Title | DNA degradation test predicts success in whole-genome amplification from diverse clinical samples. |
Publication Type | Journal Article |
Year of Publication | 2007 |
Authors | Wang F, Wang L, Briggs C, Sicinska E, Gaston SM, Mamon H, Kulke MH, Zamponi R, Loda M, Maher E, Ogino S, Fuchs CS, Li J, Hader C, G Makrigiorgos M |
Journal | J Mol Diagn |
Volume | 9 |
Issue | 4 |
Pagination | 441-51 |
Date Published | 2007 Sep |
ISSN | 1525-1578 |
Keywords | Base Sequence, DNA Fragmentation, DNA, Neoplasm, Formaldehyde, Genome, Human, Glyceraldehyde-3-Phosphate Dehydrogenases, Humans, Male, Nucleic Acid Amplification Techniques, Polymerase Chain Reaction, Temperature, Time Factors, Tissue Fixation |
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 ( approximately 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. |
DOI | 10.2353/jmoldx.2007.070004 |
Alternate Journal | J Mol Diagn |
PubMed ID | 17690213 |
PubMed Central ID | PMC1975106 |
Grant List | 5P50 CA90381 / CA / NCI NIH HHS / United States 5T32 CA09078 / CA / NCI NIH HHS / United States P50 CA090381 / CA / NCI NIH HHS / United States 1R21 CA111994-01 / CA / NCI NIH HHS / United States R21 CA115439 / CA / NCI NIH HHS / United States R21 CA111994 / CA / NCI NIH HHS / United States 1R21 CA115439-01A1 / CA / NCI NIH HHS / United States T32 CA009078 / CA / NCI NIH HHS / United States P01 CA089021 / CA / NCI NIH HHS / United States |
Related Faculty:
Massimo Loda, M.D.