Investigating CSI – Background material Table of Contents I ...

D. Creating a DNA Profile
RFLP Method - Restriction Fragment Length Polymorphism
RFLP (often pronounced "rif lip", as if it were a word) is a method used by
molecular biologists to follow a particular sequence of DNA as it is passed on to
other cells. RFLPs can be used in many different settings to accomplish different
objectives. Some uses include in paternity cases or criminal cases to determine
the source of a DNA sample, to determine the disease status of an individual, or
to measure recombination rates which can lead to a genetic map with the
distance between RFLP loci measured in centiMorgans.
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
single-stranded 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 seuqence
GAATTC and cuts the double-stranded DNA as shown:
In the segement of DNA shown below, you can see the elements of an RFLP; a
target sequence flanked by a pair of restriction sites. When this segment of DNA
is cut by EcoR I, three restriction fragments are produced, but only one contains
the target sequence which can be bound by the complementary probe
sequence(purple).
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