phusion  (New England Biolabs)


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    Structured Review

    New England Biolabs phusion
    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. <t>Phusion;</t> 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.
    Phusion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "In vitro synthesis of gene-length single-stranded DNA"

    Article Title: In vitro synthesis of gene-length single-stranded DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24677-5

    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.
    Figure Legend Snippet: ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Techniques Used: Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Produced, Labeling, Marker, Purification, Molecular Weight, Staining

    2) Product Images from "Dissecting and Tuning Primer Editing by Proofreading Polymerases"

    Article Title: Dissecting and Tuning Primer Editing by Proofreading Polymerases

    Journal: bioRxiv

    doi: 10.1101/2021.05.11.443694

    Tuning of primer editing using phosphorothioate protection. Effect of incorporating phosphorothioate bonds into E. coli -specific 515F primers on extent of primer editing observed when the primer editing standards are amplified using A) KAPA HiFi polymerase (n=3, error bars = +/-S.E.M.); B) NEB Q5 polymerase (n=3, error bars = +/-S.E.M.); C) Phusion polymerase (n=3, error bars = +/-S.E.M.).
    Figure Legend Snippet: Tuning of primer editing using phosphorothioate protection. Effect of incorporating phosphorothioate bonds into E. coli -specific 515F primers on extent of primer editing observed when the primer editing standards are amplified using A) KAPA HiFi polymerase (n=3, error bars = +/-S.E.M.); B) NEB Q5 polymerase (n=3, error bars = +/-S.E.M.); C) Phusion polymerase (n=3, error bars = +/-S.E.M.).

    Techniques Used: Amplification

    3) Product Images from "Solid-phase cloning for high-throughput assembly of single and multiple DNA parts"

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv036

    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.
    Figure Legend Snippet: Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Techniques Used: Activity Assay, Selection, Construct

    4) Product Images from "Dissecting and tuning primer editing by proofreading polymerases"

    Article Title: Dissecting and tuning primer editing by proofreading polymerases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab471

    Tuning of primer editing using phosphorothioate protection. Effect of incorporating phosphorothioate bonds into E. coli -specific 515F primers on extent of primer editing observed when the primer editing standards are amplified using A) KAPA HiFi polymerase ( n = 3, error bars = ± S.E.M.); B) NEB Q5 polymerase ( n = 3, error bars = ± S.E.M.); C) Phusion polymerase ( n = 3, error bars = ± S.E.M.).
    Figure Legend Snippet: Tuning of primer editing using phosphorothioate protection. Effect of incorporating phosphorothioate bonds into E. coli -specific 515F primers on extent of primer editing observed when the primer editing standards are amplified using A) KAPA HiFi polymerase ( n = 3, error bars = ± S.E.M.); B) NEB Q5 polymerase ( n = 3, error bars = ± S.E.M.); C) Phusion polymerase ( n = 3, error bars = ± S.E.M.).

    Techniques Used: Amplification

    5) Product Images from "In situ 10-cell RNA sequencing in tissue and tumor biopsy samples"

    Article Title: In situ 10-cell RNA sequencing in tissue and tumor biopsy samples

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41235-9

    A blend of Taq–Phusion polymerases improves selective poly(A) amplification of cDNA and reduces AL1 primer requirements. Cells were obtained by LCM from a human breast biopsy and split into 10-cell equivalent amplification replicates. ( A ) Poly(A) PCR was performed with 15 µg of AL1 primer with Taq alone (10 units), Phusion alone (4 units) or Taq/Phusion combination (3.75 units/1.5 units). ( B ) Poly(A) PCR was performed with either 25, 5, 2.5, or 0.5 µg of AL1 primer and the Taq–Phusion blend from (A). Above—Relative abundance for the indicated genes and preamplification conditions was measured by quantitative PCR (qPCR). Data are shown as the median inverse quantification cycle (40–Cq) ± range from n = 3 amplification replicates and were analysed by two-way (A) or one-way (B) ANOVA with replication. Below—Preamplifications were analysed by agarose gel electrophoresis to separate poly(A)-amplified cDNA from nonspecific, low molecular-weight concatemer (n.s.). Qualitatively similar results were obtained separately three times. Lanes were cropped by poly(A) PCR cycles for display but were electrophoresed on the same agarose gel and processed identically. The uncropped image is shown in Supplementary Fig. S13A .
    Figure Legend Snippet: A blend of Taq–Phusion polymerases improves selective poly(A) amplification of cDNA and reduces AL1 primer requirements. Cells were obtained by LCM from a human breast biopsy and split into 10-cell equivalent amplification replicates. ( A ) Poly(A) PCR was performed with 15 µg of AL1 primer with Taq alone (10 units), Phusion alone (4 units) or Taq/Phusion combination (3.75 units/1.5 units). ( B ) Poly(A) PCR was performed with either 25, 5, 2.5, or 0.5 µg of AL1 primer and the Taq–Phusion blend from (A). Above—Relative abundance for the indicated genes and preamplification conditions was measured by quantitative PCR (qPCR). Data are shown as the median inverse quantification cycle (40–Cq) ± range from n = 3 amplification replicates and were analysed by two-way (A) or one-way (B) ANOVA with replication. Below—Preamplifications were analysed by agarose gel electrophoresis to separate poly(A)-amplified cDNA from nonspecific, low molecular-weight concatemer (n.s.). Qualitatively similar results were obtained separately three times. Lanes were cropped by poly(A) PCR cycles for display but were electrophoresed on the same agarose gel and processed identically. The uncropped image is shown in Supplementary Fig. S13A .

    Techniques Used: Amplification, Laser Capture Microdissection, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight

    6) Product Images from "In situ 10-cell RNA sequencing in tissue and tumor biopsy samples"

    Article Title: In situ 10-cell RNA sequencing in tissue and tumor biopsy samples

    Journal: bioRxiv

    doi: 10.1101/444182

    A blend of Taq–Phusion polymerases improves selective poly(A) amplification of cDNA and reduces AL1 primer requirements. Cells were obtained by LCM from a human breast biopsy and split into 10-cell equivalent amplification replicates. (A) Poly(A) PCR was performed with 15 μg of AL1 primer with Taq alone (10 units), Phusion alone (4 units) or Taq/Phusion combination (3.75 units/1.5 units). (B) Poly(A) PCR was performed with either 25, 5, 2.5 or 0.5 μg of AL1 primer and the Taq–Phusion blend from (A). Above—Relative abundance for the indicated genes and preamplification conditions was measured by quantitative PCR (qPCR). Data are shown as the median inverse quantification cycle (40–Cq) ± range from n = 3 amplification replicates and were analyzed by two-way (A) or one-way (B) ANOVA with replication. Below—Preamplifications were analyzed by agarose gel electrophoresis to separate poly(A)-amplified cDNA from nonspecific, low molecular-weight concatemer (n.s.). Qualitatively similar results were obtained separately three times.
    Figure Legend Snippet: A blend of Taq–Phusion polymerases improves selective poly(A) amplification of cDNA and reduces AL1 primer requirements. Cells were obtained by LCM from a human breast biopsy and split into 10-cell equivalent amplification replicates. (A) Poly(A) PCR was performed with 15 μg of AL1 primer with Taq alone (10 units), Phusion alone (4 units) or Taq/Phusion combination (3.75 units/1.5 units). (B) Poly(A) PCR was performed with either 25, 5, 2.5 or 0.5 μg of AL1 primer and the Taq–Phusion blend from (A). Above—Relative abundance for the indicated genes and preamplification conditions was measured by quantitative PCR (qPCR). Data are shown as the median inverse quantification cycle (40–Cq) ± range from n = 3 amplification replicates and were analyzed by two-way (A) or one-way (B) ANOVA with replication. Below—Preamplifications were analyzed by agarose gel electrophoresis to separate poly(A)-amplified cDNA from nonspecific, low molecular-weight concatemer (n.s.). Qualitatively similar results were obtained separately three times.

    Techniques Used: Amplification, Laser Capture Microdissection, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight

    7) Product Images from "RF-Cloning.org: an online tool for the design of restriction-free cloning projects"

    Article Title: RF-Cloning.org: an online tool for the design of restriction-free cloning projects

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks396

    RF-Cloning.org output page. (1) A unique 32 byte hash code is generated for all new projects, and is present in the URL for bookmarking purposes. (2) The hybrid primers are color coded, blue for sequence complementary to the plasmid, and green for the insert. The length of the primers can be adjusted by clicking on the arrow buttons if the user wishes to alter the annealing temperature. (3) If the insert site needs to be adjusted, the user can use the provided arrow buttons. (4) The secondary PCR conditions are optimized for iProof or Phusion as the polymerase, so the user should follow manufacturer’s instructions if using another high fidelity enzyme. ‘Insert’ refers to the mega-primer purified from the primary PCR reaction. (5) The entire sequence of the new plasmid is output, with insert in green and parental plasmid in blue. (6) The plasmid map can be drawn by specifying the positions of markers manually, or by auto-finding common features. Restriction enzyme cut sites can also be specified or automatically identified. If desired, the plasmid can be exported as a genbank file. (7) All projects are automatically saved, but making changes to the output page will activate the save button so those changes can be uploaded to the database. If the user has registered an account to access the plasmid management system, the save button will attach the project to their profile. (8) After the project has been completed and sent for sequencing, the sequencing results can be copied into a popup window for BLAST2 sequence alignment.
    Figure Legend Snippet: RF-Cloning.org output page. (1) A unique 32 byte hash code is generated for all new projects, and is present in the URL for bookmarking purposes. (2) The hybrid primers are color coded, blue for sequence complementary to the plasmid, and green for the insert. The length of the primers can be adjusted by clicking on the arrow buttons if the user wishes to alter the annealing temperature. (3) If the insert site needs to be adjusted, the user can use the provided arrow buttons. (4) The secondary PCR conditions are optimized for iProof or Phusion as the polymerase, so the user should follow manufacturer’s instructions if using another high fidelity enzyme. ‘Insert’ refers to the mega-primer purified from the primary PCR reaction. (5) The entire sequence of the new plasmid is output, with insert in green and parental plasmid in blue. (6) The plasmid map can be drawn by specifying the positions of markers manually, or by auto-finding common features. Restriction enzyme cut sites can also be specified or automatically identified. If desired, the plasmid can be exported as a genbank file. (7) All projects are automatically saved, but making changes to the output page will activate the save button so those changes can be uploaded to the database. If the user has registered an account to access the plasmid management system, the save button will attach the project to their profile. (8) After the project has been completed and sent for sequencing, the sequencing results can be copied into a popup window for BLAST2 sequence alignment.

    Techniques Used: Clone Assay, Polyacrylamide Gel Electrophoresis, Generated, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Purification

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    New England Biolabs phusion polymerase
    Sequencing ultra-short reads on the MinION. a) 1) Molecular inversion probes (MIPs) are annealed to the target sequence (blue) at anchor site 1 (AS1, orange) and anchor site 2 (AS2, green). <t>Phusion</t> polymerase copies the target sequence into the MIP; the lack of 5’ → 3’ exonuclease activity ensures that extension halts when the polymerase reaches AS2. 2) Ampligase ligates the extended template to the phosphorylated 5’ end of the MIP, generating circular ssDNA. Linear ss- or dsDNA fragments are degraded by a combination of exonuclease I and exonuclease III. 3) The circular DNA is subjected to RCA to generate tandem repeats of the original target, yielding ultra-long, concatemerized ssDNA. 4) The RCA product is converted to dsDNA with Taq polymerase and subjected to ONT library preparation. 5) Sequencing reads are collected from a new MinION R9.4 flow-cell run for 24 hrs. b) The raw sequences are compiled and analyzed. The identified repeats have poor accuracy in isolation, but since the sequencing errors vary across repeats, they can be aligned together to produce a high-fidelity consensus sequence.
    Phusion Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion polymerase/product/New England Biolabs
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    99
    New England Biolabs phusion high fidelity dna polymerase
    Cloned pre-mir-122 stem-loop region sequences from HepG2 <t>DNA</t> show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with <t>Phusion</t> high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    New England Biolabs phusion buffer
    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, <t>Phusion</t> and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.
    Phusion Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs phusion
    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. <t>Phusion;</t> 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.
    Phusion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Sequencing ultra-short reads on the MinION. a) 1) Molecular inversion probes (MIPs) are annealed to the target sequence (blue) at anchor site 1 (AS1, orange) and anchor site 2 (AS2, green). Phusion polymerase copies the target sequence into the MIP; the lack of 5’ → 3’ exonuclease activity ensures that extension halts when the polymerase reaches AS2. 2) Ampligase ligates the extended template to the phosphorylated 5’ end of the MIP, generating circular ssDNA. Linear ss- or dsDNA fragments are degraded by a combination of exonuclease I and exonuclease III. 3) The circular DNA is subjected to RCA to generate tandem repeats of the original target, yielding ultra-long, concatemerized ssDNA. 4) The RCA product is converted to dsDNA with Taq polymerase and subjected to ONT library preparation. 5) Sequencing reads are collected from a new MinION R9.4 flow-cell run for 24 hrs. b) The raw sequences are compiled and analyzed. The identified repeats have poor accuracy in isolation, but since the sequencing errors vary across repeats, they can be aligned together to produce a high-fidelity consensus sequence.

    Journal: bioRxiv

    Article Title: High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences

    doi: 10.1101/552224

    Figure Lengend Snippet: Sequencing ultra-short reads on the MinION. a) 1) Molecular inversion probes (MIPs) are annealed to the target sequence (blue) at anchor site 1 (AS1, orange) and anchor site 2 (AS2, green). Phusion polymerase copies the target sequence into the MIP; the lack of 5’ → 3’ exonuclease activity ensures that extension halts when the polymerase reaches AS2. 2) Ampligase ligates the extended template to the phosphorylated 5’ end of the MIP, generating circular ssDNA. Linear ss- or dsDNA fragments are degraded by a combination of exonuclease I and exonuclease III. 3) The circular DNA is subjected to RCA to generate tandem repeats of the original target, yielding ultra-long, concatemerized ssDNA. 4) The RCA product is converted to dsDNA with Taq polymerase and subjected to ONT library preparation. 5) Sequencing reads are collected from a new MinION R9.4 flow-cell run for 24 hrs. b) The raw sequences are compiled and analyzed. The identified repeats have poor accuracy in isolation, but since the sequencing errors vary across repeats, they can be aligned together to produce a high-fidelity consensus sequence.

    Article Snippet: For this reaction, we employ the Phusion polymerase (New England Biolabs), which lacks 5’ → 3’ exonuclease activity, thereby ensuring that extension halts at the 5’ end of the second hybridization site, where ligation is to occur.

    Techniques: Sequencing, Activity Assay, Isolation

    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Journal: PLoS ONE

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    doi: 10.1371/journal.pone.0122471

    Figure Lengend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Article Snippet: The observed error rate was ~14-fold higher than that reported for Phusion High-Fidelity DNA Polymerase (GC Buffer) (New England Biolabs; ) which, if this error rate is correct, suggests that these changes are unlikely to have arisen solely as a result of PCR errors.

    Techniques: Clone Assay, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Journal: Nucleic Acids Research

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    doi: 10.1093/nar/gkv036

    Figure Lengend Snippet: Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Article Snippet: Extension An extension mix containing 17.25 μl water, 2.5 μl Phusion buffer (10×), 2.5 μl dNTPs (2 mM) and 0.25 μl Phusion (2 U/ μl New England Biolabs, Ipswich, MA, USA) was pre-heated at 65°C before used to resuspend the beads from the hybridization.

    Techniques: Activity Assay, Selection, Construct

    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Journal: Scientific Reports

    Article Title: In vitro synthesis of gene-length single-stranded DNA

    doi: 10.1038/s41598-018-24677-5

    Figure Lengend Snippet: ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Article Snippet: The following enzymes were purchased from the respective commercial providers to test enzymatic production of ssDNA: AccuStart™, AccuStart™ II, and AccuStart™ HiFi from Quantabio; Q5® hot start HiFi, Phusion®, LongAmp®, Deep Vent®, and Deep Vent® (exo-) from New England BioLabs Inc. (NEB); AccuPrime™, Platinum™ SuperFi™, Tth and DreamTaq™ from ThermoFisher Scientific Inc.; GoTaq® from Promega (Promega corp.); and LA Taq ® from Takara Bio.

    Techniques: Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Produced, Labeling, Marker, Purification, Molecular Weight, Staining