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

Gustavo Barra,Lídia Abdalla Nery,Camila Nobre,Ticiane Santa Rita,Pedro Mesquita,Daniella Jardim,Rafael Jácomo

doi:10.3390/diagnostics9010009

Gustavo Barra, Lídia Abdalla Nery + Show 5 more

Open Access

https://doi.org/10.3390/diagnostics9010009

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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.

Highlights

The cell-free genomic DNA concentration in serum ranges from 1500 to 7500 copies/mL within 2 h after phlebotomy (6–24 times the concentration observed in plasma) because the blood clotting process disrupts some leukocytes releasing their content into the liquid phase of this specimen.the gDNA concentration in serum increases with storage of the collection tube at 4 ◦ C, indicating that there is a continuous release of gDNA from the blood clot to serum [1]
7 days after blood collection (Figure 1). These results suggest that PCR targets with small amplicon sizes (
The observed results demonstrated that PCR products of different sizes could be amplified using genomic DNA extracted from serum as the template

Summary

Introduction

The gDNA concentration in serum increases with storage of the collection tube at 4 ◦ C, indicating that there is a continuous release of gDNA from the blood clot to serum [1]. This high gDNA yield found in serum would allow its direct use for genotyping of clinically relevant single. Regarding the genotyping of SNPs directly from serum, Ulvik and colleagues described a protocol with that purpose in 2001 [9], the specimen needed to be dried before the qPCR to avoid the complete inhibition of the reaction. The direct use of crude serum for SNP genotyping would be more feasible because the qPCR master mixes were added by components and/or modified enzymes that diminish the impact of inhibitors over the reaction [6,7,10,11]

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