scholarly journals Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis

2015 ◽  
Vol 6 ◽  
Author(s):  
Yanbin Hong ◽  
Manish K. Pandey ◽  
Ying Liu ◽  
Xiaoping Chen ◽  
Hong Liu ◽  
...  
Keyword(s):  
High Resolution ◽  
Single Nucleotide Polymorphisms ◽  
Arachis Hypogaea ◽  
High Resolution Melting ◽  
Amplicon Sequencing ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Hrm Analysis ◽  
Arachis Hypogaea L

scholarly journals Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons

2004 ◽  
Vol 50 (7) ◽  
pp. 1156-1164 ◽  
Author(s):  
Michael Liew ◽  
Robert Pryor ◽  
Robert Palais ◽  
Cindy Meadows ◽  
Maria Erali ◽  
...  
Keyword(s):  
High Resolution ◽  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Nucleotide Polymorphisms ◽  
Clinical Protocols ◽  
Single Nucleotide ◽  
Base Exchange ◽  
Closed Tube

Abstract Background: High-resolution melting of PCR amplicons with the DNA dye LCGreen™ I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (≤50 bp). Methods: Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and β-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification. Results: In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (Tms) that differ by 0.8–1.4 °C. However, in ∼16% of SNPs, the bases only switch strands and preserve the base pair, producing very small Tm differences between homozygotes (<0.4 °C). Although most of these cases can be genotyped by Tm, one-fourth (4% of total SNPs) show nearest-neighbor symmetry, and, as predicted, the homozygotes cannot be resolved from each other. In these cases, adding 15% of a known homozygous genotype to unknown samples allows melting curve separation of all three genotypes. This approach was used for the HFE 187C>G protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols. Conclusions: SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation. The method is closed-tube, performed without probes or real-time PCR, and can be completed in less than 2 min after completion of PCR.

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