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    New England Biolabs bst dna polymerase large fragment
    Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, <t>Bst</t> <t>DNA</t> polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).
    Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs large fragment bst dna polymerase
    Evaluation of <t>DNA</t> 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 <t>Bst</t> 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.
    Large Fragment Bst Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs large fragment bst 2 0 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.
    Large Fragment Bst 2 0 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs bst large fragment 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.
    Bst Large Fragment Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bst 2 0 warmstart 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.
    Bst 2 0 Warmstart Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 473 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    New England Biolabs enzyme solution
    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.
    Enzyme Solution, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Journal: PLoS ONE

    Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli

    doi: 10.1371/journal.pone.0230881

    Figure Lengend Snippet: Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

    Article Snippet: The CPA assay was conducted in a reaction mixture of 30 μL total volume containing 6 μL 1s (10 mM), 1.5 μL 2a/3a (10 mM), 0.5 μL 4s/5a (10 mM), 2.5 μL (1.0 mM) dNTPs (Takara Bio, Dalian, CN), 3 μL 10 × Thermol-Pol reaction buffer, 1.5 μL Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA, USA), 3 μL (1 mM) MgCl2 (Sigma-Aldrich, St. Louis, MO, USA), 2 μL (0.8 M) betaine (Sigma-Aldrich) and 2 μL of an appropriate concentration of DNA template (E . coli C83920).

    Techniques: Amplification, Selection, Incubation, Concentration Assay, Marker, Negative Control

    Effect of dNTPs, ATP, PPi and APS on light output in BART. Simulation of effects of different ingredients on the light output in LAMP-BART in a “deficient mix” lacking primers and Bst polymerase but containing all other components as described in each case below. (A) Light output detected using varying concentrations of an equimolar mixture of four dNTPs. Light output peaks at 500 µM total dNTP concentration. (B) Light output detected using varying concentrations of ATP in the presence of 250 µM equimolar dNTPs. Light output is higher than in panel (A) and reaches saturation at 100 µM ATP, showing greater sensitivity to ATP. (C) Inhibitory effect of different concentrations of PPi on the light emission in the presence of 250 µM dNTPs and 100 µM ATP. (D) Stimulatory effect of increasing concentrations of APS on the light emission in the presence of 250 µM dNTPs and 100 µM PPi. (E) Effect of different concentrations of APS on BART curves in complete LAMP-BART formulation with 10 7 ChAT target DNA (red – 100 µM, navy – 200 µM, brown – 500 µM, green – 750 µM, blue - 1000 µM). As APS concentration is increased, there is little effect on peaking time but more PP i is converted to ATP resulting in a lower rate of inhibition of luciferase and a slower “switch off” of light output.

    Journal: PLoS ONE

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

    doi: 10.1371/journal.pone.0014155

    Figure Lengend Snippet: Effect of dNTPs, ATP, PPi and APS on light output in BART. Simulation of effects of different ingredients on the light output in LAMP-BART in a “deficient mix” lacking primers and Bst polymerase but containing all other components as described in each case below. (A) Light output detected using varying concentrations of an equimolar mixture of four dNTPs. Light output peaks at 500 µM total dNTP concentration. (B) Light output detected using varying concentrations of ATP in the presence of 250 µM equimolar dNTPs. Light output is higher than in panel (A) and reaches saturation at 100 µM ATP, showing greater sensitivity to ATP. (C) Inhibitory effect of different concentrations of PPi on the light emission in the presence of 250 µM dNTPs and 100 µM ATP. (D) Stimulatory effect of increasing concentrations of APS on the light emission in the presence of 250 µM dNTPs and 100 µM PPi. (E) Effect of different concentrations of APS on BART curves in complete LAMP-BART formulation with 10 7 ChAT target DNA (red – 100 µM, navy – 200 µM, brown – 500 µM, green – 750 µM, blue - 1000 µM). As APS concentration is increased, there is little effect on peaking time but more PP i is converted to ATP resulting in a lower rate of inhibition of luciferase and a slower “switch off” of light output.

    Article Snippet: Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA).

    Techniques: Concentration Assay, Inhibition, Luciferase

    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

    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