Custom Mutation Assays

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Method Comparison - a brief introduction

There are many methods to choose from for mutation detection with different advantages and shortfalls.  Choosing the right one for your project is a critical step. 

The traditional Sanger sequencing method offers the advantage of detecting all the potential mutations at any mutation site, but has a low sensitivity of 20-50% mutant (mutant X gene has to be more than 20-50% of the total X gene) depending on whether the results are read by software or manually. 

Pyrosequencing or next-gen sequencing has a higher sensitivity of about 1%, but is much more expensive; the readout is easily affected by deletion/insertion mutations, and is not quantitative (unable to determine gene copy numbers).  

Mutant-specific real time PCR is an affordable way of detecting mutations.  It has a sensitivity of about 1-5% mutant DNA, and is quantitative.  It's sensitivity can be enhanced by using a WT DNA PCR blocker to ~0.1%.  This approach works fine when the mutation site is less variable.  However, when it comes to a complex mutation where the mutation site has several potential mutations (such as KRas codon 12/13 mutations), it can become complicated or impractical to assemble such "assay kits" because each possible mutation requires a dedicated assay.  One also has to overcome the cross-reactivity or "mis-priming" issues with a complex mutation.  A typical example is that most of the BRaf V600E commercial assays often cannot distinguish V600E from V600D and V600K mutations, leading to false positives.

Non-"mutant-specific" amplification followed by probe-based melting analysis offers the advantage of differentiating multiple mutations using a single probe in a single reaction.  Multiplex melting can be used to tackle more complex situations.  This approach is especially useful when you have limited amount of sample DNA.

Get it right the first time

There are two main reasons why our clients choose our custom mutation assay service. 

First, we have substantial experience and expertise in mutation assay development and validation.  Unlike other general service CROs, we have active R&D programs in early detection & diagnosis that are supported by government and private fundings.  Our expertise is also demonstrated by our mutation assay kits

Second, we are excellence-driven.  We take pains to improve assay sensitivity and specificity, as well as the capability to distinguish different mutation variants at a single mutation site.  For example, BRaf V600E mutation is not the only mutation at codon 600; other mutations at codon 600 may have distinct effects on BRaf kinase activities.  Our BRaf V600E qPCR mutation assay is the only BRaf mutation kit on the market that can distinguish V600E from V600D and V600K mutations, and detect other codon 600 mutations such as V600A, V600G, V600L, V600M and V600R.  

Get it right the first time by using our expertise; you will save tremendous amount of time, money and resources.  Establishing a perfect assay is like tackling the Rubik's Cube; we know exactly how to do it, and we can do it very fast!



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