Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.
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, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.
Fehér
Liliána Z
LZ
Laboratory of Functional Genomics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
Balázs
Margit
M
Kelemen
János Z
JZ
Zvara
Agnes
A
Németh
István
I
Varga-Orvos
Zoltán
Z
Puskás
László G
LG
D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis* / Q000737:chemistry; D015894:Genome, Human; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D015342:DNA Probes; D004273:DNA, Neoplasm