Improved DOP-PCR–Based Representational Whole-Genome Amplification Using Quantitative Real-Time PCR Lilia ´na Z. Fehe ´r, MSc,* Margit Bala ´zs, PhD, DSc,w Ja ´nos Z. Kelemen, MSc,* A ´ gnes Zvara, PhD,* Istva ´n Ne ´meth, MD,z Zolta ´n Varga-Orvos, MD,* and La ´szlo ´ G. Puska ´s, PhD* 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 demon- strate 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. Key Words: degenerate oligonucleotide-primed PCR (DOP- PCR), genome amplification, quantitative real-time PCR, copy number changes (Diagn Mol Pathol 2006;15:43–48) H igh-throughput genomic analysis requires large amounts of template; however, the typical yield of DNA from individual samples can be often limited. The primary drawback of the use of laser capture microscopy in DNA analysis is that microdissections yield insufficient nucleic acids due to low genomic DNA recovered from small sample sizes. With samples such as these, the ability to conduct sample amplification becomes imperative, to ensure that enough material is available for genetic analysis. Not only the amount but also the quality of genomic DNA obtained from various samples is impedi- ment for further studies. For example, paraffin-embedded tissues often provide partially degraded low-quality DNA. Therefore, developing a whole-genome amplifica- tion method which is not entirely dependent on high- quality DNA is eminently important to provide an extensive supply of DNA for large-scale genetic studies 1 such as metaphase comparative genomic hybridization (CGH), 2 array CGH, 3 single-nucleotide polymorphism (SNP) genotyping, 4 microsatellite genotyping, 5 single- stranded conformational polymorphism (SSCP), 6 loss of heterozygosity (LOH), 7 and sequence analysis. 8 Gene amplifications and deletions are common in tumors and can influence tumorigenesis and metastatic potential. Traditional CGH can detect aneuploidies, deletions, and unbalanced translocations. There are 3 basic techniques to amplify the entire genomic DNA: PCR-based ap- proaches, 5–15 strand or multiple displacement amplifica- tion (SDA or MDA), 16 and T7-based linear amplification (TLAD). 17 The SDA and MDA isothermal methods employ the unique biochemical properties of Phi29 DNA poly- merase to amplify linear DNA. These techniques are based on the rolling circle amplification by which circular DNA molecules such as plasmids or viruses frequently replicate. These rolling circle amplification methods were initially adapted for the amplification of large circular DNA templates 18,19 and more recently for the amplifica- tion of genomic DNA. 16 The drawback of SDA is that the efficiency of amplification depends on the length of the template. TLAD is applicable for the amplification of genomic DNA of varying fragment size and quality, and Copyright r 2006 by Lippincott Williams & Wilkins From the *Laboratory of Functional Genomics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary; wDepart- ment of Preventive Medicine, School of Public Health, Medical and Health Science Center, University of Debrecen, Hungary; and zDepartment of Pathology, University of Szeged, Szeged, Hungary. Supported by grants from the Hungarian Medical Research Council (ETT 400/2003) and a bilateral grant between the Hungarian Prime Minister’s Office and Hungarian Academy of Sciences (40.0499/ 2004). Reprints: La´ szlo´ G. Puska´s, Laboratory of Functional Genomics, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, Szeged, H-6701, Hungary (e-mail: pusi@brc.hu). ORIGINAL ARTICLE Diagn Mol Pathol  Volume 15, Number 1, March 2006 43