Genomic DNA, extracted from whole blood samples, is a key element for all genotyping workflows. When stored serum or plasma is the only source of DNA available, the main problem to overcome is the low quantity and poor quality of the DNA obtained, irrespective of the isolation procedure applied. The prevalence of artifacts, such as unbalanced amplification of alleles at specific sites (allelic dropout), is typically associated with PCR amplification of low quality/quantity DNA template, which is known to promote genotyping errors. The aim of this study was to determine whether the quality of genomic DNA from plasma samples may affect genotyping results. The ABCB1 c.3435C>T polymorphism was determined with two different real-time PCR assays, LightSNiP and TaqMan assays. We observed higher signal fluorescence values with DNA isolated from whole blood samples than with those from fresh and frozen plasma samples, due to reduced DNA concentration in the second ones. Despite the signal strength, a 100% concordance of genotyping data was however obtained in both assay types, regardless of the method of extraction. Our results show that, regardless of the lower DNA yield, extraction from plasma samples can still represent a valid alternative for real-time PCR genotyping application.
DNA extraction from fresh and frozen plasma: an alternative for real-time PCR genotyping in pharmacogenetics
Clementi E;
2020
Abstract
Genomic DNA, extracted from whole blood samples, is a key element for all genotyping workflows. When stored serum or plasma is the only source of DNA available, the main problem to overcome is the low quantity and poor quality of the DNA obtained, irrespective of the isolation procedure applied. The prevalence of artifacts, such as unbalanced amplification of alleles at specific sites (allelic dropout), is typically associated with PCR amplification of low quality/quantity DNA template, which is known to promote genotyping errors. The aim of this study was to determine whether the quality of genomic DNA from plasma samples may affect genotyping results. The ABCB1 c.3435C>T polymorphism was determined with two different real-time PCR assays, LightSNiP and TaqMan assays. We observed higher signal fluorescence values with DNA isolated from whole blood samples than with those from fresh and frozen plasma samples, due to reduced DNA concentration in the second ones. Despite the signal strength, a 100% concordance of genotyping data was however obtained in both assay types, regardless of the method of extraction. Our results show that, regardless of the lower DNA yield, extraction from plasma samples can still represent a valid alternative for real-time PCR genotyping application.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
