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Development and validation of a T7 based linear amplification for genomic DNA.

Liu CL, Schreiber SL, Bernstein BE - BMC Genomics (2003)

Bottom Line: Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis.Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach.Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Chemical Biology, and Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA. clliu@fas.harvard.edu

ABSTRACT

Background: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail.

Results: We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach.

Conclusion: We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

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Comparisons of microarray data collected using direct labeling, IVT or R-PCR methods. (A) Bar graph showing correlations between replicates collected using the same protocol, and between the averaged datasets determined using different protocols. (B) Venn diagrams showing overlap between sets of features with the highest Cy5/Cy3 ratios.
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Figure 3: Comparisons of microarray data collected using direct labeling, IVT or R-PCR methods. (A) Bar graph showing correlations between replicates collected using the same protocol, and between the averaged datasets determined using different protocols. (B) Venn diagrams showing overlap between sets of features with the highest Cy5/Cy3 ratios.

Mentions: First, we assessed the reproducibility of the IVT protocol. Three Alu I / Rsa I datasets were independently generated using the IVT protocol. Each dataset contains 4,481 ratios that each reflect the relative abundance of DNA corresponding to a specific array feature (yeast ORF) in the two digests. We found the IVT protocol to be highly reproducible: correlation coefficients calculated between the replicate datasets averaged 0.98. This reproducibility is maintained even when only 5 ng starting material are used (correlation of 0.97). The direct labeling and R-PCR protocols are also highly reproducible, with mean correlations of 0.93 and 0.91, respectively (Fig 3A).


Development and validation of a T7 based linear amplification for genomic DNA.

Liu CL, Schreiber SL, Bernstein BE - BMC Genomics (2003)

Comparisons of microarray data collected using direct labeling, IVT or R-PCR methods. (A) Bar graph showing correlations between replicates collected using the same protocol, and between the averaged datasets determined using different protocols. (B) Venn diagrams showing overlap between sets of features with the highest Cy5/Cy3 ratios.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161798&req=5

Figure 3: Comparisons of microarray data collected using direct labeling, IVT or R-PCR methods. (A) Bar graph showing correlations between replicates collected using the same protocol, and between the averaged datasets determined using different protocols. (B) Venn diagrams showing overlap between sets of features with the highest Cy5/Cy3 ratios.
Mentions: First, we assessed the reproducibility of the IVT protocol. Three Alu I / Rsa I datasets were independently generated using the IVT protocol. Each dataset contains 4,481 ratios that each reflect the relative abundance of DNA corresponding to a specific array feature (yeast ORF) in the two digests. We found the IVT protocol to be highly reproducible: correlation coefficients calculated between the replicate datasets averaged 0.98. This reproducibility is maintained even when only 5 ng starting material are used (correlation of 0.97). The direct labeling and R-PCR protocols are also highly reproducible, with mean correlations of 0.93 and 0.91, respectively (Fig 3A).

Bottom Line: Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis.Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach.Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Chemical Biology, and Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA. clliu@fas.harvard.edu

ABSTRACT

Background: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail.

Results: We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach.

Conclusion: We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

Show MeSH
Related in: MedlinePlus