LM-PCR permits highly representative whole genome amplification of DNA isolated from small number of cells and paraffin-embedded tumor tissue sections

Dongxin Liu, Cheng Liu, Sandy De Vries, Frederic Waldman, Richard J Cote, Ram Datar

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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 μg 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.

Original languageEnglish
Pages (from-to)105-115
Number of pages11
JournalDiagnostic Molecular Pathology
Volume13
Issue number2
DOIs
StatePublished - Jun 1 2004
Externally publishedYes

Fingerprint

Paraffin
Ligation
Cell Count
Genome
Polymerase Chain Reaction
DNA
Neoplasms
Endonucleases
Single-Stranded Conformational Polymorphism
Comparative Genomic Hybridization
Point Mutation
Formaldehyde
Cultured Cells
Lasers

Keywords

  • Comparative genome hybridization
  • Formalin-fixed paraffin-embedded tissue sections
  • Genetic changes
  • Global genome amplification
  • Laser-captured microdissection
  • Ligation-mediated PCR

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

LM-PCR permits highly representative whole genome amplification of DNA isolated from small number of cells and paraffin-embedded tumor tissue sections. / Liu, Dongxin; Liu, Cheng; De Vries, Sandy; Waldman, Frederic; Cote, Richard J; Datar, Ram.

In: Diagnostic Molecular Pathology, Vol. 13, No. 2, 01.06.2004, p. 105-115.

Research output: Contribution to journalArticle

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