DNA extraction from fresh and frozen plasma: an alternative for real-time PCR genotyping in pharmacogenetics
- PDF / 1,012,496 Bytes
- 5 Pages / 595.276 x 790.866 pts Page_size
- 28 Downloads / 209 Views
SHORT COMMUNICATION
DNA extraction from fresh and frozen plasma: an alternative for real‑time PCR genotyping in pharmacogenetics Stefania Cheli1 · Alessandra Napoli2 · Emilio Clementi2,3 · Cristina Montrasio1 Received: 29 April 2020 / Accepted: 11 July 2020 © Springer Nature B.V. 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. Keywords DNA extraction · Plasma · Pharmacogenetics · Polymorphisms · Real time PCR
Introduction DNA extraction is the initial step of laboratory procedures required for almost every molecular biology technique and particularly genetic analysis. In diagnostic applications above all, the gold standard is DNA extraction from ethylenediaminetetraacetic acid (EDTA) whole blood samples, with automated systems, by now a simple, fast and standardized method that allows the achievement of a large quantity and high quality DNA. Besides this, there are several other sources of DNA, such as dried blood spots, buccal swabs, frozen or fresh plasma/serum [1]. Genomic DNA isolation has to be efficient and reliable for downstream genetic * Cristina Montrasio cristina.montrasio@asst‑fbf‑sacco.it 1
Unit of Clinical Pharmacology, Department of Laboratory Medicine, ASST Fatebenefratelli-Sacco, Milan, Italy
2
Unit of Clinical Pharmacology, Department of Biomedical and Clinical Sciences, Consiglio Nazionale delle Ricerche, Institute of Neuroscience, “Luigi Sacco” University Hospital, Università di Milano, Milan, Italy
3
Scientific Institute, IRCCS E. Medea, Lecco, Italy
analysis. The choice of input DNA depends on application fields and therefore on yield and purity required. Previous studies demonstrated that plasma or serum may be employed as an appropriate source of genomic DNA for some genetic analysis [2–4]. When working with extracellular nucleic acid
Data Loading...
