TY - JOUR
T1 - DNA degradation test predicts success in whole-genome amplification from diverse clinical samples
AU - Wang, Fengfei
AU - Wang, Lilin
AU - Briggs, Christine
AU - Sicinska, Ewa
AU - Gaston, Sandra M.
AU - Mamon, Harvey
AU - Kulke, Matthew H.
AU - Zamponi, Raffaella
AU - Loda, Massimo
AU - Maher, Elizabeth
AU - Ogino, Shuji
AU - Fuschs, Charles S.
AU - Li, Jin
AU - Hader, Carlos
AU - Makrigiorgos, G. Mike
N1 - Funding Information:
Supported by the National Cancer Institute ( Innovative Molecular Analysis Technologies program grants 1R21 CA111994-01 and 1R21CA115439-01A1 and training grant 5 T32 CA09078 ), the Joint Center for Radiation Therapy Foundation, the National Institutes of Health (SPORE 5P50CA90381 and PO1 CA089021 to M.L. ), the Department of Defense ( PC051271 to M.L. ), and the Prostate Cancer Foundation (to M.L.).
PY - 2007/9
Y1 - 2007/9
N2 - 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.
AB - 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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U2 - 10.2353/jmoldx.2007.070004
DO - 10.2353/jmoldx.2007.070004
M3 - Article
C2 - 17690213
AN - SCOPUS:34548722702
SN - 1525-1578
VL - 9
SP - 441
EP - 451
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 4
ER -