bst dna polymerase Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    New England Biolabs bst dna polymerase buffer
    Bst Dna Polymerase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase buffer/product/New England Biolabs
    Average 90 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase buffer - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    92
    Eiken Chemical bst dna polymerase
    Bst Dna Polymerase, supplied by Eiken Chemical, used in various techniques. Bioz Stars score: 92/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase/product/Eiken Chemical
    Average 92 stars, based on 101 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-02
    92/100 stars
      Buy from Supplier

    89
    Promega bst dna polymerase
    Bst Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase/product/Promega
    Average 89 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-02
    89/100 stars
      Buy from Supplier

    89
    Bio-Rad bst dna polymerase
    Bst Dna Polymerase, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase/product/Bio-Rad
    Average 89 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-02
    89/100 stars
      Buy from Supplier

    95
    New England Biolabs bst dna polymerase
    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu <t>DNA</t> ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using <t>Bst</t> DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.
    Bst Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase/product/New England Biolabs
    Average 95 stars, based on 1187 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs bst dna polymerase lg frag
    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu <t>DNA</t> ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using <t>Bst</t> DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.
    Bst Dna Polymerase Lg Frag, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase lg frag/product/New England Biolabs
    Average 95 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase lg frag - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs bst2 0 warmstart polymerase
    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated <t>Bst2.0</t> <t>WarmStart</t> polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.
    Bst2 0 Warmstart Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst2 0 warmstart polymerase/product/New England Biolabs
    Average 95 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    bst2 0 warmstart polymerase - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    89
    Lucigen Corp bst dna polymerase
    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated <t>Bst2.0</t> <t>WarmStart</t> polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.
    Bst Dna Polymerase, supplied by Lucigen Corp, used in various techniques. Bioz Stars score: 89/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase/product/Lucigen Corp
    Average 89 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-02
    89/100 stars
      Buy from Supplier

    89
    Thermo Fisher bst dna polymerase
    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated <t>DNA</t> ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by <t>Bst</t> polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.
    Bst Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase/product/Thermo Fisher
    Average 89 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-02
    89/100 stars
      Buy from Supplier

    95
    New England Biolabs bst dna polymerase large fragment
    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 <t>DNA</t> amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U <t>Bst</t> polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).
    Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase large fragment/product/New England Biolabs
    Average 95 stars, based on 264 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase large fragment - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs dna polymerase
    Species-specificity of <t>Hha</t> I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 <t>DNA</t> polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 3856 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/New England Biolabs
    Average 95 stars, based on 3856 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    77
    New England Biolabs strand displacing bst dna polymerase enzyme
    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a <t>DNA</t> extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the <t>Bst</t> strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.
    Strand Displacing Bst Dna Polymerase Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strand displacing bst dna polymerase enzyme/product/New England Biolabs
    Average 77 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    strand displacing bst dna polymerase enzyme - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    80
    New England Biolabs bacillus stearothermophilus bst dna polymerase
    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a <t>DNA</t> extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the <t>Bst</t> strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.
    Bacillus Stearothermophilus Bst Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bacillus stearothermophilus bst dna polymerase/product/New England Biolabs
    Average 80 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    bacillus stearothermophilus bst dna polymerase - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    75
    New England Biolabs oligonucleotides bst dna polymerase large fragment
    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a <t>DNA</t> extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the <t>Bst</t> strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.
    Oligonucleotides Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligonucleotides bst dna polymerase large fragment/product/New England Biolabs
    Average 75 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    oligonucleotides bst dna polymerase large fragment - by Bioz Stars, 2020-02
    75/100 stars
      Buy from Supplier

    78
    Roche l restriction endonuclease bst 1107
    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a <t>DNA</t> extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the <t>Bst</t> strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.
    L Restriction Endonuclease Bst 1107, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l restriction endonuclease bst 1107/product/Roche
    Average 78 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    l restriction endonuclease bst 1107 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    81
    Thermo Fisher bst xi eco ri adaptor
    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a <t>DNA</t> extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the <t>Bst</t> strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.
    Bst Xi Eco Ri Adaptor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst xi eco ri adaptor/product/Thermo Fisher
    Average 81 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    bst xi eco ri adaptor - by Bioz Stars, 2020-02
    81/100 stars
      Buy from Supplier

    84
    New England Biolabs restriction enzyme bst ni
    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a <t>DNA</t> extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the <t>Bst</t> strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.
    Restriction Enzyme Bst Ni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme bst ni/product/New England Biolabs
    Average 84 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme bst ni - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    95
    New England Biolabs bst ui
    Promoter CpG methylation correlates with lack of TUSC3 gene expression. (A) Schematic of TUSC3 locus showing the regions investigated by bisulfite pyrosequencing on the promoter (−105 to −57 relative to the transcriptional start site according to NM_006765) and genotyping assays of the 5′ untranslated region. PCR primers for <t>DNA</t> genotyping are indicated by black arrows while RT-PCR primers for mRNA genotyping are indicated by arrows highlighted in white. Enzyme recognition sites for <t>Bst</t> UI are indicated by “ B ”. (B) Methylation status of TUSC3 promoter region studied by bisulfite pyrosequencing. A similar methylation level of every CpG within each sample is observed and the gene follows “on-or-off” methylation pattern. Each circle represents a CpG site in a sample. Area shaded in black is proportional to the methylation level of the CpG site indicated by pyrosequencing. (C) Validation of complete methylation-sensitive restriction enzyme digestion on unmethylated molecules. Genomic DNA was predigested with Bst UI followed by PCR amplification with TUSC3 and ID2 specific primers ( Table S2 ). Bst UI digestion sites within the ID2 region were unmethylated ( Figure S6 ) and, therefore, no PCR product was generated after enzyme digestion. (D) Allele-specific methylation of TUSC3 on the fragment containing SNP rs12550009 demonstrated by enzyme digestion pyrosequencing. The “Simplex” diagrams (top) show the reference pyrograms by genotype. A heterozygous CT in the methylated samples (PM55 and PM123) displays a homozygous T pattern after Bst UI digestion indicating predominant methylation of the T allele. Allele-specific mRNA expression is concordant with allele-specific methylation on the same SNP rs1250009. Predominant expression of C alleles was observed in the cDNAs generated by RNA specific primers (bottom). RT+ and RT− represent assays with Reverse Transcriptase and without Reverse Transcriptase, respectively.
    Bst Ui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst ui/product/New England Biolabs
    Average 95 stars, based on 335 article reviews
    Price from $9.99 to $1999.99
    bst ui - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    77
    New England Biolabs restriction endonuclease bst u1
    Promoter CpG methylation correlates with lack of TUSC3 gene expression. (A) Schematic of TUSC3 locus showing the regions investigated by bisulfite pyrosequencing on the promoter (−105 to −57 relative to the transcriptional start site according to NM_006765) and genotyping assays of the 5′ untranslated region. PCR primers for <t>DNA</t> genotyping are indicated by black arrows while RT-PCR primers for mRNA genotyping are indicated by arrows highlighted in white. Enzyme recognition sites for <t>Bst</t> UI are indicated by “ B ”. (B) Methylation status of TUSC3 promoter region studied by bisulfite pyrosequencing. A similar methylation level of every CpG within each sample is observed and the gene follows “on-or-off” methylation pattern. Each circle represents a CpG site in a sample. Area shaded in black is proportional to the methylation level of the CpG site indicated by pyrosequencing. (C) Validation of complete methylation-sensitive restriction enzyme digestion on unmethylated molecules. Genomic DNA was predigested with Bst UI followed by PCR amplification with TUSC3 and ID2 specific primers ( Table S2 ). Bst UI digestion sites within the ID2 region were unmethylated ( Figure S6 ) and, therefore, no PCR product was generated after enzyme digestion. (D) Allele-specific methylation of TUSC3 on the fragment containing SNP rs12550009 demonstrated by enzyme digestion pyrosequencing. The “Simplex” diagrams (top) show the reference pyrograms by genotype. A heterozygous CT in the methylated samples (PM55 and PM123) displays a homozygous T pattern after Bst UI digestion indicating predominant methylation of the T allele. Allele-specific mRNA expression is concordant with allele-specific methylation on the same SNP rs1250009. Predominant expression of C alleles was observed in the cDNAs generated by RNA specific primers (bottom). RT+ and RT− represent assays with Reverse Transcriptase and without Reverse Transcriptase, respectively.
    Restriction Endonuclease Bst U1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction endonuclease bst u1/product/New England Biolabs
    Average 77 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    restriction endonuclease bst u1 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    99
    New England Biolabs restriction endonuclease bst ui
    Methylation of the 5′-associated region of VWA2 in normal and tumor tissues of CRC patients. The scale on top is valid for the three panels and refers to the distance (in bp) to the VWA2 transcriptional start site. Panel a: Scheme of the DNA region containing the first exon of VWA2 (exon 1, in red). The yellow diamond indicates the location of the Not I site originally found hypomethylated by the MS-AFLP analyses. The first track (CpGs) shows the CpG sites (black vertical bars) and the promoter-associated CpG island (in pink). The second track (PCRs) shows the four products analyzed by bisulfite-based <t>PCR</t> (light blue squares) and the <t>Bst</t> UI sites studied by COBRA (dark blue vertical bars). The third track (HM450K) indicates the position of the 10 probes of the Illumina HM450K methylation arrays within this region. Panel b: methylation status in normal (N) and tumor (T) tissues from CRC patients 725, 726, 727, 828, 829, 830, 831 and 832. Methylation was estimated by direct Sanger’s sequencing of the four PCR products shown in panel a. Panel c: average methylation level of normal (yellow) and tumor (orange) tissues from the eight patients shown in panel b, at every CpG site within the studied region. The solid lines represent the locally weighted moving average.
    Restriction Endonuclease Bst Ui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction endonuclease bst ui/product/New England Biolabs
    Average 99 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    restriction endonuclease bst ui - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    88
    Eiken Chemical dna amplification kit
    Methylation of the 5′-associated region of VWA2 in normal and tumor tissues of CRC patients. The scale on top is valid for the three panels and refers to the distance (in bp) to the VWA2 transcriptional start site. Panel a: Scheme of the DNA region containing the first exon of VWA2 (exon 1, in red). The yellow diamond indicates the location of the Not I site originally found hypomethylated by the MS-AFLP analyses. The first track (CpGs) shows the CpG sites (black vertical bars) and the promoter-associated CpG island (in pink). The second track (PCRs) shows the four products analyzed by bisulfite-based <t>PCR</t> (light blue squares) and the <t>Bst</t> UI sites studied by COBRA (dark blue vertical bars). The third track (HM450K) indicates the position of the 10 probes of the Illumina HM450K methylation arrays within this region. Panel b: methylation status in normal (N) and tumor (T) tissues from CRC patients 725, 726, 727, 828, 829, 830, 831 and 832. Methylation was estimated by direct Sanger’s sequencing of the four PCR products shown in panel a. Panel c: average methylation level of normal (yellow) and tumor (orange) tissues from the eight patients shown in panel b, at every CpG site within the studied region. The solid lines represent the locally weighted moving average.
    Dna Amplification Kit, supplied by Eiken Chemical, used in various techniques. Bioz Stars score: 88/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna amplification kit/product/Eiken Chemical
    Average 88 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    dna amplification kit - by Bioz Stars, 2020-02
    88/100 stars
      Buy from Supplier

    95
    New England Biolabs nebnext end repair module
    Methylation of the 5′-associated region of VWA2 in normal and tumor tissues of CRC patients. The scale on top is valid for the three panels and refers to the distance (in bp) to the VWA2 transcriptional start site. Panel a: Scheme of the DNA region containing the first exon of VWA2 (exon 1, in red). The yellow diamond indicates the location of the Not I site originally found hypomethylated by the MS-AFLP analyses. The first track (CpGs) shows the CpG sites (black vertical bars) and the promoter-associated CpG island (in pink). The second track (PCRs) shows the four products analyzed by bisulfite-based <t>PCR</t> (light blue squares) and the <t>Bst</t> UI sites studied by COBRA (dark blue vertical bars). The third track (HM450K) indicates the position of the 10 probes of the Illumina HM450K methylation arrays within this region. Panel b: methylation status in normal (N) and tumor (T) tissues from CRC patients 725, 726, 727, 828, 829, 830, 831 and 832. Methylation was estimated by direct Sanger’s sequencing of the four PCR products shown in panel a. Panel c: average methylation level of normal (yellow) and tumor (orange) tissues from the eight patients shown in panel b, at every CpG site within the studied region. The solid lines represent the locally weighted moving average.
    Nebnext End Repair Module, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebnext end repair module/product/New England Biolabs
    Average 95 stars, based on 357 article reviews
    Price from $9.99 to $1999.99
    nebnext end repair module - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs polymerase
    Methylation of the 5′-associated region of VWA2 in normal and tumor tissues of CRC patients. The scale on top is valid for the three panels and refers to the distance (in bp) to the VWA2 transcriptional start site. Panel a: Scheme of the DNA region containing the first exon of VWA2 (exon 1, in red). The yellow diamond indicates the location of the Not I site originally found hypomethylated by the MS-AFLP analyses. The first track (CpGs) shows the CpG sites (black vertical bars) and the promoter-associated CpG island (in pink). The second track (PCRs) shows the four products analyzed by bisulfite-based <t>PCR</t> (light blue squares) and the <t>Bst</t> UI sites studied by COBRA (dark blue vertical bars). The third track (HM450K) indicates the position of the 10 probes of the Illumina HM450K methylation arrays within this region. Panel b: methylation status in normal (N) and tumor (T) tissues from CRC patients 725, 726, 727, 828, 829, 830, 831 and 832. Methylation was estimated by direct Sanger’s sequencing of the four PCR products shown in panel a. Panel c: average methylation level of normal (yellow) and tumor (orange) tissues from the eight patients shown in panel b, at every CpG site within the studied region. The solid lines represent the locally weighted moving average.
    Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 833 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polymerase/product/New England Biolabs
    Average 95 stars, based on 833 article reviews
    Price from $9.99 to $1999.99
    polymerase - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    84
    OptiGene Ltd loopamp dna amplification kit
    Methylation of the 5′-associated region of VWA2 in normal and tumor tissues of CRC patients. The scale on top is valid for the three panels and refers to the distance (in bp) to the VWA2 transcriptional start site. Panel a: Scheme of the DNA region containing the first exon of VWA2 (exon 1, in red). The yellow diamond indicates the location of the Not I site originally found hypomethylated by the MS-AFLP analyses. The first track (CpGs) shows the CpG sites (black vertical bars) and the promoter-associated CpG island (in pink). The second track (PCRs) shows the four products analyzed by bisulfite-based <t>PCR</t> (light blue squares) and the <t>Bst</t> UI sites studied by COBRA (dark blue vertical bars). The third track (HM450K) indicates the position of the 10 probes of the Illumina HM450K methylation arrays within this region. Panel b: methylation status in normal (N) and tumor (T) tissues from CRC patients 725, 726, 727, 828, 829, 830, 831 and 832. Methylation was estimated by direct Sanger’s sequencing of the four PCR products shown in panel a. Panel c: average methylation level of normal (yellow) and tumor (orange) tissues from the eight patients shown in panel b, at every CpG site within the studied region. The solid lines represent the locally weighted moving average.
    Loopamp Dna Amplification Kit, supplied by OptiGene Ltd, used in various techniques. Bioz Stars score: 84/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/loopamp dna amplification kit/product/OptiGene Ltd
    Average 84 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    loopamp dna amplification kit - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    Image Search Results


    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.

    Journal: Frontiers in Microbiology

    Article Title: Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

    doi: 10.3389/fmicb.2015.01385

    Figure Lengend Snippet: Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.

    Article Snippet: Circularized probes were amplified with replication primers RCA1 and RCA2 with Bst DNA polymerase.

    Techniques: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Hybridization, DNA Synthesis, Nucleic Acid Electrophoresis, SYBR Green Assay

    Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.

    Journal: PLoS ONE

    Article Title: Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pone.0139286

    Figure Lengend Snippet: Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.

    Article Snippet: LAMP Assays LAMP reactions contained 1.6 μM each of primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM each of LF and FB, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of Bst 2.0 DNA Polymerase (New England Biolabs) mixed with one of several template DNAs, or 10 mM Tris-HCl (pH 8), 0.1 mM EDTA for non-template controls (NTC), in a total volume of 25 μl.

    Techniques: Lamp Assay, Amplification, Concentration Assay, Standard Deviation

    Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.

    Journal: PLoS ONE

    Article Title: Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pone.0139286

    Figure Lengend Snippet: Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.

    Article Snippet: LAMP Assays LAMP reactions contained 1.6 μM each of primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM each of LF and FB, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of Bst 2.0 DNA Polymerase (New England Biolabs) mixed with one of several template DNAs, or 10 mM Tris-HCl (pH 8), 0.1 mM EDTA for non-template controls (NTC), in a total volume of 25 μl.

    Techniques: DNA Extraction

    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Journal: MethodsX

    Article Title: NGS-based deep bisulfite sequencing

    doi: 10.1016/j.mex.2015.11.008

    Figure Lengend Snippet: Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Article Snippet: The current protocol also uses a mixture of T4 ligase and Bst2.0 WarmStart polymerase with two stepwise incubations, a 25-min incubation at 25 °C for the ligation reaction by T4 ligase and another 30-min incubation at 65 °C for the extension/displacement reaction by Bst2.0 WarmStart polymerase (available from NEB).

    Techniques: Next-Generation Sequencing, Methylation Sequencing, Flow Cytometry, Ligation, Polymerase Chain Reaction, Derivative Assay, Activity Assay

    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Journal: Nucleic Acids Research

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

    doi: 10.1093/nar/gkp037

    Figure Lengend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Article Snippet: Nick repair Nicks at the 3′-junctions between DNA fragments and adaptors were repaired by the strand-displacement activity of Bst DNA polymerase, Large Fragment (Fermentas).

    Techniques: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

    Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Journal: Nucleic Acids Research

    Article Title: Loop-mediated isothermal amplification of DNA

    doi:

    Figure Lengend Snippet: Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

    Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

    Techniques: Amplification, Incubation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Journal: Nucleic Acids Research

    Article Title: Loop-mediated isothermal amplification of DNA

    doi:

    Figure Lengend Snippet: Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

    Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

    Techniques: Southern Blot, Hybridization, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Staining, Marker

    Evaluation of DNA amplification bias using array–CGH on human cDNA microarrays. ( A ) To assess the extent of dynamic range compression, three microarray–CGH experiments were performed in duplicate using genomic female DNA (46, XX) versus genomic male DNA, or DNA from cell lines containing 3 X-chromosomes (47, XXX) and 5 X-chromosomes (49, XXXXX) with a normal number of autosomes against genomic male DNA. Autosomal genes located in Chromosomes 1 and 2 are compared with genes located in Chromosome X for the same set of experiments. Ratio values correspond to the average of two independent experiments, and are displayed in a log 2 scale. Averaging the log 2 ratios for the X-linked probes in the three different experiments gives values of 0.234 ± 0.143 (1.176 in linear scale, average of 112 probes), 0.423 ± 0.220 (1.341 in linear scale, average of 113 probes), and 0.681 ± 0.290 (1.603 in linear scale, average of 115 probes) for the 2X, 3X, and 5X experiments, respectively. Dynamically compressed ratios can be converted to actual ratios by fitting log 2 . ( B ) Comparison of DNA polymerase-induced representational distortion using human DNA samples. Normal human DNA was amplified with either φ29 or Bst DNA polymerase, labeled with Cy3, and hybridized against similarly amplified human DNA labeled with Cy5. Plots for Chromosomes 1 and 2 are shown in the same scale as the plot in A . ( C ) Confidence limits for array–CGH analysis of human DNA. Plots correspond to unamplified human female versus male DNAs and whole genome Bst -amplified human female versus Bst -amplified male DNAs. Average log 2 fluorescence ratios for replicate spots are ordered according to the chromosome number and the position in the chromosome. Ratio values for X-linked genes show a similar distribution to that observed for the 2X dosage in A . Confidence limits (horizontal dashed lines) for 99.9% of data for autosomal genes are between −0.262 and 0.262 (0.833 and 1.199 when expressed as linear ratios) for the unamplified experiment. The same confidence bounds calculated for the unamplified experiment are replicated in the plot of ratios generated by microarray analysis of amplified DNA.

    Journal: Genome Research

    Article Title: Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array-CGH

    doi: 10.1101/gr.377203

    Figure Lengend Snippet: Evaluation of DNA amplification bias using array–CGH on human cDNA microarrays. ( A ) To assess the extent of dynamic range compression, three microarray–CGH experiments were performed in duplicate using genomic female DNA (46, XX) versus genomic male DNA, or DNA from cell lines containing 3 X-chromosomes (47, XXX) and 5 X-chromosomes (49, XXXXX) with a normal number of autosomes against genomic male DNA. Autosomal genes located in Chromosomes 1 and 2 are compared with genes located in Chromosome X for the same set of experiments. Ratio values correspond to the average of two independent experiments, and are displayed in a log 2 scale. Averaging the log 2 ratios for the X-linked probes in the three different experiments gives values of 0.234 ± 0.143 (1.176 in linear scale, average of 112 probes), 0.423 ± 0.220 (1.341 in linear scale, average of 113 probes), and 0.681 ± 0.290 (1.603 in linear scale, average of 115 probes) for the 2X, 3X, and 5X experiments, respectively. Dynamically compressed ratios can be converted to actual ratios by fitting log 2 . ( B ) Comparison of DNA polymerase-induced representational distortion using human DNA samples. Normal human DNA was amplified with either φ29 or Bst DNA polymerase, labeled with Cy3, and hybridized against similarly amplified human DNA labeled with Cy5. Plots for Chromosomes 1 and 2 are shown in the same scale as the plot in A . ( C ) Confidence limits for array–CGH analysis of human DNA. Plots correspond to unamplified human female versus male DNAs and whole genome Bst -amplified human female versus Bst -amplified male DNAs. Average log 2 fluorescence ratios for replicate spots are ordered according to the chromosome number and the position in the chromosome. Ratio values for X-linked genes show a similar distribution to that observed for the 2X dosage in A . Confidence limits (horizontal dashed lines) for 99.9% of data for autosomal genes are between −0.262 and 0.262 (0.833 and 1.199 when expressed as linear ratios) for the unamplified experiment. The same confidence bounds calculated for the unamplified experiment are replicated in the plot of ratios generated by microarray analysis of amplified DNA.

    Article Snippet: The reaction mixture was then brought up to 30 μL containing 400 μM dNTPs in 1× buffer and the polymerase. φ29 was added at a final concentration of 0.1 units/μL, and large fragment Bst DNA polymerase (New England Biolabs) at 0.35 units/μL.

    Techniques: Amplification, Microarray, Labeling, Fluorescence, Generated

    Evaluation of amplification bias using array–CGH on yeast cDNA microarrays. Microarrays contained 6135 unique yeast ORFs. Fluorescence ratios were measured and plotted against the order of the genes in the genome, starting from Chromosome I to Chromosome XVI. ( Upper left panel) Analysis of a microarray hybridized with the same DNA, labeled with Cy3 and Cy5. ( Upper right panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Lower left panel) DNA from the yeast KO strain was amplified using Bst DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center left panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase for only 2 h, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center right panel) Equivalent experiment using Bst DNA polymerase. ( Lower right panel) DNAs from the two different yeast strains were amplified to the same extent using Bst and hybridized together. The three genes known to be deleted appear as outlier data points indicated by arrows. The other two outlier data points, near genes GIN4 and CLA4 , have abnormally low area values of 48 and 21 according to the Spot analysis software, compared with the average of 255 for all the spots in the array. This abnormality could be produced by a fluorescent speckle over the spot, resulting in unreliable ratios.

    Journal: Genome Research

    Article Title: Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array-CGH

    doi: 10.1101/gr.377203

    Figure Lengend Snippet: Evaluation of amplification bias using array–CGH on yeast cDNA microarrays. Microarrays contained 6135 unique yeast ORFs. Fluorescence ratios were measured and plotted against the order of the genes in the genome, starting from Chromosome I to Chromosome XVI. ( Upper left panel) Analysis of a microarray hybridized with the same DNA, labeled with Cy3 and Cy5. ( Upper right panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Lower left panel) DNA from the yeast KO strain was amplified using Bst DNA polymerase, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center left panel) DNA from the yeast KO strain was amplified using φ29 DNA polymerase for only 2 h, labeled with Cy3, and hybridized against unamplified (Cy5) DNA from the same strain. ( Center right panel) Equivalent experiment using Bst DNA polymerase. ( Lower right panel) DNAs from the two different yeast strains were amplified to the same extent using Bst and hybridized together. The three genes known to be deleted appear as outlier data points indicated by arrows. The other two outlier data points, near genes GIN4 and CLA4 , have abnormally low area values of 48 and 21 according to the Spot analysis software, compared with the average of 255 for all the spots in the array. This abnormality could be produced by a fluorescent speckle over the spot, resulting in unreliable ratios.

    Article Snippet: The reaction mixture was then brought up to 30 μL containing 400 μM dNTPs in 1× buffer and the polymerase. φ29 was added at a final concentration of 0.1 units/μL, and large fragment Bst DNA polymerase (New England Biolabs) at 0.35 units/μL.

    Techniques: Amplification, Fluorescence, Microarray, Labeling, Software, Produced

    Gel electrophoresis analysis of amplified DNA. ( A ) Control reactions were incubated for 5 h in a 30-μL volume containing no DNA, or 7.5 ng of human DNA. Samples representing 5% of the reaction were denatured in alkaline buffer and analyzed on a 0.5% alkaline agarose gel, and stained with SYBR-green II (Molecular Probes). Lanes labeled M contained phage λ DNA digested with restriction endonuclease Hin dIII. Reactions catalyzed by φ29 polymerase were incubated with (+) or without (−) input of denatured human DNA. Random heptamers contained standard (−) DNA, or were modified by the addition of two nitroindole groups (+) at the 5′ end. ( B ) Time-course reactions for φ29 and Bst DNA polymerases. Reactions were performed using nitroindole-modified primers. Every hour (from 1–5 h), 1.5 μL was removed, denatured in alkaline buffer, and analyzed in 0.5% alkaline agarose gel. Lanes labeled C correspond to control samples incubated for 5 h without input DNA. The lane labeled G corresponds to a gel load of genomic DNA equivalent to 100× the original DNA input of the amplification reactions. ( C ) Plots under gel images display the time course (fold amplification vs. time) of both polymerase reactions, generated by quantification of DNA yield with the PicoGreen Quantitation Kit. Background fluorescence at time 0 was subtracted for all time points. Each point represents the mean (±1 SD) of four independent analyses.

    Journal: Genome Research

    Article Title: Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array-CGH

    doi: 10.1101/gr.377203

    Figure Lengend Snippet: Gel electrophoresis analysis of amplified DNA. ( A ) Control reactions were incubated for 5 h in a 30-μL volume containing no DNA, or 7.5 ng of human DNA. Samples representing 5% of the reaction were denatured in alkaline buffer and analyzed on a 0.5% alkaline agarose gel, and stained with SYBR-green II (Molecular Probes). Lanes labeled M contained phage λ DNA digested with restriction endonuclease Hin dIII. Reactions catalyzed by φ29 polymerase were incubated with (+) or without (−) input of denatured human DNA. Random heptamers contained standard (−) DNA, or were modified by the addition of two nitroindole groups (+) at the 5′ end. ( B ) Time-course reactions for φ29 and Bst DNA polymerases. Reactions were performed using nitroindole-modified primers. Every hour (from 1–5 h), 1.5 μL was removed, denatured in alkaline buffer, and analyzed in 0.5% alkaline agarose gel. Lanes labeled C correspond to control samples incubated for 5 h without input DNA. The lane labeled G corresponds to a gel load of genomic DNA equivalent to 100× the original DNA input of the amplification reactions. ( C ) Plots under gel images display the time course (fold amplification vs. time) of both polymerase reactions, generated by quantification of DNA yield with the PicoGreen Quantitation Kit. Background fluorescence at time 0 was subtracted for all time points. Each point represents the mean (±1 SD) of four independent analyses.

    Article Snippet: The reaction mixture was then brought up to 30 μL containing 400 μM dNTPs in 1× buffer and the polymerase. φ29 was added at a final concentration of 0.1 units/μL, and large fragment Bst DNA polymerase (New England Biolabs) at 0.35 units/μL.

    Techniques: Nucleic Acid Electrophoresis, Amplification, Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, Labeling, Modification, Generated, Quantitation Assay, Fluorescence

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Article Snippet: For the evaluation of the Hha I LAMP primer set with either Bst 2.0 DNA polymerase or Bst 2.0 WarmStart DNA polymerase (New England Biolabs), reactions were set up and performed as described above, except 50 mM KCl was used.

    Techniques: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Article Snippet: For the evaluation of the Hha I LAMP primer set with either Bst 2.0 DNA polymerase or Bst 2.0 WarmStart DNA polymerase (New England Biolabs), reactions were set up and performed as described above, except 50 mM KCl was used.

    Techniques: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Article Snippet: For the evaluation of the Hha I LAMP primer set with either Bst 2.0 DNA polymerase or Bst 2.0 WarmStart DNA polymerase (New England Biolabs), reactions were set up and performed as described above, except 50 mM KCl was used.

    Techniques: Lamp Assay, Infection, Isolation

    mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a DNA extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the Bst strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.

    Journal: Investigative Genetics

    Article Title: DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

    doi: 10.1186/2041-2223-4-26

    Figure Lengend Snippet: mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a DNA extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the Bst strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.

    Article Snippet: The strand-displacing Bst DNA polymerase enzyme (large fragment, New England Biolabs) was used to release library DNA from the DNA-capture probe (immobilised to beads on the magnet).

    Techniques: Hybridization, Polymerase Chain Reaction, Generated, Ligation, Amplification, Blocking Assay, Concentration Assay, Next-Generation Sequencing

    Promoter CpG methylation correlates with lack of TUSC3 gene expression. (A) Schematic of TUSC3 locus showing the regions investigated by bisulfite pyrosequencing on the promoter (−105 to −57 relative to the transcriptional start site according to NM_006765) and genotyping assays of the 5′ untranslated region. PCR primers for DNA genotyping are indicated by black arrows while RT-PCR primers for mRNA genotyping are indicated by arrows highlighted in white. Enzyme recognition sites for Bst UI are indicated by “ B ”. (B) Methylation status of TUSC3 promoter region studied by bisulfite pyrosequencing. A similar methylation level of every CpG within each sample is observed and the gene follows “on-or-off” methylation pattern. Each circle represents a CpG site in a sample. Area shaded in black is proportional to the methylation level of the CpG site indicated by pyrosequencing. (C) Validation of complete methylation-sensitive restriction enzyme digestion on unmethylated molecules. Genomic DNA was predigested with Bst UI followed by PCR amplification with TUSC3 and ID2 specific primers ( Table S2 ). Bst UI digestion sites within the ID2 region were unmethylated ( Figure S6 ) and, therefore, no PCR product was generated after enzyme digestion. (D) Allele-specific methylation of TUSC3 on the fragment containing SNP rs12550009 demonstrated by enzyme digestion pyrosequencing. The “Simplex” diagrams (top) show the reference pyrograms by genotype. A heterozygous CT in the methylated samples (PM55 and PM123) displays a homozygous T pattern after Bst UI digestion indicating predominant methylation of the T allele. Allele-specific mRNA expression is concordant with allele-specific methylation on the same SNP rs1250009. Predominant expression of C alleles was observed in the cDNAs generated by RNA specific primers (bottom). RT+ and RT− represent assays with Reverse Transcriptase and without Reverse Transcriptase, respectively.

    Journal: PLoS ONE

    Article Title: Human Placental-Specific Epipolymorphism and its Association with Adverse Pregnancy Outcomes

    doi: 10.1371/journal.pone.0007389

    Figure Lengend Snippet: Promoter CpG methylation correlates with lack of TUSC3 gene expression. (A) Schematic of TUSC3 locus showing the regions investigated by bisulfite pyrosequencing on the promoter (−105 to −57 relative to the transcriptional start site according to NM_006765) and genotyping assays of the 5′ untranslated region. PCR primers for DNA genotyping are indicated by black arrows while RT-PCR primers for mRNA genotyping are indicated by arrows highlighted in white. Enzyme recognition sites for Bst UI are indicated by “ B ”. (B) Methylation status of TUSC3 promoter region studied by bisulfite pyrosequencing. A similar methylation level of every CpG within each sample is observed and the gene follows “on-or-off” methylation pattern. Each circle represents a CpG site in a sample. Area shaded in black is proportional to the methylation level of the CpG site indicated by pyrosequencing. (C) Validation of complete methylation-sensitive restriction enzyme digestion on unmethylated molecules. Genomic DNA was predigested with Bst UI followed by PCR amplification with TUSC3 and ID2 specific primers ( Table S2 ). Bst UI digestion sites within the ID2 region were unmethylated ( Figure S6 ) and, therefore, no PCR product was generated after enzyme digestion. (D) Allele-specific methylation of TUSC3 on the fragment containing SNP rs12550009 demonstrated by enzyme digestion pyrosequencing. The “Simplex” diagrams (top) show the reference pyrograms by genotype. A heterozygous CT in the methylated samples (PM55 and PM123) displays a homozygous T pattern after Bst UI digestion indicating predominant methylation of the T allele. Allele-specific mRNA expression is concordant with allele-specific methylation on the same SNP rs1250009. Predominant expression of C alleles was observed in the cDNAs generated by RNA specific primers (bottom). RT+ and RT− represent assays with Reverse Transcriptase and without Reverse Transcriptase, respectively.

    Article Snippet: For Bst UI predigestion assay followed by pyrosequencing on TUSC3 , 200 ng of genomic DNA was digested with 100 units of Bst UI (New England Biolabs) for 18 hours.

    Techniques: CpG Methylation Assay, Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Methylation, Amplification, Generated

    Methylation of the 5′-associated region of VWA2 in normal and tumor tissues of CRC patients. The scale on top is valid for the three panels and refers to the distance (in bp) to the VWA2 transcriptional start site. Panel a: Scheme of the DNA region containing the first exon of VWA2 (exon 1, in red). The yellow diamond indicates the location of the Not I site originally found hypomethylated by the MS-AFLP analyses. The first track (CpGs) shows the CpG sites (black vertical bars) and the promoter-associated CpG island (in pink). The second track (PCRs) shows the four products analyzed by bisulfite-based PCR (light blue squares) and the Bst UI sites studied by COBRA (dark blue vertical bars). The third track (HM450K) indicates the position of the 10 probes of the Illumina HM450K methylation arrays within this region. Panel b: methylation status in normal (N) and tumor (T) tissues from CRC patients 725, 726, 727, 828, 829, 830, 831 and 832. Methylation was estimated by direct Sanger’s sequencing of the four PCR products shown in panel a. Panel c: average methylation level of normal (yellow) and tumor (orange) tissues from the eight patients shown in panel b, at every CpG site within the studied region. The solid lines represent the locally weighted moving average.

    Journal: Scientific Reports

    Article Title: Epigenetic and transcriptional dysregulation of VWA2 associated with a MYC-driven oncogenic program in colorectal cancer

    doi: 10.1038/s41598-018-29378-7

    Figure Lengend Snippet: Methylation of the 5′-associated region of VWA2 in normal and tumor tissues of CRC patients. The scale on top is valid for the three panels and refers to the distance (in bp) to the VWA2 transcriptional start site. Panel a: Scheme of the DNA region containing the first exon of VWA2 (exon 1, in red). The yellow diamond indicates the location of the Not I site originally found hypomethylated by the MS-AFLP analyses. The first track (CpGs) shows the CpG sites (black vertical bars) and the promoter-associated CpG island (in pink). The second track (PCRs) shows the four products analyzed by bisulfite-based PCR (light blue squares) and the Bst UI sites studied by COBRA (dark blue vertical bars). The third track (HM450K) indicates the position of the 10 probes of the Illumina HM450K methylation arrays within this region. Panel b: methylation status in normal (N) and tumor (T) tissues from CRC patients 725, 726, 727, 828, 829, 830, 831 and 832. Methylation was estimated by direct Sanger’s sequencing of the four PCR products shown in panel a. Panel c: average methylation level of normal (yellow) and tumor (orange) tissues from the eight patients shown in panel b, at every CpG site within the studied region. The solid lines represent the locally weighted moving average.

    Article Snippet: For COBRA, after amplification, the PCR products were subjected to digestion with the restriction endonuclease Bst UI (New England Biolabs, Ipswich, MA) or its isoschizomer Bsh12361 (Thermofisher Scientific), and subsequently resolved on agarose gels.

    Techniques: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Combined Bisulfite Restriction Analysis Assay, Sequencing