3 assays presented in A (standard deviation =22.07). C) PCR product visualised on a 1% agarose gel stained with SYBR safe. Left panel (1): Petri dishes, right panel (2): micro-plate cultures, ML: Molecular ladder. All amplifications were carried out using Platinium Taq.

    Article Snippet: With the Platinium Taq DNA polymerase (Invitrogen), the PCR mix was performed with final concentrations of: 1x manufacturer-supplied buffer, 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.3 mM primers, 2.4% DMSO, 0.5 U of the Hot Start enzyme, H2 O to 20 μL.

    Techniques: Amplification, Modification, Standard Deviation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Positron Emission Tomography

    Data from the longitudinal study of A431 xenografts: a Changes in tumor volumes with the days since inoculation. b TACs of the differing uptakes in one A431 xenograft of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 ( black curve, day 8) and targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 ( red , blue , and green curves on days 8, 19, and 21, respectively). c SUV mean for the uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 measured in the A431 tumors vs. days since inoculation. d Variations in specific uptake (SUV mean(ZEGFR:2377) –SUV mean(ZTaq:3638) ) with the growth of all ten xenografts from inoculation. e Variations in specific uptake (SUV mean of Z EGFR:2377 –SUV mean of Z Taq:3638 ) of all ten xenografts as their volumes increased. f Variations in specific uptake, SUV mean , in individual xenografts as their volumes increased

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: Data from the longitudinal study of A431 xenografts: a Changes in tumor volumes with the days since inoculation. b TACs of the differing uptakes in one A431 xenograft of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 ( black curve, day 8) and targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 ( red , blue , and green curves on days 8, 19, and 21, respectively). c SUV mean for the uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 measured in the A431 tumors vs. days since inoculation. d Variations in specific uptake (SUV mean(ZEGFR:2377) –SUV mean(ZTaq:3638) ) with the growth of all ten xenografts from inoculation. e Variations in specific uptake (SUV mean of Z EGFR:2377 –SUV mean of Z Taq:3638 ) of all ten xenografts as their volumes increased. f Variations in specific uptake, SUV mean , in individual xenografts as their volumes increased

    Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques:

    a Cell-binding assay of non-blocked ( red ) and blocked ( blue , tenfold Z EGFR:2377 ) Z EGFR:2377 -ST-[DyLight 488] and controls Z Taq:3638 -ST-[DyLight 488] ( green ) and DyLight 488 dye ( black ) using FACS analysis in A431 ( left ), FaDu ( middle ), and MDA-MB-453 ( right ) cells with high, medium, and low/no expressions of EGFR, respectively. b Fluorescent microscopy images of tumor (A431), liver, and kidney. Fluorescence in the tumor is only observed with the Z EGFR:2377 probe. High autofluorescence of the liver is observed with all probes. Fluorescence from both the Z EGFR:2377 and Z Taq:3638 probes is observed in the kidney. c western blots of A431, FaDu, and MDA from cell ( left ) and tumor ( right ) lysates using an antibody against human EGFR. The protein concentration of the cell lysates was determined by Bradford protein assay and Ponceau S staining of the membranes was used as loading control. The arrow indicates full-length EGFR

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: a Cell-binding assay of non-blocked ( red ) and blocked ( blue , tenfold Z EGFR:2377 ) Z EGFR:2377 -ST-[DyLight 488] and controls Z Taq:3638 -ST-[DyLight 488] ( green ) and DyLight 488 dye ( black ) using FACS analysis in A431 ( left ), FaDu ( middle ), and MDA-MB-453 ( right ) cells with high, medium, and low/no expressions of EGFR, respectively. b Fluorescent microscopy images of tumor (A431), liver, and kidney. Fluorescence in the tumor is only observed with the Z EGFR:2377 probe. High autofluorescence of the liver is observed with all probes. Fluorescence from both the Z EGFR:2377 and Z Taq:3638 probes is observed in the kidney. c western blots of A431, FaDu, and MDA from cell ( left ) and tumor ( right ) lysates using an antibody against human EGFR. The protein concentration of the cell lysates was determined by Bradford protein assay and Ponceau S staining of the membranes was used as loading control. The arrow indicates full-length EGFR

    Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Cell Binding Assay, FACS, Multiple Displacement Amplification, Microscopy, Fluorescence, Western Blot, Protein Concentration, Bradford Protein Assay, Staining

    Biodistribution of [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 and [methyl- 11 C]-Z Taq:3638 -ST-CH 3 in healthy Balb/c mice ( black lines Z EGFR:2377 and blue lines Z Taq:3638 (means ± SD, n = 4 mice): a PET images (supine, summed from 30–60 min, 3-D volume rendering) and b time activity curves (SUV) in arterial and venous blood, skeletal muscle, liver, and kidney over 60 min after injection

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: Biodistribution of [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 and [methyl- 11 C]-Z Taq:3638 -ST-CH 3 in healthy Balb/c mice ( black lines Z EGFR:2377 and blue lines Z Taq:3638 (means ± SD, n = 4 mice): a PET images (supine, summed from 30–60 min, 3-D volume rendering) and b time activity curves (SUV) in arterial and venous blood, skeletal muscle, liver, and kidney over 60 min after injection

    Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Mouse Assay, Positron Emission Tomography, Activity Assay, Injection

    PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Positron Emission Tomography