An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.
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.
Lee
Cheryl I P
CI
Division of Medical Sciences, National Cancer Centre 11 Hospital Drive, Singapore 169610.
Leong
Siew Hong
SH
Png
Adrian E H
AE
Choo
Keng Wah
KW
Syn
Christopher
C
Lim
Dennis T H
DT
Law
Hai Yang
HY
Kon
Oi Lian
OL
D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization; D020080:Tandem Repeat Sequences
D017931:DNA Primers; D004247:DNA