P097 Quantity, integrity & purity: A comparative study of different genomic dna assessment methods

Abstract

Aim DNA quantity and quality are critical for the performance of downstream applications. There are different methods to determine these characteristics and each provide data to evaluate the DNA present in the sample. Methods In this study, four methods were used to evaluate gDNA from 20 blood and buccal swab sources: measurement by Nanodrop 2000 (Thermo Fisher), quantitation by Qubit 3.0 (Thermo Fisher), evaluation by the Tapestation 4200 (Agilent Technologies), and assessment by the Promega ProNex DNA QC Assay (Promega). Results The Nanodrop provides the quantity of DNA, but can overestimate or underestimate the quantity in an unpredictable fashion. The Qubit accurately provides quantity of DNA present. Download : Download high-res image (158KB) Download : Download full-size image We utilized the TapeStation to measure fragment sizes and to obtain a DNA integrity number (DIN) that reflects the level of degradation. Download : Download high-res image (117KB) Download : Download full-size image The ProNex DNA QC assay determines varying amplifiable gDNA sequences of 75 bp, 150 bp, and 300 bp, providing information about quantity and quality of gDNA present in the sample. There is also an internal positive control which can test for the presence of a PCR inhibitor. Four samples (2,3,6, and 9) exhibited degradation with the TapeStation assay, which was also reflected in the qPCR ratios. Download : Download high-res image (195KB) Download : Download full-size image Conclusions All four methods evaluated provide varied information about the quantity, quality and purity of DNA specimens. Ultimately, the appropriateness of assay selected depends on the downstream application.