Optimization of degenerate oligonucleotide primed PCR for amplification of microdissected chromosome segments

Publication Year:
Usage 52
Abstract Views 50
Downloads 2
Repository URL:
Mahjoubi, Frouzandeh
School of Biological Sciences
thesis / dissertation description
The degenerate oligonucleotide primer of sequence, 5' CCGACTCGAGNNNNNNATGTGG 3', contains a specific 3' hexanucleotide , a central random hexanucleotide and an Xhol site towards it's 5' end. This degenerate primer (Telenius et al, 1992) has been developed for the amplification and subsequent cloning of any source of target DNA, particularly the human chromosome, in a process termed degenerate oligonucleotide primed PGR (DOP-PCR). In the present study, bacteriophage lambda DNA was employed as a model target DNA to test a variety of DOPPCR procedures. To achieve the highest efficiency, different concentrations of primer and Taq DNA polymerase and also a variety of PGR program cycles were tested. After achieving the optimal concentration of the DOP primer and Taq DNA polymerase, two different protocols were investigated. The first reUes on low-temperature pre-amplification of the target DNA by modified T7 DNA polymerase (Sequenase) and uses the amphfied segments for a subsequent amplification catalysed by Taq DNA polymerase. With this protocol, amplified DNA was obtained with down to 0.2 pg DNA of template. The other procedure employs Taq DNA polymerase for all stages of amphfication. We have developed a modified form of this strategy by boosting Taq DNA polymerase in the middle of the amphfication program. This modification increases the efficiency and sensitivity of the procedure and enables us to ampUfy as little as 0.02 pg DNA. The latter method should be of considerable value for amphfication of DNA from a wide variety of sources, particularly human chromosomes, since it should reduce to a minimum the number of dissected chromosome fragments required. In fact, following the completion of this work, the method has been successfully applied to the analysis of a human chromosome translocation in collaboration