title: DNA degradation test predicts success in whole-genome amplification from diverse clinical samples.
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.
authors: Department of Radiation Oncology, Dana Farber Cancer Institute, Boston, MA 02115, USA.
authors: Fengfei
authors: F
authors: Wang
authors: Lilin
authors: L
authors: Wang
authors: Christine
authors: C
authors: Briggs
authors: Ewa
authors: E
authors: Sicinska
authors: Sandra M
authors: SM
authors: Gaston
authors: Harvey
authors: H
authors: Mamon
authors: Matthew H
authors: MH
authors: Kulke
authors: Raffaella
authors: R
authors: Zamponi
authors: Massimo
authors: M
authors: Loda
authors: Elizabeth
authors: E
authors: Maher
authors: Shuji
authors: S
authors: Ogino
authors: Charles S
authors: CS
authors: Fuchs
authors: Jin
authors: J
authors: Li
authors: Carlos
authors: C
authors: Hader
authors: G Mike
authors: GM
authors: Makrigiorgos
mesh_terms: 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
chemical_list: D004273:DNA, Neoplasm; D005557:Formaldehyde; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
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