DNA degradation test predicts success in whole-genome amplification from diverse clinical samples.
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.
Wang
Fengfei
F
Department of Radiation Oncology, Dana Farber Cancer Institute, Boston, MA 02115, USA.
Wang
Lilin
L
Briggs
Christine
C
Sicinska
Ewa
E
Gaston
Sandra M
SM
Mamon
Harvey
H
Kulke
Matthew H
MH
Zamponi
Raffaella
R
Loda
Massimo
M
Maher
Elizabeth
E
Ogino
Shuji
S
Fuchs
Charles S
CS
Li
Jin
J
Hader
Carlos
C
Makrigiorgos
G Mike
GM
D001483:Base Sequence; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000097:blood; D005557:Formaldehyde; D015894:Genome, Human / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors; D016707:Tissue Fixation
D004273:DNA, Neoplasm; D005557:Formaldehyde; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
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