Chemistry choice
KBiosciences uses both its own novel form of competitive allele specific PCR system (KASPar) and Taqman™ chemistries for genotyping. Both work well in our hands. We will typically choose the chemistry to support the project, however should you have a preference then please let us know and we'll be happy to accommodate your request. In practice due to the high cost of setup and mediocre conversion rate for new assays for the Taqman Chemistry > 99% of our customers specify the use of KASParMore information on these chemistries can be found at the links below.
The KASPar chemistry is a proprietary KBioscience invented genotyping chemistry. KASPar reagents are also available for customers who wish to conduct their genotyping in-house.
Taqman is supplied by Applied Biosystems™
Assay validation setup
This is the process whereby we design & order oligos to the SNP
of interest. We then test the assay with a random set of DNA samples to ensure that it is polymorphic
(not all Database SNPs are). KBioscience will select the most appropriate
technology for your study unless you have a preference.
If your SNP is listed in ABI's Assay on demand™ service or you already have Taqman™ Primers & Probes then you may send us the Primers & Probes instead of us doing the validation setup therefore no validation charge will be applied. Using our miniaturisation strategy we require at least 4 fold less than ABI recommend. If you have a large population this equates to a very large cost saving for you. If you need more clarification then please give us a call.
| Technology | KASPar | Taqman™ |
| Options | We are able to design and develop indirect and direct KASPar assays. Direct KASPar assays are suited best in real-time applications, whereas indirect KASPar assays are suited well for end-point determinations. | If you have a preference, we can design and validate Taqman™ assays, either as standard Taqman™ assays or as Minor Groove Binders. |
| Success Rates | Typically we are able to convert approximately >80% of database SNPs (such as the TSC SNP set) to assays. Should the SNP be known to exist in the population under test this number increases significantly to >90%. | Typically we are able to convert 55% of database SNPs (such as the TSC SNP set) to assays. Should the SNP be known to exist in the population under test this number increases significantly to >80%. |
All our assays can be run on the ABI 7700, the ABI 7900 and any other fluorescent plate reader capable of distinguishing the fluorophores used.
Quality control criteria
For KBiosciences to deem an assay successful it must possess;
- Three distinct clusters
- Water controls must be negative
- Number of genotypes callable must be >90%
- Minor allele frequency should be greater than 2% unless the SNP is known to be very low frequency (KBiosciences is happy to accept your positive controls should you wish to include these).
We will of course show you the data for you approval should you be interested in this service. We will also perform a Hardy Weinberg Equilibrium Test to help you decide on the suitability of the chosen SNP for your Genotyping if appropriate.
Genotyping your samples
Using miniaturisation techniques, KBiosciences is able to keep the cost of
Genotyping to an absolute minimum whilst maintaining the highest levels of quality.
KBiosciences has built internal pipelines that accommodates small projects and
those with thousands of SNPs and millions of genotpyes.
Success rates
Generally, an accuracy greater than 99% is achieved. To date this has been assessed
in a number of blinded trials from customers and the error rate has been shown
to always be <0.3%.
Quality control criteria
KBiosciences performs routine quality control measures on all its Genotyping.
These criteria are shown below:
- Water controls must be negative
- Inter plate duplicate testing of a known DNA
- Intra plate testing of a known DNA
- Percentage success rate of calls must be greater than a customer determined level
- Clusters are clear and distinct