Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies - PubMed (original) (raw)

Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies

Jennifer Frenck Holbrook et al. J Biomol Tech. 2005 Jun.

Abstract

The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.

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Figures

FIGURE 1

FIGURE 1

Agarose (0.8%) gel electrophoresis of genomic DNAs amplified using the GenomiPhi Kit. M: 1-Kb molecular weight markers. Lanes 1–10 show typical profiles of human genomic DNA post amplification. Lane 11: Water control depicting random hexamer-mediated amplification after overnight incubation with Phi29.

FIGURE 2

FIGURE 2

Typical genotyping profiles of human DNA pre- and post-GenomiPhi amplification using two microsatellites—D7S661 (blue) and D8S284 _(green)_—included in panel 12 of the ABI Prism Linkage Mapping Set v2.5. Depleted stock of sample 7-20 (see Table 1) was successfully replenished by WGA as indicated by identical genotypes for pre- and post-genomic amplification. The black arrow points to the 322-bp differentially amplified allele of D7S661.

FIGURE 3

FIGURE 3

Allelic discrimination plot obtained for rs2294714 after screening adult human DNA using ABI Assays-On-Demand. Red circles indicate GP-DNAs 7-20 (aunt), 7-30 (niece) and 7-31 (nephew) (see Table 1). The individuals in the study cluster in three groups (blue and red are homozygous samples, green are heterozygous C/T). Negative (water and no template) controls and failed samples all cluster at the bottom left area of the plot.

FIGURE 4

FIGURE 4

Screening of SNPs in genomic and GP samples from related individuals using pyrosequencing. A: Theoretical profiles of the WDR8 gene SNP rs3818330 for the homozygous C/C and heterozygous C/A genotypes. B: Experimental results of an individual with genotypes C/C (7-31, nephew) and C/A (7-20, aunt) using GP-DNA. The SNP site is highlighted in yellow. Additional sequence information obtained on the sample pyrograms (TAGAG) show 100% concordance with the known sequence adjacent to the SNP site.

FIGURE 5

FIGURE 5

Sequencing profiles of a region of FGFR4 in pediatric samples. A 489-bp PCR product was generated using genomic DNA before and after the genome amplification procedure (GP) and sequenced. SNP rs393923 is indicated by a red arrow. A blue arrow indicates an additional heterozygous site in 35. No mutation was detected in the GP-DNA samples when compared with the original genomic DNA templates (Table 2).

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