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An efficient procedure for genotyping single nucleotide polymorphisms - PubMed

  • ️Mon Jan 01 2001

An efficient procedure for genotyping single nucleotide polymorphisms

S Ye et al. Nucleic Acids Res. 2001.

Abstract

Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.

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Figures

Figure 1
Figure 1

Schematic presentation of the tetra-primer ARMS-PCR method. The single nucleotide polymorphism used here as an example is a G→A substitution, but the method can be used to type other types of single base substitutions. Two allele-specific amplicons are generated using two pairs of primers, one pair (indicated by pink and red arrows, respectively) producing an amplicon representing the G allele and the other pair (indicated by indigo and blue arrows, respectively) producing an amplicon representing the A allele. Allele specificity is conferred by a mismatch between the 3′-terminal base of an inner primer and the template. To enhance allelic specificity, a second deliberate mismatch (indicated by an asterisk) at position –2 from the 3′-terminus is also incorporated in the inner primers. The primers are 26 nt or longer, so as to minimize the difference in stability of primers annealed to the target and non-target alleles, ensuring that allele specificity results from differences in extension rate, rather than hybridisation rate. By positioning the two outer primers at different distances from the polymorphic nucleotide, the two allele-specific amplicons differ in length, allowing them to be discriminated by gel electrophoresis.

Figure 2
Figure 2

(Above and previous two pages) Results of tetra-primer ARMS-PCR and validation using the restriction fragment length polymorphism typing method. (A) the TNF gene –308G→A polymorphism; (B) the IL6 gene –174G→C polymorphism; (C) the AGTR1 gene 1166A→C polymorphism.

Figure 2
Figure 2

(Above and previous two pages) Results of tetra-primer ARMS-PCR and validation using the restriction fragment length polymorphism typing method. (A) the TNF gene –308G→A polymorphism; (B) the IL6 gene –174G→C polymorphism; (C) the AGTR1 gene 1166A→C polymorphism.

Figure 2
Figure 2

(Above and previous two pages) Results of tetra-primer ARMS-PCR and validation using the restriction fragment length polymorphism typing method. (A) the TNF gene –308G→A polymorphism; (B) the IL6 gene –174G→C polymorphism; (C) the AGTR1 gene 1166A→C polymorphism.

Figure 3
Figure 3

Tetra-primer ARMS-PCR assays with different concentrations of the outer primers. Shown here are tetra-primer ARMS-PCR assays for the AGTR1 gene 1166A→C polymorphism, with the same amounts of inner primers but different amounts of outer primers (i.e. the ratios of outer primers to inner primers in terms of their final concentration being 1:20, 1:10, 1:5, 1:3 or 1:1, respectively).

Figure 4
Figure 4

Results of tetra-primer ARMS-PCR reactions with the TNF gene –308G→A polymorphism, with touchdown or normal cycling conditions.

Figure 5
Figure 5

Analysis of tetra-primer ARMS-PCR products using the MADGE system. (A) PCR products for the IL6 gene –174G→C polymorphism were subjected to MADGE gel electrophoresis. The gel was then stained with Vistra green and scanned using a fluorimager. (B) The MADGE gel image in (A) was analysed using the Phoretix 1D Advanced computer software in which all 96 lanes are laid out in tandem to facilitate genotype scoring. Lanes 11H and 12H, negative controls where no DNA template was included in the PCR reactions; lanes 6H, 7H and 12H, PCR failed.

Figure 6
Figure 6

Flow chart of the tetra-primer ARMS-PCR primer design program.

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