SNPs

SNP Detection - Jacqueline Batley

Agricultural applications of SNPs, for example in genetic mapping projects or breeding programs, involves the analysis of a large number of samples, and therefore requires rapid, inexpensive, and highly automated methods to genotype the sequence variants. Towards this, we have applied a high throughput Single Nucleotide Primer Extension assay The SNuPE (Single nucleotide primer extension) method is essentially a minisequencing reaction where only a single base (the SNP) is sequenced. The SNuPE method can be broken down into two stages. Firstly, template DNA from the SNP containing genomic region is amplified by PCR using marker specific primers. This template then undergoes a single base extension reaction. A detection primer anneals to the nucleic acid sequence immediately 3' of the nucleotide position to be analysed. This primer is then extended by one base, incorporating one fluorescently-labelled ddNTP. Using different labels for each ddNTP allows the identification of the specific nucleotide incorporated at that position. ddNTPs are used over dNTPs as they terminate the reaction and do not allow the incorporation of further nucleotides. The annealing primer determines the position of the nucleotide being detected and the fluorescent label on the incorporated ddNTP identifies the nucleotide at that site. DNA sequencing instruments allow sensitive detection of SNuPE primers extended with fluorescent ddNTPs. With the use of capillary-array gel electrophoresis, up to 13 injections of 96 loci can be identified using the same matrix. By separating primers by size and multiplexing these for each injection, several markers can be analysed in the same injection, further increasing the throughput of the reaction.

Step 1: PCR
The locus or gene containing the SNP(s) of interest is amplified from genomic DNA

Step 2: Purification of the PCR product
Unincorporated primers and dNTPs are removed from the PCR product. If PCR primers or single stranded DNA are present in the extension reaction they can get extended along with the extension primer and confound the experimental results.

Step 3: Single base extension reaction
A primer which anneals immediately adjacent to the SNP is extended, using Thermosequenase, by one base using a fluorescently labelled ddNTP. Two nucleotides are used (corresponding to the 2 alleles present at the SNP to be genotyped), labelled with two different dyes. No non-terminator nucleotides are present.

Step 4: Purification of the single base extension reaction product
Unincorporated ddNTPs are removed from the PCR product. This ensures they do not produce extra peaks during electrophoresis and obscure the allele of interest.

Step 5: Electrophoresis of samples on a MegaBACE capillary sequencer
The samples are electrophoresed on an automated sequencer (13x96-well plates per run). A marker is used to identify each injection, and the data is analysed to using genotypic analysis to determine the SNP alleles present.

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Maintained by Gary Barker Last updated Dec 2001