Debra Nickson looks at an error-free genotyping tool used to analyse genetic variation. Genotypes can be identified by simply looking for a colour change. Results are available instantaneously.
Single nucleotide polymorphisms (SNPs) represent the most common form of genetic variability. They are distinguished from rare mutations in that the least abundant allele is present at a frequency of >1 per cent. SNP genotyping applications are wide-ranging. SNP-based markers provide high resolution for mapping studies, and SNP scoring serves as an effective means to conduct population genetics studies.
The SureScore SNP Genotyping technology from Invitrogen is a tool to analyse genetic variation. The key to accurate SureScore genotyping is SNP-IT (SNP Identification Technology), a highly robust single-base primer extension SNP scoring method (Fig. 1). The reaction involves three main elements: u Your templatea PCR-amplified, phosphorothioate-protected, single-stranded DNA containing the polymorphic site of interest. u DNA polymeraseto accurately distinguish and identify nucleotides at the SNP site. u SNP-IT capture primer custom-designed with SureScore software for each genotyping assay.
The SNP-IT capture primer hybridises to your templateaat the base directly adjacent to the SNP site. DNA polymerase uses an antibody conjugate-labelled terminating nucleotide to extend the 3´ end of the capture primer by one base, and precisely denotes the base at the SNP site. Genotype calling is error-free, and your results are as accurate as with direct DNA sequencing.
Design a pair of PCR amplification primers and a SNP-IT capture primer for your genotyping assay by visiting Invitrogen's website at www.invitrogen.com/surescore. Submit your target sequence containing the SNP, and their user-friendly, web-based SureScore primer design software will generate recommended sequences for all three primers. Once you have designed your primers, you can then choose the right kit for your genotyping experiment. Select the SureScore Complete Kit if you are studying multiple types of SNPs.
To identify your SNP genotype, simply look for the colour change. The ELISA-based colourimetric reaction provides instantaneous resultsathe biotin-labelled nucleotide yields a blue colour; the fluorescein-labelled nucleotide yields a yellow colour (Fig. 2). To further your capabilities, you can use a plate reader in conjunction with SureScore Data Analysis Software. The software is accessible on-line with kit purchase. It allows you to automate genotype calling, perform cluster analysis, and generate reports.
Using the kit, you perform your SNP genotyping assay using standard lab equipment that you already have. Conduct a colourimetric assay, and score your SNPs by simple visualisation.
Unlike many other SNP scoring products, you do not need to invest in expensive and specialised equipment. The kit comes in a 96-well format, enabling you to conduct genotyping on up to 96 genomic DNA samples on a single SureScore strip-well plate.
SureScore makes error-free SNP genotyping accessible to any laboratory. It is a suitable system for low- to medium-throughput SNP genotyping studies. u
ENQUIRY No 93
Debra Nickson is with Invitrogen Ltd, Paisley, UK. www.invitrogen.com/surescore
