Published online before print August 9, 2007

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Journal of Molecular Diagnostics 2007, Vol. 9, No. 4
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2007.070004

Technical Advance

DNA Degradation Test Predicts Success in Whole-Genome Amplification from Diverse Clinical Samples

Fengfei Wang^*, Lilin Wang^*, Christine Briggs, Ewa Sicinska, Sandra M. Gaston^¶, Harvey Mamon^*, Matthew H. Kulke, Raffaella Zamponi, Massimo Loda, Elizabeth Maher, Shuji Ogino, Charles S. Fuchs, Jin Li^*, Carlos Hader^* and G. Mike Makrigiorgos^*

From the Departments of Radiation Oncology * and Medical Oncology, Center for Molecular Oncologic Pathology, Dana Farber Cancer Institute, Boston, the Department of Pathology, Brigham and Women’s Hospital, Boston; the Molecular Genetics Core Facility, Children’s Hospital, Boston; and the Department of Surgery, ^¶ Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Abstract

The need to apply modern technologies to analyze DNA from diverse clinical samples often stumbles on suboptimal sample quality. We developed a simple approach to assess DNA fragmentation in minute clinical samples of widely different origin and the likelihood of success of degradation-tolerant whole genome amplification (restriction and circularization-aided rolling circle amplification, RCA-RCA) and subsequent polymerase chain reaction (PCR). A multiplex PCR amplification of four glyceraldehyde-3-phosphate dehydrogenase amplicons of varying sizes was performed using genomic DNA from clinical samples, followed by size discrimination on agarose gel or fluorescent denaturing high-performance liquid chromatography (dHPLC). RCA-RCA followed by real-time PCR was also performed, for correlation. Even minimal quantities of longer PCR fragments (300 to 400 bp), visible via high-sensitivity fluorescent dHPLC or agarose gel, were essential for the success of RCA-RCA and subsequent PCR-based assays. dHPLC gave a more accurate correlation between DNA fragmentation and sample quality than agarose gel electrophoresis. Multiplex-PCR-dHPLC predicted correctly the likelihood of assay success in formalin-fixed, paraffin-embedded samples fixed under controlled conditions and of different ages, in laser capture microdissection samples, in tissue print micropeels, and plasma-circulating DNA. Estimates of the percent information retained relative to snap-frozen DNA are derived for real-time PCR analysis. The assay is rapid and convenient and can be used widely to characterize DNA from any clinical sample of unknown quality.

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