Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.

Resource Type: 
Publication
Publication Type: 
Journal Article
Title: 
Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.
Authors: 
Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C
Series Name: 
Clinical chemistry
Journal Abbreviation: 
Clin Chem
Volume: 
50
Issue: 
7
Page Numbers: 
1156-64
Publication Year: 
2004
Publication Date: 
2004 Jul
ISSN: 
0009-9147
PISSN: 
0009-9147
Cross Reference: 
PMIDLoading content
Citation: 
Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.. Clinical chemistry. 2004 Jul; 50(7):1156-64.
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 (

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 beta-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 (T(m)s) that differ by 0.8-1.4 degrees C. However, in approximately 16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T(m) differences between homozygotes (<0.4 degrees C). Although most of these cases can be genotyped by T(m), 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.

Publication Model: 
Print
Language: 
English
Language Abbr: 
eng
Journal Country: 
England