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.

Figure 1

Procedure to assess DNA quality via a multiplex PCR step followed by dHPLC or agarose gel analysis, using ∼1 ng of genomic DNA from diverse clinical samples. Samples of optimal quality are amplified via rolling circle-based WGA and processed for downstream assays. Samples of suboptimal quality are not processed further.

Figure 2

Multiplex PCR performed on varying amounts of human male genomic DNA (reference DNA). A: PCR products examined via ethidium bromide-stained agarose gel electrophoresis. B: PCR products examined via dHPLC equipped with a high-sensitivity fluorescent detector.

Figure 3

Evaluation of the real-time PCR threshold difference (amplification efficiency) before and after WGA via RCA-RCA for 20 randomly chosen genes, using reference DNA. The anti-primer quenching method was used for real-time PCR quantification. For selected genes (HER2, GAPDH, TOP1, and HB-EGF) the TaqMan real-time PCR method was also used, for comparison (columns with pattern). Dotted line is the average threshold difference observed between all 20 genes.

Figure 4

Examination of the effects on DNA sample quality of wait-time period at room temperature after surgery and time of fixation in formalin. Extracted genomic DNA was amplified via WGA (RCA-RCA) and tested for amplification efficiency at 20 gene loci. A: Amplification efficiency versus sample preparation conditions applied. B: Ethidium bromide gel-based multiplex PCR test of the extracted genomic DNA. C: dHPLC-based multiplex PCR test of the extracted genomic DNA.

Figure 5

Examination of DNA quality in a range of glioblastoma FFPE samples, ∼5 years old, using three different approaches: direct observation of extracted DNA on ethidium bromide gel (A), ethidium bromide gel-based examination of multiplex-PCR products (B), and fluorescence detector-based dHPLC screening of multiplex-PCR products (C). The number of sites that amplified successfully via real-time PCR after RCA-RCA WGA from these samples is also depicted in C (aQRT-PCR score).

Figure 6

Examination of DNA quality in a range of colon FFPE samples, ∼10 to 12 years old, using three different approaches: direct observation of extracted DNA on ethidium bromide gel (A), ethidium bromide gel-based examination of multiplex-PCR products (B), and fluorescence detector-based dHPLC screening of multiplex PCR products (C). The number of sites that amplified successfully via real-time PCR after RCA-RCA WGA from these samples is also depicted in C (aQRT-PCR score). The individual samples in C are listed in the order of decreasing aQRT-PCR score.

Figure 7

Examination of DNA quality in a range of clinical samples using fluorescence detector-based dHPLC screening of multiplex PCR products. A: Plasma-circulating DNA from four different colon cancer patients. B: 1 and 2, Breast cancer snap-frozen and corresponding FFPE; 3 and 4, prostate cancer micropeel (snap-frozen) and corresponding FFPE; and 5 and 6, prostate cancer LCM and manually dissected specimens.