scholarly journals 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 ◽  
Vol 9 (1) ◽  
pp. 9 ◽  
Author(s):  
Gustavo Barra ◽  
Ticiane Santa Rita ◽  
Daniella Jardim ◽  
Pedro Mesquita ◽  
Camila Nobre ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Base Pair ◽  
Genomic Dna ◽  
Room Temperature ◽  
Capillary Blood ◽  
Snp Genotyping ◽  
Nucleotide Polymorphisms ◽  
Base Pairs ◽  
Single Nucleotide ◽  
Allele Specific

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.

A fully integrated SNP genotyping system for hereditary hearing-loss detection

Lab on a Chip ◽  
2022 ◽  
Author(s):  
Nan Li ◽  
Yuanyue Zhang ◽  
Minjie Shen ◽  
Youchun Xu
Keyword(s):  
Hearing Loss ◽  
Early Intervention ◽  
Single Nucleotide Polymorphisms ◽  
Snp Genotyping ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Fully Integrated ◽  
Hereditary Hearing Loss

Hereditary hearing loss is one of the most common human neurosensory disorder, and there is a great need for early intervention methods such as genetically screening newborns. Single nucleotide polymorphisms...

scholarly journals A novel procedure for genotyping of single nucleotide polymorphisms in trisomy with genomic DNA and the invader assay

2008 ◽  
Vol 36 (22) ◽  
pp. e145-e145 ◽  
Author(s):  
K. J. Duffy ◽  
J. Littrell ◽  
A. Locke ◽  
S. L. Sherman ◽  
M. Olivier
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Genomic Dna ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Invader Assay

Fabrication and Application of Single Nucleotide Polymorphisms Library on Magnetic Nanoparticles Using Adaptor PCR

2008 ◽  
Vol 8 (1) ◽  
pp. 405-409 ◽  
Author(s):  
Hongna Liu ◽  
Song Li ◽  
Meiju Ji ◽  
Libo Nie ◽  
Jianrong Chen ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Single Nucleotide Polymorphisms ◽  
Reliable Method ◽  
Nucleotide Polymorphisms ◽  
Probe Signal ◽  
Single Nucleotide ◽  
Novel Approach ◽  
Mismatch Probe ◽  
Allele Specific ◽  
Agt Gene

We have developed a novel approach to fabricate single nucleotide polymorphisms (SNPs) library on magnetic nanoparticles (MNPs) based on adaptor PCR. Each SNP locus in the library was interrogated by hybridization with a pair of allele specific dual-color fluorescence (Cy3, Cy5) probes to determine SNP. Two SNPs loci (M235T and A-6G) associated with essential hypertension in the angiotensinogen (AGT) gene were detected by this method and their fluorescent signals were quantified. The fluorescent ratios (match probe: mismatch probe signal) of homozygous genotypes were over 3.0, whereas heterozygous genotypes had ratios near to 1.0. Without any complex multiplex PCR procedure, it is a simple, efficient and reliable method for the multiplex SNPs detection using limited amount of DNA samples from individuals.

scholarly journals Analysis of Clinically Relevant Single-Nucleotide Polymorphisms by Use of Microelectronic Array Technology

2002 ◽  
Vol 48 (12) ◽  
pp. 2124-2130 ◽  
Author(s):  
Rosa Santacroce ◽  
Antonia Ratti ◽  
Francesco Caroli ◽  
Barbara Foglieni ◽  
Alessandro Ferraris ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Factor Vii ◽  
Restriction Enzyme Digestion ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Ret Protooncogene ◽  
Mismatched Dna ◽  
Specific Pcr ◽  
Pcr Products ◽  
Allele Specific

Abstract Background: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. Methods: Primer pairs, with one containing a 5′-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, β-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with β-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. Results: Analysis of amplified DNA required 4–6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. Conclusions: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.

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