Biomedical Engineering Reference
In-Depth Information
main task of the assay design is to identify a short sequence of approx
60-80 bp within the DNA sequence of interest that satisfies the following
criteria (
see
Note 1
).
1.
The target sequence should preferably not have a high GC content so that it is
easily amplifiable by PCR.
2.
Two PCR primers (forward and reverse primers) that anneal to the two ends of
the sequence. The PCR primers are typically 20 bases long and have a melting
temperature (Tm) above 58°C. A 10-base tag (5'-ACGTTGGATG-3') is added to
the 5' end of the PCR primers so that these primers will not interfere in mass
spectra.
3.
A G/C (or C/G) mutation can be introduced approximately in the middle of the
sequence.
4.
Directly adjacent to the mutation site (either on the forward or reverse strand), an
extension primer (
see
Note 2
) of 16-25 bases (approx 4800 to 7500 Da) with a
Tm of 55°C or higher can be identified.
3.1.2. rcPCR for DNA Mutation Detection and Quantification
This design applies to situations in which both a wild-type and a mutant
DNA sequence may be present and a researcher wants to detect and quantify
both DNA sequences (allele-specific quantification). Assay designs are slightly
more complicated here, because three extension products (from the DNA stan-
dard, the mutant DNA, and the wild-type DNA) may be produced. In addition,
one or more pausing products may also be present. In
Fig. 1
, if only G is added
to the extension primer (instead of GddA), we will obtain a pausing product
(
Fig. 2
). The pausing product will interfere in the mass spectrum if its molecu-
lar weight is close to one of the normal extension products. Under
Subheading
3.1.1.
, because the G/C (or C/G) mutation is chosen, there will be no pausing
product. However, natural mutations are not always G/C (or C/G) mutations.
There are two possible options for DNA mutation detection and quantification.
1.
Only the relative abundance between a wild type DNA and a mutant DNA is quan-
tified. The assay design is very similar to that under
Subheading 3.1.1.
, except
that we do not add an oligonucleotide standard with an artificial mutation. Because
a natural mutation is not necessarily a G/C mutation, extra care must be taken to
ensure that the pausing product does not have molecular weight that is very simi-
lar to the extension products.
2.
The absolute concentrations of a wild-type and a mutant DNA are quantified. An
example is given in
Fig. 2
.
The wild-type DNA has a C whereas the mutant DNA
has a G at the polymorphic site. The oligonucleotide standard (competitor) is
designed to have a T/A mutation next to the G/C polymorphism. These three
DNA sequences are co-amplified by PCR. For base extension reaction, a ddC/
ddG/ddT/dA mixture is used to produce three extension products and a possible
pausing product. The synthetic mutation in the oligonucleotide standard is
