Primer-Extension Preamplified DNA Is a Reliable Template for Genotyping
2002
A common constraint faced by genetic studies is the limited amount of DNA available from study participants. It is often not practical or possible to either collect a large amount of tissue such as liver or blood from individuals or to ask for a second sample, particularly if the critical individuals died since the first sample was taken. Immortalization of white blood cells by use of Epstein–Barr virus in theory provides an unlimited source of material. The immortalization protocol itself, however, must be carried out within 48 h of blood collection and is time-consuming.
DNA linkage studies in which microsatellite or other polymorphic DNA markers are used for a genome scan require appreciable amounts of DNA. As multiple PCRs are carried out with each sample, valuable genomic DNA is easily depleted. To prevent loss of irreplaceable samples and to maximize the available genetic materials for DNA linkage and genetic association studies as well as for future studies of screening for mutations in candidate genes, we used the primer-extension preamplification (PEP) protocol (1) to carry out whole-genome amplification.
The advantages of using the PEP protocol are that only a small amount of genomic DNA is needed for the entire genome scan and the remaining genomic DNA can be stored for future analyses. One possible caveat is that PEP may not amplify both parental copies of an individual’s DNA or may not amplify them equally at all loci. In turn, genotyping inaccuracies may potentially obscure allele sharing. To investigate the reliability of genotyping results when PEP was used to produce PCR templates, we compared the results for PCR products from PEP with those obtained with genomic DNA templates. All experiments were carried out according to protocols approved by the Institutional Review Board of Wayne State University School of Medicine.
Before PEP, 30–250 …
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