Agriculture Reference
In-Depth Information
markers known so far (Gupta
et al.,
2001). SNPs, at a particular site in a DNA
molecule should in principle involve four possible nucleotides, but in actual practice
only two of these four possibilities have been observed at a specific site in the
population (Brookes, 1999). Consequently, SNP is a biallelic marker system as
against the polyallelic marker systems such as RFLPs and microsatellites.
Identification of SNPs within a genetic locus can be achieved by using different
methods. Prior to the detection of polymorphism, the sequence of the locus for a
reference genotype needs to be determined. Once determined, this sequence can be
used in designing oligonucleotide primers for use in the PCR, which forms the
cornerstone of all subsequent SNP-based technology (Erlich, 1989).
Direct
sequencing
is the most common and direct way of identifying SNPs; however, it is
also the most time-consuming and expensive method. Sequencing errors are also a
restrain and if not detected in early stages, would result in a considerable waste of
resources in both designing allele-specific nucleotides and carrying out SNP assays.
Single-strand conformation polymorphism (SSCP)
uses variation in the mobility
of small polymorphic single-stranded DNA fragments in non-denaturing acrylamide
gels (Jordan
et al.,
1998). SSCP is based on the assumption that the mobility of any
DNA fragment will vary based on the exact sequence, and even a single base change
can modify the mobility. This technique works well with small fragments (100-400
bp) that can be generated by PCR. Any change in mobility across the genotype
would indicate a sequence change that could be targeted by direct sequencing.
Etscheid and Riesner (1998) proposed several modifications and improvements to
the SSCP technique and at present temperature gradient gel electrophoresis and
denaturing gradient gel electrophoresis are the most widely used for SNP detection.
Chemical cleavage of mismatches (CCM)
relies on the ability of certain chemicals
like piperidine (Prosser, 1993), to cleave a chemically modified heteroduplex
precisely at mismatched bases. This technique involves a reference sample that is
labelled with radioactivity and a sample under question. Both samples can be
generated by PCR and after denaturation, the two samples are brought together and
allowed to anneal to form homoduplexes and heteroduplexes. When a heteroduplex
with sequence difference has been formed, it is chemically modified using osmium
tetraoxide and then subjected to peperdine treatment that cleaves the mismatch. The
cleaved and uncleaved products are then run on a denaturing polyacrylamide gel and
autoradiographed. CCM is capable of scanning regions of up to 3 kb with cent per
cent accuracy despite the disadvantages of being time-consuming and the handling
of hazardous chemicals. To overcome the latter, a technique called
Enzyme
mismatch cleavage (EMC)
has been introduced for SNP identification. EMC is
very similar in principle to CCM except osmium tetraoxide is replaced by the
enzyme 'resolvase'. Similar to CCM, the amplified DNA from either an individual
plant or two individual plants is heat-denatured and allowed to re-anneal to form
homo- and heteroduplexes which are then labelled with radioactivity. Any
heteroduplex structures present are then subjected to resolvase enzyme treatment
that cleaves the mismatch; this is then examined on a denaturing polyacrylamide gel.
EMC has been found to be more efficient in scanning for SNPs (several kilo bases
of DNA) and is gaining importance in SNP identification (Edwards and Mogg,
2001). All the methods listed above are used for the fingerprinting of two or more
