Founder Effects and Exotic Variability - Alien Species and Evolution - page 35

R ESTRICTION F RAGMENT L ENGTH P OLYMORPHISM (RFLP)
The RFLP procedure is most suited to analysis of variation within a
species or among closely related species.The objective usually is to deter-
mine if allelic variation exists for a particular gene.To detect allelic differ-
ences, the products of digestion by restriction enzymes are hybridized
with a radioactively labeled DNA probe. This probe is a short sequence
of DNA, chosen to match a portion of the gene under analysis, combined
with a label such as a radioisotope.The DNA fragments created by use of
restriction enzymes are separated by gel electrophoresis and hybridized
with the probe.Then the positions of the hybridized fragments are iden-
tified on an x-ray film. If genetic variations exist that affect the length of
DNA fragments containing the gene, bands will appear at slightly differ-
ent positions on the electrophoretic gel.
A MPLIFIED F RAGMENT L ENGTH P OLYMORPHISM (AFLP)
In the AFLP technique, double-stranded DNA is digested with two
restriction enzymes, one that cuts at a specific six-nucleotide section and
one at a specific four-nucleotide section. Specific double-stranded DNA
adapters, usually about 25-30 base pairs in length, are then ligated to the
ends of restriction fragments. DNA primers, complementary to the
adapters except for a specific extension of three nucleotides, are then used
to prepare a subset of the restriction fragments for amplification by the
enzyme DNA polymerase, a procedure termed polymerase chain reaction
(PCR). These primers attach to the fragments with the complementary
adapter and extension sequence.The DNA region amplified thus depends
on the primers. These may be specific to a particular region of the
genome, such as a specific gene. Only the fragments that hybridize with
the primers are amplified. Amplification involves the annealing of the
double-stranded DNA, that is, its separation into individual strands, and
its replication by DNA polymerase.This process is repeated 30-40 times,
and the fragments with attached primers are increased enormously in
abundance. The amplified DNA fragments are then separated by elec-
trophoresis on a polyacrylamide gel.The locations of these fragments are
then determined by staining or application of a radioactive probe.
R ANDOM A MPLIFIED P OLYMORPHIC DNA A NALYSIS (RAPD)
The RAPD technique is similar to the AFLP technique but usually does
not involve the use of restriction enzymes (Williams et al. 1990). The
primers used in RAPD are only 10 bases in length and consequently find