DNA degradation test predicts success in whole-genome amplification from diverse clinical samples Academic Article uri icon

Overview

MeSH Major

  • DNA Fragmentation
  • Genome, Human
  • Nucleic Acid Amplification Techniques

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.

publication date

  • January 2007

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC1975106

Digital Object Identifier (DOI)

  • 10.2353/jmoldx.2007.070004

PubMed ID

  • 17690213

Additional Document Info

start page

  • 441

end page

  • 51

volume

  • 9

number

  • 4