Abstract
BackgroundGenomic 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.ResultsWe 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.ConclusionWe 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.
Highlights
Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure
Microarray-based genome mapping protocols utilize the technique of chromatin immunoprecipitation (ChIP) to isolate genomic DNA associated in vivo with a transcription factor of interest, or with histones exhibiting a particular post-translational modification
An anchored primer adapter was designed that contains a 5' T7 promoter and a 3' polyA (18 bps in length) stretch terminating in a single C, G, or T base. This adapter is annealed to the genomic DNA fragments, and Klenow is added for second strand synthesis
Summary
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. 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. Microarray-based genome mapping protocols utilize the technique of chromatin immunoprecipitation (ChIP) to isolate genomic DNA associated in vivo with a transcription factor of interest, or with histones exhibiting a particular post-translational modification. Microarrays have proven valuable for the unbiased analysis and identification of DNA isolated by ChIP
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.