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Frequently Asked Questions

What are the components of the kit?
Forward and reverse primers for both genomic DNA and heteroduplex reagent, the heteroduplex reagent, wild type and mutant controls for all known allelles, dNTPs, PCR buffer and instruction for use booklet containing an example of the expected gel patterns. The freeze dried reagents are easily reconstituted in their tubes using the PCR buffer supplied. The user has only to supply their preferred Taq polymerase and sample of genomic DNA.

How many DNA samples does each kit test?
Two sizes of kit are available to test 10 or 25 DNA samples. Each kit also contains sufficient reagents to include a wild type and mutant control with each DNA test if required.

Do you need to extract genomic DNA before the PCR reaction or can raw blood/tissue be added to the tube?
We recommend extracted DNA but crude lysates of blood, buffy coat or any nucleated cell preparation should produce a result.

If the DNA is extracted do you need to quantify it in order to know how much needs to be added to the PCR reaction to achieve enough amplification to analyse?
We recommend 100ng-500ng per reaction but there is considerable flexibility. Concentrations between 10ng and 1ug per reaction have been successfully used in over 24,000 clinical samples.

Can kits investigate several SNPs or diseases at the same time?
Several of our kits are examples of multiplexed reactions where 2 or more SNPs on the same region of the genome are detected (eg. Fibroblast growth factor 3 R248C/S249C/p250R) or when SNP?s on separate genomes are detected simultaneously (eg. AAT-Pi*S/AAT-Pi*Z). In addition we have ?screening? kits able to analyse heteroduplex products from separate PCR reactions in a single lane of a polyacrylamide gel (eg. Coagulation screen).

How long does a test take, start to finish?
Assuming DNA is isolated and amplified, it takes about 15 minutes for the crossmatch, 60-90 minutes for minigel PAGE and 5 minutes for the stain. Total approximately 80-110 minutes. Different parameters are used for capillary electrophoresis and fluorescent detection, depending upon the end user?s equipment.

How many PCR cycles are required? Is the PCR reaction a standard, universal one which applies to all the kits?
The standard recommended is 30-32 cycles. This depends completely on the thermal cycler used, and can take 30 minutes (light cycler) to 3 hours. PCR conditions will vary from kit to kit but we try to accommodate most in a single program.

What percentage gel is required for analysis? What size gel is required? How long will separation take and at what voltage?
We recommend 15% nondenaturing PAGE. Any size gel can be used but separation can be achieved in 60-90 minutes on a 7cm height gel or above. On a 7cm gel, we would use 200V (for a 10cm gel width) or 300V (for a 25cm gel width).

Can Heteroduplex Analysis make kits to order?
Yes, Heteroduplex Analysis can make bespoke products. Please contact us on 01904 435261.


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Multiple SNP Genotyping


Cell Analysis Ltd • The Innovation Centre • Innovation Way • York  YO10 5DG  United Kingdom  Tel: +44 (0) 1904 435261    Fax: +44 (0) 1904 435126