Limits...
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.

Show MeSH
Hierarchical clustering of replicate datasets generated by direct labeling, IVT and R-PCR. Each thin bar represents a single datapoint. Red bars correspond to enrichment in the Cy5-labeled Alu I probe, while green bars correspond to enrichment in the Cy3-labeled Rsa I probe. The dendrograms (top) indicate clustering relationships among the sample replicates. The lengths of the branches represent the degree of similarity between the samples (shorter indicates higher similarity). Purple stripes to the right of the diagram highlight discordant areas (log ratios with opposite signs) in the R-PCR replicates relative to the direct labeling and IVT samples.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC161798&req=5

Figure 5: Hierarchical clustering of replicate datasets generated by direct labeling, IVT and R-PCR. Each thin bar represents a single datapoint. Red bars correspond to enrichment in the Cy5-labeled Alu I probe, while green bars correspond to enrichment in the Cy3-labeled Rsa I probe. The dendrograms (top) indicate clustering relationships among the sample replicates. The lengths of the branches represent the degree of similarity between the samples (shorter indicates higher similarity). Purple stripes to the right of the diagram highlight discordant areas (log ratios with opposite signs) in the R-PCR replicates relative to the direct labeling and IVT samples.

Mentions: Hierarchical clustering, shown in Fig 5, was used as an additional means to identify differences between the amplification protocols [20]. Replicates for a given method cluster closely, confirming the high reproducibility observed for each method. Consistent with the correlation analysis, the individual replicates for the IVT and direct labeling protocol cluster together, while the R-PCR replicates cluster in a separate arm of the dendrogram. Most of the 4,481 features shown in the cluster diagram exhibit consistent coloring across the diagram, indicating that the direction of enrichment is consistent for all replicates. However, the cluster diagram highlights two groups of genes (purple stripes) whose ratios in the R-PCR dataset are inconsistent with their ratios in the direct labeling and IVT datasets.


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

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

Hierarchical clustering of replicate datasets generated by direct labeling, IVT and R-PCR. Each thin bar represents a single datapoint. Red bars correspond to enrichment in the Cy5-labeled Alu I probe, while green bars correspond to enrichment in the Cy3-labeled Rsa I probe. The dendrograms (top) indicate clustering relationships among the sample replicates. The lengths of the branches represent the degree of similarity between the samples (shorter indicates higher similarity). Purple stripes to the right of the diagram highlight discordant areas (log ratios with opposite signs) in the R-PCR replicates relative to the direct labeling and IVT samples.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Hierarchical clustering of replicate datasets generated by direct labeling, IVT and R-PCR. Each thin bar represents a single datapoint. Red bars correspond to enrichment in the Cy5-labeled Alu I probe, while green bars correspond to enrichment in the Cy3-labeled Rsa I probe. The dendrograms (top) indicate clustering relationships among the sample replicates. The lengths of the branches represent the degree of similarity between the samples (shorter indicates higher similarity). Purple stripes to the right of the diagram highlight discordant areas (log ratios with opposite signs) in the R-PCR replicates relative to the direct labeling and IVT samples.
Mentions: Hierarchical clustering, shown in Fig 5, was used as an additional means to identify differences between the amplification protocols [20]. Replicates for a given method cluster closely, confirming the high reproducibility observed for each method. Consistent with the correlation analysis, the individual replicates for the IVT and direct labeling protocol cluster together, while the R-PCR replicates cluster in a separate arm of the dendrogram. Most of the 4,481 features shown in the cluster diagram exhibit consistent coloring across the diagram, indicating that the direction of enrichment is consistent for all replicates. However, the cluster diagram highlights two groups of genes (purple stripes) whose ratios in the R-PCR dataset are inconsistent with their ratios in the direct labeling and IVT datasets.

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