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

BioMed Central
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  • Methodology Article
  • Biology


07-Genome-ch7-cpp CHAPTER 7 DNA linear amplification Chih Long Liu, Bradley E. Bernstein and Stuart L. Schreiber Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, Massachusetts 02138, USA Amplification of nucleic acids has become a mainstay of molecular biology. It permits genomic and transcriptional analysis when the amount of tissue or number of cells being studied becomes limiting, and is invaluable in studies where the biology of the specimens being investigated severely limits the amount of nucleic acids available. It is also particularly important for large-scale, high- throughput genomic studies, since these studies typically use microarrays or other high-throughput assays that often require microgram amounts of nucleic acids. Furthermore, these studies may employ a large matrix of many different conditions and/or time points, which may make it prohibitively expensive to generate sufficient amounts of unamplified material. One example of such a genomics application is the ChIP–chip method, where DNA recovered from chromatin immunoprecipitation (ChIP) of cell lysate is used for subsequent analysis on DNA microarrays. This method (for review, see 1) is typically used to identify transcription factor binding sites and to map histone variants, histone post-translational modification patterns, or any other interesting epitope within the genome. Fig. 1 shows an example of the ChIP–chip method using spotted microarrays. ChIP, however, typically yields DNA in the nanogram range, which is insufficient for most DNA microarray applications. Laboratories using the ChIP–chip technique typically employ an exponential amplification method, such as ligation-mediated PCR (LM–PCR) (2) or random PCR (R–PCR) (3–5), to obtain the quantities necessary for microarray analysis. R–PCR involves the annealing of primer adaptors with a 5′ conserved end and a 3′ degenerate end to the template DNA, followed by extension and subsequent PCR with primers complementary

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