scholarly journals Rapid Detection of Aneuploidy (Trisomy 21) by Allele Quantification Combined with Melting Curves Analysis of Single-Nucleotide Polymorphism Loci

2003 ◽  
Vol 49 (7) ◽  
pp. 1087-1094 ◽  
Author(s):  
Genevieve Pont-Kingdon ◽  
Elaine Lyon
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Trisomy 21 ◽  
Tandem Repeats ◽  
Melting Curve ◽  
Curve Analysis ◽  
Chromosome 21 ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Melting Curves

Abstract Background: Molecular approaches for the detection of chromosomal abnormalities will allow the development of rapid, cost-effective screening strategies. We present here a molecular alternative for the detection of aneuploidies and, more specifically, trisomy 21. Methods: We used the quantitative value of melting curve analysis of heterozygous genetic loci to establish a relative allelic count. The two alleles of a given single-nucleotide polymorphism (SNP) were differentiated by thermodynamic stability with a fluorescently labeled hybridization probe and were quantified by relative areas of derivative melting curves detected after fluorescence resonance energy transfer. Heterozygous SNPs provided internal controls for the assay. Results: We selected six SNPs, heterozygous in at least 30% of a random population, to form a panel of informative loci in the majority of a random population. After normalization to a heterozygous control, samples segregated into three categories; nontrisomic samples had mean allele ratios of 0.96–1.09, whereas trisomic samples had mean ratios of 1.84–2.09 or 0.46–0.61, depending on which allele was duplicated. Within-run mean CVs of ratios were 6.5–27%, and between-assay mean CVs were 13–24%. Conclusions: The use of melting curve analysis of multiple SNPs is an alternative to the use of small tandem repeats for the detection of trisomies. Because of the high density of SNPs, the approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome; it is also applicable to aneuploidies other than trisomy 21 and to specimens that are not amenable to cytogenetic analysis.

scholarly journals Synergy of Total PLAC4 RNA Concentration and Measurement of the RNA Single-Nucleotide Polymorphism Allelic Ratio for the Noninvasive Prenatal Detection of Trisomy 21

2010 ◽  
Vol 56 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Nancy BY Tsui ◽  
Ranjit Akolekar ◽  
Rossa WK Chiu ◽  
Katherine CK Chow ◽  
Tak Y Leung ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Real Time Pcr ◽  
Trisomy 21 ◽  
Digital Pcr ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Allelic Ratio ◽  
Mrna Concentration

Abstract Background: Maternal plasma mRNA encoded by the PLAC4 gene (placenta-specific 4), which is transcribed from chromosome 21 in placental cells, is a potential marker for the noninvasive assessment of chromosome 21 dosage in the fetus. We evaluated the diagnostic sensitivities and specificities of 2 trisomy 21–screening approaches that use maternal plasma PLAC4 mRNA. Methods: We studied maternal plasma samples from 153 pregnant women carrying euploid and trisomy 21 fetuses. For the samples in which the fetuses were heterozygous for the studied PLAC4 single-nucleotide polymorphism (SNP), we measured the ratio between 2 alleles of the SNP in maternal plasma PLAC4 mRNA (RNA-SNP) by mass spectrometric (MS) and digital PCR methods. For pregnancies involving fetuses homozygous for the SNP, we quantified the total PLAC4 mRNA concentration in maternal plasma by real-time PCR and digital PCR. Results: For the RNA-SNP approach, we achieved a diagnostic sensitivity and specificity of 100% (95% CI, 40.2%–100%) and 89.7% (95% CI, 78.8%–96.1%), respectively, for both the MS and the digital PCR methods. For the mRNA-quantification approach, the areas under the ROC curves were 0.859 (95% CI, 0.741–0.903) and 0.833 (95% CI, 0.770–0.923) for plasma PLAC4 mRNA concentrations measured by the real-time PCR and the digital PCR methods, respectively. Conclusions: For prenatal screening of trisomy 21, the quantification of the total PLAC4 mRNA concentration can be used in a synergistic manner with the RNA-SNP allelic ratio approach to increase the population coverage of cases in which diagnostic information can be obtained.

scholarly journals Sub-7-second genotyping of single-nucleotide polymorphism by high-resolution melting curve analysis on a thermal digital microfluidic device

Lab on a Chip ◽  
2016 ◽  
Vol 16 (4) ◽  
pp. 743-752 ◽  
Author(s):  
Tianlan Chen ◽  
Yanwei Jia ◽  
Cheng Dong ◽  
Jie Gao ◽  
Pui-In Mak ◽  
...  
Keyword(s):  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Dna Melting ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
High Resolution Melting Curve ◽  
Digital Microfluidic ◽  
Multiple Samples

A novel thermal digital microfluidic (T-DMF) device enables precise thermal modulation and pipelined measurement of multiple samples. Ultrafast DNA melting curve analysis is achieved in less than 7 seconds, with the resolution adequate for single-nucleotide discrimination.

scholarly journals Automated melting curve analysis in droplet microfluidics for single nucleotide polymorphisms (SNP) genotyping

RSC Advances ◽  
2017 ◽  
Vol 7 (8) ◽  
pp. 4646-4655 ◽  
Author(s):  
F.-W. Liu ◽  
S.-T. Ding ◽  
E.-C. Lin ◽  
Y.-W. Lu ◽  
J.-S. R. Jang
Keyword(s):  
Melting Curve ◽  
Automated Analysis ◽  
Curve Analysis ◽  
Snp Genotyping ◽  
Droplet Microfluidics ◽  
Melting Curve Analysis ◽  
Dna Melting ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

An integrated microchip platform with automated analysis capability for DNA melting curves is developed for Single Nucleotide Polymorphism (SNP) genotyping applications.

scholarly journals Rapid base-specific calling of SARS-CoV-2 variants of concern using combined RT-PCR melting curve screening and SIRPH technology

2021 ◽  
Author(s):  
Sascha Tierling ◽  
Kathrin Kattler ◽  
Markus Vogelgesang ◽  
Thorsten Pfuhl ◽  
Stefan Lohse ◽  
...  
Keyword(s):  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Single Nucleotide ◽  
Specific Identification ◽  
Rp Hplc ◽  
Novel Variants ◽  
Reliable Detection ◽  
Cost Efficient

The emergence of novel variants of concern of SARS-CoV-2 demands a fast and reliable detection of such variants in local populations. Here we present a cost-efficient and fast workflow combining a pre-screening of SARS-CoV-2 positive samples using RT-PCR melting curve analysis with multiplexed IP-RP-HPLC-based single nucleotide primer extensions (SIRPH). The entire workflow from positive SARS-CoV-2 testing to base-specific identification of variants requires about 24 h. We applied the sensitive method to monitor the local VOC outbreaks in a few hundred positive samples collected in a confined region of Germany.

scholarly journals Detection of Single Nucleotide Polymorphism rs2013162 of IRF6 Gene in Patient with Cleft Lip and Palate

2019 ◽  
Vol 3 (1) ◽  
pp. 28-40
Author(s):  
Husnain Shehzad ◽  
Osheen Shehzad
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Cleft Lip ◽  
Cleft Lip And Palate ◽  
Melting Curve ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Pakistani Population ◽  
Irf6 Gene ◽  
High Resolution Melting Curve ◽  
Hardy Weinberg Equilibrium

Abstract: Background: Cleft lip and palate are congenital disorders which induce affected individuals medically, socially and psychologically. The objective of this study was to investigate the association of Single Nucleotide Polymorphism(SNP); rs2013162 of IRF6 Gene in Patient with Cleft Lip and Palate. Materials and Methods: Fifty patients with non-syndromic CL/P were included in present study alongwith fifty individuals with no psychiatric history as controls. In all of the these individuals, search for Single nucleotide polymorphism was carried out by designing sequence specific primers. The sequence was amplified by using Real time PCR and products were investigated by visualizing high resolution melting curve upon HRM-PCR. Results: The logistic regression and Hardy-Weinberg equilibrium were applied to investigate the association of IRF6 SNP rs2013162 with disease. Results revealed no association of this polymorphism with non-syndromic CL/P. Conclusion: We found no association of IRF6 SNP rs2013162 in patients with non-syndromic CL/P. Further study is required with larger sample size to validate the findings of the present study in Pakistani population and along with this SNP other polymorphisms of the same gene should be analyzed to find out the association with the non-syndromic CL/P.

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