Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlex whole genome amplified DNA generated from multiple DNA sources.

Author(s): Bergen AW, Haque KA, Qi Y, Beerman MB, Garcia-Closas M, Rothman N, Chanock SJ

Publication: Hum Mutat, 2005, Vol. 26, Page 262-70

PubMed ID: 16086324 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of DNA source, WGA method, assay type and GC content on genotyping success rates. Blood was transported at room temperature, stored at 4 degrees C and separated the next day after which buffy coat specimens were frozen at -80 degrees C. Buccal cells were obtained by swishing 10 mL of mouth wash around the upper mouth for 45 sec. Mouthwash specimens were stored at room temperature for 3 days, pelleted, and then the cells were stored at -80 degrees C prior to DNA extraction.

   Summary of Findings:

   The highest median increase in DNA concentration (5,100 fold) was observed when DNA was amplified using the REPLI-G kit. Similar median increases in DNA concentration were observed for the GenomiPhi and OmniPlex methods (1,500 and 1,700 fold, respectively), and when DNA was amplified using OmniPlex, the increase in concentration was highest using buffy coat. The OmniPlex method produced 66% ssDNA, while the REPL-G and GenomiPhi kits produced 20% and 8% ssDNA, respectively. In all cases, the percentage of ssDNA was highest when amplified from buffy coat as opposed to buccal cells or lymphoblasts. Although genotyping was possible from all genomic DNA specimens using AmpFlSTR, there were several amplification failures and discordant genotypes found when WGA DNA was used as the template. The authors state that there was no significant difference in genotyping success using AmpFlSTR between the WGA methods, but DNA amplified with the GenomiPhi and REPLI-G kits showed higher amplification and concordance rates then DNA amplified by the OmniPlex method. Buffy coat DNA generally had a higher amplification success rate in the AmpFlSTR genotyping kit than buccal cell or lymphoblast DNA using the AmpFLSTR kit. Genotyping using TaqMan resulted in genotyping failures in 1-3% of reactions using unamplified genomic DNA or WGA DNA as template. There were fewer determined genotypes by TaqMan assay with OmniPlex WGA template DNA rather than GenomiPhi or REPLI-g WGA DNA. The local GC content was positively correlated with discordant genotypes obtained by TaqMan from unamplified DNA (p=0.028), REPLI-G WGA DNA (p=0.040), and OmniPlex WGA DNA (p=0.04) and with the number of undetermined genotypes obtained from GenomiPhi WGA DNA (p=0.027) and OmniPlex WGA DNA (p<0.001).

   Biospecimens

   • Cell - Buccal
   • Cell - White Cells
   • Bodily Fluid - Buffy Coat
   • Bodily Fluid - Saliva

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	SNP assay
   DNA	Spectrophotometry
   DNA	Whole genome amplification
   DNA	Microsatellite analysis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Whole genome amplification Specific	Nucleic acid amplification	GenomiPhi REPLI-g OmniPlex
   SNP assay Specific	Targeted nucleic acid	Various %GC content
   Biospecimen Acquisition	Biospecimen location	Buffy coat Lymphoblasts Buccal cells

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