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DNA Research Advance Access originally published online on March 27, 2006
DNA Research 2006 13(2):77-88; doi:10.1093/dnares/dsi029
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

An Isothermal Method for Whole Genome Amplification of Fresh and Degraded DNA for Comparative Genomic Hybridization, Genotyping and Mutation Detection

Cheryl I. P. Lee, Siew Hong Leong, Adrian E. H. Png1, Keng Wah Choo1, Christopher Syn2, Dennis T. H. Lim3, Hai Yang Law4 and Oi Lian Kon*

Division of Medical Sciences, National Cancer Centre 11 Hospital Drive, Singapore 169610
1 Bioinformatics Group, Nanyang Polytechnic 180 Ang Mo Kio Avenue 8, Singapore 569830
2 Centre for Forensic Science, Health Sciences Authority 11 Outram Road, Singapore 169078
3 Department of General Surgery, Singapore General Hospital Outram Road, Singapore 169608
4 Department of Paediatric Medicine, KK Women's & Children's Hospital 100 Bukit Timah Road, Singapore 229899

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self–self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.

Key words: whole genome amplification; isothermal; comparative genomic hybridization; genotyping; mutations

*To whom correspondence should be addressed. Tel. +65-6436-8319/8307, Fax. +65-6372-0161, Email: dmskol{at}nccs.com.sg

Communicated by Michio Oishi


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