LM-PCR permits highly representative whole genome amplification of DNA isolated from small number of cells and paraffin-embedded tumor tissue sections.
Analysis of genetic changes is often hampered by insufficient starting DNA from limited clinical tissue specimens. We employed ligation-mediated PCR (LM-PCR) for global amplification of the genome to overcome this limitation, generating up to 5 microg of representative amplicons of genomic DNA from as little as one cell. We demonstrate successful global genome amplification in high-quality starting DNA source like laser-captured cultured cells, as well as partially degraded starting DNA from old formalin-fixed paraffin-embedded tissue sections. This process generates adaptor-tailed templates that can be repeatedly amplified almost ad infinitum. We have further modified this technique such that, instead of a single endonuclease digest, we can achieve higher amplicon coverage by combining 3 endonuclease digests prior to LM-PCR. As tested by examining amplification of STS sequences scattered genome-wide, the coverage was improved from the published 70% to 96%. The faithful representation of global losses and gains in the amplified genomic DNA was confirmed by array-comparative genomic hybridization. Further, we exemplify the utility of this technique for finer p53 point mutation analysis by PCR-SSCP. This technique is thus a clinically useful tool for globally amplifying and archiving DNA from finite sources like paraffin tissue sections, providing a potentially unlimited resource for genetic analyses.
Liu
Dongxin
D
Department of Pathology, University of Southern California, Keck School of Medicine, Los Angeles, 90033, USA.
Liu
Cheng
C
DeVries
Sandy
S
Waldman
Frederic
F
Cote
Richard J
RJ
Datar
Ram H
RH
D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D016678:Genome; D006801:Humans; D008297:Male; D042282:Microdissection; D009693:Nucleic Acid Hybridization; D016612:Paraffin Embedding; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53