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Diagnostics 2019, 9(1), 9; https://doi.org/10.3390/diagnostics9010009

Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge

1
Sabin Laboratory, Brasília 70632-300, Brazil
2
Post-Graduation of Health Science, University of Brasília, Brasilia 70910-900, Brazil
*
Author to whom correspondence should be addressed.
These authors contributed equally to the study.
Received: 15 November 2018 / Revised: 27 December 2018 / Accepted: 29 December 2018 / Published: 11 January 2019
(This article belongs to the Special Issue Novel Point-of-Care Technologies in Diagnostics 2018)
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Abstract

The cell-free genomic DNA (gDNA) concentration in serum ranges from 1500 to 7500 copies/mL within 2 h after phlebotomy (6–24 times the concentration observed in plasma). Here, we aimed to evaluate the gDNA size distribution in serum with time after coagulation and to test if crude serum can be directly used as a source of gDNA for qPCR. Next, we investigated if single nucleotide polymorphisms (SNPs) could be genotyped directly from the crude serum isolated from capillary blood using a hand-powered paper centrifuge. All tested PCR targets (65, 100, 202 and 688 base pairs) could be successfully amplified from DNA extracted from serum, irrespective of their amplicon size. The observed qPCR quantitation cycles suggested that the genomic DNA yield increased in serum with incubation at room temperature. Additionally, only 65 and 101 base pair qPCR targets could be amplified from crude serum soon after the coagulation. Incubation for 4 days at room temperature was necessary for the amplification of PCR targets of 202 base pairs. The 688 base pair qPCR target could not be amplified from serum directly. Lastly, serum was successfully separated from capillary blood using the proposed paper centrifuge and the genotypes were assigned by testing the crude serum using allele-specific qPCR, producing small amplicon sizes in complete agreement with the genotypes assigned by testing the DNA extracted from whole blood. The serum can be used directly as the template in qPCR for SNP genotyping, especially if small amplicon sizes are applied. This shortcut in the SNP genotyping process could further molecular point-of-care diagnostics due to elimination of the DNA extraction step. View Full-Text
Keywords: single nucleotide polymorphism; real-time polymerase chain reaction; serum single nucleotide polymorphism; real-time polymerase chain reaction; serum
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Barra, G.B.; Santa Rita, T.H.; Jardim, D.P.; Mesquita, P.G.; Nobre, C.S.; Jácomo, R.H.; Abdalla Nery, L.F. Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge. Diagnostics 2019, 9, 9.

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