Biology Reference
In-Depth Information
Table 13.3: A Simplified Method for Constructing Microsatellite-Enriched Libraries.
l
Genomic DNA is digested in four separate reactions using two endonucleases that produce blunt ends to
produce DNA fragments 200-800 bp in length.
l
Digested DNA is A-tailed to facilitate ligation to adapters.
l
Two random adapters 20-23 nt in length are designed with a 3
′
T overhang to facilitate ligation to
genomic DNA fragments having an A overhang at the 3
′
end.
l
Genomic DNA and the adapter sequences are ligated, then cleaned.
l
DNA (with the adapters at each end) from the ligation step is amplified by the PCR using the adapter
oligo as a primer.
l
Cleaned DNA from the PCR is hybridized to biotinylated oligo repeats.
l
Magnetic beads are prepared to bind the biotinylated DNA.
l
Beads are washed, releasing single-stranded DNA.
l
Single-stranded DNA is amplified by the PCR.
l
The PCR products are cloned into a T-A vector and transformed into cells. Plasmids are extracted and
sequenced.
l
Sequenced clones are analyzed to identify sequence repeats with 1-8-bp motifs.
l
Specific PCR primers are designed based on the motifs and tested to determine whether they produce
useful PCR products for analysis.
Adapted from Techen et al. (2010).
13.6.3 RAPD-PCR
RAPD-PCR bands are considered as dominant loci in diplo-diploid organisms, and
scored as present or absent (
Hadrys et al. 1992
).
Kambhampati et al. (1992)
dis-
cussed appropriate statistical methods for analysis of data. It appears that RAPD-
PCR loci can be used to determine paternity, kinship, and hybridization, as well
as to estimate population heterozygosity, effective population size, identify bio-
types and cryptic species, genetic distance between populations, and interpopu-
lation diversity (
Table 13.1
).
13.6.4 RFLPs
Visualization and interpretation of RFLP data was described by
Aquadro et al.
(1992)
and
Dowling et al. (1990)
. Restriction patterns can be compared either by
the lengths of the fragments or by comparing actual restriction sites. Restriction
patterns can be classified as haplotypes and a measure of diversity can be
derived as a function of the frequency of the different haplotypes (
Hoelzel and
Bancroft 1992
). The term
haplotype
is a contraction of
haplo
id and geno
type
and describes the combination of linked alleles in a cluster of related genes.
Likewise, genetic distance is measured as an estimate of the number of base
substitutions per nucleotide separating the two populations. Interpopulation
diversity (G
ST
) can be estimated in a manner similar to that for allozyme data,
