RFLP's- SNP's and Microsatellites
An RFLP – is a mutation that results in a change in the pattern in restriction fragments generated
when a DNA molecule is treated with a restriction enzyme.
Restriction enzymes cut DNA at precise points producing
 a collection of DNA fragments of precisely defined length.
 These can be separated by electrophoresis, with the smaller fragments migrating farther than
the larger fragments.
 One or more of the fragments can be visualized with a "probe" — a molecule of singlestranded DNA that is
 complementary to a run of nucleotides in one or more of the restriction fragments
and is
 radioactive (or fluorescent).
RFLP Production
Each organism inherits its DNA from its parents. Since DNA is replicated with each generation, any
given sequence can be passed on to the next generation. An RFLP is a sequence of DNA that has a
restriction site on each end with a "target" sequence in between. A target sequence is any segment
of DNA that bind to a probe by forming complementary base pairs. A probe is a sequence of singlestranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be
detected. When a probe base pairs to its target, the investigator can detect this binding and know
where the target sequence is since the probe is detectable. RFLP produces a series of bands when a
Southern blot is performed with a particular combination of restriction enzyme and probe sequence.
For example, let's follow a particular RFLP that is defined by the restriction enzyme EcoR I and the
target sequence of 20 bases GCATGCATGCATGCATGCAT. EcoR I binds to its recognition
sequ
ence
GAA
TTC
and
cuts
the
doub
lestran
ded
DN
A.
SNP
's
A
Sing
Michelle Murphy 13-46
le Nucleotide Polymorphism, or SNP (pronounced "snip"), is a small genetic change, or variation,
that can occur within a person's DNA sequence. The genetic code is specified by the four
nucleotide "letters" A (adenine), C (cytosine), T (thymine), and G (guanine). SNP variation occurs
when a single nucleotide, such as an A, replaces one of the other three nucleotide letters—C, G, or
T.
Each person's genetic material contains a unique SNP pattern that is made up of many different
genetic variations. Researchers have found that most SNPs are not responsible for a disease state.
Instead, they serve as biological markers for pinpointing a disease on the human genome map,
because they are usually located near a gene found to be associated with a certain disease.
Occasionally, a SNP may actually cause a disease and, therefore, can be used to search for and
isolate the disease-causing gene.
SNP's as molecular markers
Because SNPs occur frequently throughout the genome and tend to be relatively stable genetically,
they serve as excellent biological markers. Biological markers are segments of DNA with an
identifiable physical location that can be easily tracked and used for constructing a chromosome
map that shows the positions of known genes, or other markers, relative to each other.
Micro
satelli
tes
SSR's
Micro
satelli
tes are
simpl
e
seque
nce
repeat
s
(SSRs
), 1 to
6
nucle
otides
in
length
,
which
show
a high
degre
e of
polym
orphism. Specific microsatellites can be isolated using hybridized probes followed by their
Michelle Murphy 13-46
sequencing. Like any DNA fragment, SSRs can be detected by specific dyes or by radiolabelling
using gel electrophoresis. The advantage of using SSRs as molecular markers is the extent of
polymorphism shown, which enables the detection of differences at multiple loci between strains
[3].Coupled with chemical and morphological data, we can identify the plant species or strain of
interest. The main advantage of using SSRs for fingerprinting is that small amounts of DNA are
required compared to the restriction fragment length polymorphisms (RFLP) method. This is due to
the large amounts of SSRs present in any genome. Further, assays involving SSRs are more robust
than random amplified polymorphic DNA (RAPDs), making them up to seven times more efficient.
A drawback to using SSRs is the need to develop separate SSR primer sets for each species. The
latest research suggests that SSRs will be involved in new methods of detection of alterations of
specific sequences in the DNA.
Michelle Murphy 13-46