PMID: 16531768Mar 15, 2006Paper

Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR

Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B
Liliána Z FehérLászló G Puskás

Abstract

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, alt...Continue Reading

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Citations

Dec 14, 2011·Pathology Oncology Research : POR·Liliána Z FehérLászló G Puskás
May 9, 2007·Nanotechnology·Jason ReedJames K Gimzewski

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