Single tube allele specific PCR: a low cost technique for molecular  screening of sickle cell anaemia in Nigeria

Abstract Abstract 1653 Background: Sickle cell disease (SCD) is a monogenic disorder with phenotypic heterogeneity, possibly determined by polymorphisms (SNPs) in genes whose products modify the pathophysiology of the disease. Priapism is one of the most common vaso-occlusive complications of SCD, and it occurred in more than 30% of males. The Klotho (KL) gene appears to be associated with vascular function and nitric oxide biology and the presence of SNPs could affect its function. Association between KL and priapism in SCD patients was suggested by Nolan et al. in 2004. However, other authors could not confirm this finding (Elliot et al., 2007). Objective: We decided to evaluate the relevance of SNPs rs2249358, rs211234 and rs9536314 to the occurrence of priapism in patients with SCD followed at Outpatient clinic at Escola Paulista de Medicina/UNIFESP. Methods: Forty male patients with SCD were enrolled, 39 (97.5%) with sickle cell anemia (SS) and one (2.5%) SC hemoglobinopathy. The manifestation of priapism was identified through analyses of medical records. The SNP rs2249358 was identified by PCR followed by restriction with XbaI. The other SNPs, rs211234 and rs9536314, were analyzed by allele specific PCR. Statistical analysis: t test, Chi2 or Fisher. This study was approved by Ethical Committee, and all patients agreed in participate. Results: The median age of the patient was 28.5 years-old (20-68 y.o.). Fourteen out of 40 patients had priapism (35%), each one with SS disease. The group of patients with priapism were older (32.5 y.o., 25–68 y.o.) than the group without this manifestation (27.5 y.o., 20–56 y.o.) (p=0.03). There was no statistical difference in the distribution of the SNPs rs211234 and rs9536314 between the two groups of patients (p=0.51 and p= 0.09, respectively). Regarding the distribution of SNP rs2249358, the group with priapism presented 8 individuals (57.1%) with GG genotype, 5 (35.7%) with AA and 1 (7.17%) with AG, whereas in the group without priapism, the distribution was different: 5 (19.2%) with GG, 7 (26.9%) with AA and 14 (53.8%) with AG genotype (p=0.0212). When we compare the presence of at least one A allele (AA or A-) with the G allele in homozygosis (GG), we observed that the A allele has a protector effect (OR: 0.1786; CI: 0.04232–0.7535) (p=0.031). Conclusions: In a relatively small group of patients with SCD, it was observed a significant proportion of individuals with priapism, which reinforces the importance of this manifestation. We also observed correlation between SNP rs2249358 of KL gene and priapism, as suggested previously. Disclosures: No relevant conflicts of interest to declare.

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Kompetitive Allele Specific PCR (KASP) Markers for Potato: An Effective Tool for Increased Genetic Gains

Agronomy ◽

10.3390/agronomy11112315 ◽

2021 ◽

Vol 11 (11) ◽

pp. 2315

Author(s):

Moctar Kante ◽

Hannele Lindqvist-Kreuze ◽

Leticia Portal ◽

Maria David ◽

Manuel Gastelo

Keyword(s):

Quality Control ◽

Low Cost ◽

Yield Reduction ◽

Genetic Gains ◽

Specific Pcr ◽

Breeding Populations ◽

Allele Specific ◽

Cultivated Potatoes ◽

Allele Specific Pcr ◽

Kasp Markers

Potato virus Y (PVY) and Phytophthora infestans (Mont.) de Bary that causes potato late blight (LB), pose serious constraints to cultivated potatoes due to significant yield reduction, and phenotyping for resistance remains challenging. Breeding operations for vegetatively propagated crops can lead to genotype mislabeling that, in turn, reduces genetic gains. Low-density and low-cost molecular marker assessment for phenotype prediction and quality control is a viable option for breeding programs. Here, we report on the development of kompetitive allele specific PCR (KASP) markers for LB and PVY resistance, and for routine quality control assessment of different breeding populations. Two KASP markers for LB resistance and two for PVY Ryadg were validated with an estimated assay power that ranged between 0.65 and 0.88. The developed QC KASP markers demonstrated the capability of discriminating tetraploid calls in breeding materials, including full-sibs and half-sibs. Routine implementation of the developed markers in a breeding program would assist with better allocation of resources and enable precise characterization of breeding material, thereby leading to increased genetic gains.

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Limits of Dideoxysequencing in the Detection of Somatic Mutations in Gastrointestinal Stromal Tumors

Acta Medica Martiniana ◽

10.1515/acm-2015-0013 ◽

2015 ◽

Vol 15 (3) ◽

pp. 13-20 ◽

Cited By ~ 1

Author(s):

K Jasek ◽

V Buzalkova ◽

P Szepe ◽

L Plank ◽

Z Lasabova

Keyword(s):

Gastrointestinal Stromal Tumors ◽

Somatic Mutations ◽

Low Cost ◽

Gene Mutations ◽

Wild Type ◽

Stromal Tumors ◽

Specific Pcr ◽

Allele Specific ◽

Detection Of Mutations ◽

Allele Specific Pcr

Abstract Detection of mutations in cancer is particularly important in terms of proper treatment and targeted therapy. The aim of this study was the comparison of two methods: allele-specific PCR (AS-PCR) and dideoxysequencing applied for the identification of BRAF gene mutations in wild-type gastrointestinal stromal tumors (WT GISTs). We have optimized the conditions for the detection V600E mutation representing the c.1799 T>A substitution by AS-PCR and have used dideoxysequencing to verify our results. In nine cases, we were able to detect the mutation by AS-PCR approach; however, the mutations have been confirmed by dideoxysequencing in four cases only. AS-PCR is fast and low cost method for the detection of V600E mutation which was validated as a sensitive assay for the identification of the most common BRAF mutation in DNA extracted from paraffin-embedded tissue of WT GISTs.

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Individual Single tube genotyping and DNA pooling by allele-specific PCR to uncover associations of polymorphisms with complex diseases

Clinica Chimica Acta ◽

10.1016/j.cca.2006.07.015 ◽

2006 ◽

Author(s):

A CASADODIAZ ◽

R CUENCAACEVEDO ◽

G DORADO ◽

J QUESADA

Keyword(s):

Complex Diseases ◽

Dna Pooling ◽

Single Tube ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Drop-In, Drop-Out Allele-Specific PCR: A Highly Sensitive, Single-Tube Method

Molecular Biotechnology ◽

10.1385/mb:28:3:171 ◽

2004 ◽

Vol 28 (3) ◽

pp. 171-174 ◽

Cited By ~ 5

Author(s):

Alice Alexander ◽

Nivedita Subramanian ◽

Joel N. Buxbaum ◽

Daniel R. Jacobson

Keyword(s):

Drop Out ◽

Single Tube ◽

Specific Pcr ◽

Highly Sensitive ◽

Allele Specific ◽

Allele Specific Pcr

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Single-tube real-time multiple allele-specific PCR for genotyping chicken Mx gene G2032A SNP

British Poultry Science ◽

10.1080/00071668.2010.499140 ◽

2010 ◽

Vol 51 (3) ◽

pp. 361-367

Author(s):

X. Ye ◽

Y. Zhang ◽

Z. Tan ◽

K. Li

Keyword(s):

Real Time ◽

Multiple Allele ◽

Single Tube ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Rapid Single-Tube Screening of the C282Y Hemochromatosis Mutation by Real-Time Multiplex Allele-specific PCR without Fluorescent Probes

Clinical Chemistry ◽

10.1093/clinchem/46.10.1540 ◽

2000 ◽

Vol 46 (10) ◽

pp. 1540-1547 ◽

Cited By ~ 24

Author(s):

Gerard G Donohoe ◽

Maija Laaksonen ◽

Kari Pulkki ◽

Tapani Rönnemaa ◽

Veli Kairisto

Keyword(s):

Accurate Determination ◽

Hereditary Hemochromatosis ◽

Pcr Amplification ◽

Single Tube ◽

Melting Temperatures ◽

Specific Pcr ◽

Pcr Products ◽

Allele Specific ◽

Dissociation Curves ◽

Allele Specific Pcr

Abstract Background: An accurate determination of the major HFE mutation (C282Y), which is associated with hereditary hemochromatosis, is important in diagnosis and risk assessment for this disease. We report a single-tube high-throughput PCR method for the detection of C282Y. Methods: We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches. Results: PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished. Conclusions: This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.

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