NDLI: Multiplex amplification coupled with COLD-PCR and high resolution melting enables identification of low-abundance mutations in cancer samples with low DNA content.

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Year: 2011 Volume: 13 Issue: 1

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Multiplex amplification coupled with COLD-PCR and high resolution melting enables identification of low-abundance mutations in cancer samples with low DNA content.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Milbury, Coren A. Chen, Clark C. Mamon, Harvey Liu, Pingfang Santagata, Sandro Makrigiorgos, G. Mike
Description	Country affiliation: United States Author Affiliation: Milbury CA ( Department of Radiation Oncology, Dana-Farber/Brigham and Women's Cancer Center, Harvard Medical School, Boston, Massachusetts 02115, USA.)
Abstract	Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification.
File Format	HTM / HTML
ISSN	15251578
e-ISSN	19437811
DOI	10.1016/j.jmoldx.2010.10.008
Journal	The Journal of Molecular Diagnostics
Issue Number	2
Volume Number	13
Language	English
Publisher	Elsevier
Publisher Date	2011-03-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Molecular Biology Dna Mutational Analysis Dna, Neoplasm Mutation Neoplasms Genetics Nucleic Acid Denaturation Polymerase Chain Reaction Freezing Molecular Sequence Data Sensitivity And Specificity Evaluation Studies Research Support, N.i.h., Extramural
Content Type	Text
Resource Type	Article
Subject	Molecular Medicine Pathology and Forensic Medicine

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