Single Nucleotide Extension (SNE)
This SNP genotyping technique is best suited for small projects with small number of samples (let's say...less than 30 samples), and a handful of SNPs.
We offer the Applied Biosystems SNaPshot kit for single nucleotide extension reactions. This is essentially a "mini-sequencing" reaction using non-fluor primers and the same Big Dye terminators used in fluorescent sequencing. The multi-step process involves 1) PCR of a region containing a known SNP, 2) ExoSap clean-up of excess dNTPs and primers, 3) SNE reaction using an interrogation primer and dye labeled ddNTP's, 4) Sap cleanup and 5) Gel electrophoresis.
The result is a single color peak for each specific nucleotide incorporated. The electropherogram below shows six multiplexed SNE reactions. Left to right, the sample 1 shows a heterozygote of T and C, sample 2 shows a homozygote A, sample 3 is a TG het, sample 4 is a G hom, sample 5 is a TC het and sample 6 is an AT het. This method is highly specific, but requires rigorous clean-up.
1 ------------- 2 --------- 3 ------------ 4 ------------- 5 --------- 6
Multiple SNE reactions can be run in the same lane (as in the picture above) if the primers are designed with tails to allow at least a 4bp separation. This results in a cost savings similar to that of multiplexed sets of STRPs.
Contact us for more information, including pricing.