Biology Reference
In-Depth Information
required for each SNP. An exception is the mitochondrial genome, where a number
of SNPs are concentrated into a small area and can be analysed in a small number
of reactions (see Chapter 13). Sequencing has also been a powerful method to type
SNPs within rapidly evolving regions of DNA in the HIV virus [25, 26].
SNP detection for forensic applications
Restriction digestion and sequence analysis are not viable methods to use for most
forensic cases that might require the analysis of 50 - 80 SNPs dispersed around the
genome. A number of methods have evolved that can be applied to the detection of
multiple SNPs. Methods that are based around the concepts of either primer extension
or primer hybridization are the most widely used.
Primer extension
Primer extension is a robust method for discriminating between different alleles
and several methodologies have been developed [27]. One of the commonly used
methods is the mini-sequencing reaction [28]. The basis of the reaction is very similar
to Sanger sequencing. The first part of the procedure is to amplify the target region
using PCR. An internal primer then anneals to the denatured PCR product; the 3 end
of the primer is adjacent to the polymorphic site. The primer is then extended by Ta q
polymerase but only ddNTPs that are labelled with fluorescent dyes are provided,
therefore the primer is only extended by one nucleotide. The extended primer can
be analysed using capillary gel electrophoresis and the colour of the detected peak
allows the SNP to be characterized (Figure 12.4). The widely used SNaPshot
kit
(Applied Biosystems) is based on this methodology.
By using different sized primers and different fluorescent tags for each of the four
bases, a large number of SNPs can be simultaneously detected [29].
Variations on the primer extension technique include pyrosequencing [30, 31];
microarrays, where the extension primers are attached to a silicon chip [32, 33]; and
allele-specific extension, when the primer is only extended if it is 100% complemen-
tary to the target sequence [34].
Allele-specific hybridization
Under stringent conditions, even one nucleotide mismatching between a template
and primer can differentiate between two alleles (Figure 12.5).
There is a large number of methods that exploit the hybridization of probes,
including Taqman MGB (minor groove binder) assays [35], GenPlex [36], reverse
dot blots [37]; LightCycler
assays [38]; molecular beacons [3, 39, 40]; and
GeneChips
[41] (see Ref. [27] for details).
Search WWH ::




Custom Search