Method Comparison - a brief
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.
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
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
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
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