In molecular biology, restriction fragment length polymorphism, or
RFLP, is a technique that exploits variations in homologous DNA
sequences. It refers to a difference between samples of homologous DNA
molecules from differing locations of restriction enzyme sites, and to
a related laboratory technique by which these segments can be
illustrated. In RFLP analysis, the
DNA sample is broken into pieces
(and digested) by restriction enzymes and the resulting restriction
fragments are separated according to their lengths by gel
electrophoresis. Although now largely obsolete due to the rise of
DNA sequencing technologies, RFLP analysis was the first
DNA profiling technique inexpensive enough to see widespread
application. RFLP analysis was an important tool in genome mapping,
localization of genes for genetic disorders, determination of risk for
disease, and paternity testing.
1 Analysis (technology)
4 See also
6 External links
The basic technique for the detection of RFLPs involves fragmenting a
DNA by a restriction enzyme, which can recognize and cut DNA
wherever a specific short sequence occurs, in a process known as a
restriction digest. The resulting
DNA fragments are then separated by
length through a process known as agarose gel electrophoresis, and
transferred to a membrane via the
Southern blot procedure.
Hybridization of the membrane to a labeled
DNA probe then determines
the length of the fragments which are complementary to the probe. An
RFLP occurs when the length of a detected fragment varies between
individuals. Each fragment length is considered an allele, and can be
used in genetic analysis.
RFLP analysis may be subdivided into single- (SLP) and multi-locus
probe (MLP) paradigms. Usually, the SLP method is preferred over MLP
because it is more sensitive, easier to interpret and capable of
DNA samples. Moreover, data can be
generated even when the
DNA is degraded (e.g. when it is found in bone
Schematic for RFLP by cleavage site loss.
Analysis and inheritance of allelic RFLP fragments (NIH).
Schematic for RFLP by VNTR length variation.
There are two common mechanisms by which the size of a particular
restriction fragment can vary. In the first schematic, a small segment
of the genome is being detected by a
DNA probe (thicker line). In
allele "A", the genome is cleaved by a restriction enzyme at three
nearby sites (triangles), but only the rightmost fragment will be
detected by the probe. In allele "a", restriction site 2 has been lost
by a mutation, so the probe now detects the larger fused fragment
running from sites 1 to 3. The second diagram shows how this fragment
size variation would look on a Southern blot, and how each allele (two
per individual) might be inherited in members of a family.
In the third schematic, the probe and restriction enzyme are chosen to
detect a region of the genome that includes a variable number tandem
repeat segment (boxes in schematic diagram). In allele "c" there are
five repeats in the VNTR, and the probe detects a longer fragment
between the two restriction sites. In allele "d" there are only two
repeats in the VNTR, so the probe detects a shorter fragment between
the same two restriction sites. Other genetic processes, such as
insertions, deletions, translocations, and inversions, can also lead
to RFLPs. RFLP tests require much bigger samples of
DNA than do short
tandem repeat (STR) tests.
Analysis of RFLP variation in genomes was a vital tool in genome
mapping and genetic disease analysis. If researchers were trying to
initially determine the chromosomal location of a particular disease
gene, they would analyze the
DNA of members of a family afflicted by
the disease, and look for RFLP alleles that show a similar pattern of
inheritance as that of the disease (see Genetic linkage). Once a
disease gene was localized, RFLP analysis of other families could
reveal who was at risk for the disease, or who was likely to be a
carrier of the mutant genes.
RFLP analysis was also the basis for early methods of genetic
fingerprinting, useful in the identification of samples retrieved from
crime scenes, in the determination of paternity, and in the
characterization of genetic diversity or breeding patterns in animal
The technique for RFLP analysis is, however, slow and cumbersome. It
requires a large amount of sample DNA, and the combined process of
DNA fragmentation, electrophoresis, blotting,
hybridization, washing, and autoradiography could take up to a month
to complete. A limited version of the RFLP method that used
oligonucleotide probes was reported in 1985. Fortunately, the
results of the
Human Genome Project
Human Genome Project have largely replaced the need for
RFLP mapping, and the identification of many single-nucleotide
polymorphisms (SNPs) in that project (as well as the direct
identification of many disease genes and mutations) has replaced the
need for RFLP disease linkage analysis (see SNP genotyping). The
analysis of VNTR alleles continues, but is now usually performed by
polymerase chain reaction (PCR) methods. For example, the standard
DNA fingerprinting involve PCR analysis of panels of
more than a dozen VNTRs.
RFLP is still a technique used in marker assisted selection. Terminal
restriction fragment length polymorphism (TRFLP or sometimes T-RFLP)
is a molecular biology technique initially developed for
characterizing bacterial communities in mixed-species samples. The
technique has also been applied to other groups including soil fungi.
TRFLP works by PCR amplification of
DNA using primer pairs that have
been labeled with fluorescent tags. The PCR products are then digested
using RFLP enzymes and the resulting patterns visualized using a DNA
sequencer. The results are analyzed either by simply counting and
comparing bands or peaks in the TRFLP profile, or by matching bands
from one or more TRFLP runs to a database of known species. The
technique is similar in some aspects to
DGGE or TGGE.
The sequence changes directly involved with an RFLP can also be
analyzed more quickly by PCR. Amplification can be directed across the
altered restriction site, and the products digested with the
restriction enzyme. This method has been called Cleaved Amplified
Polymorphic Sequence (CAPS). Alternatively, the amplified segment can
be analyzed by
Allele specific oligonucleotide (ASO) probes, a process
that can often be done by a simple Dot blot.
^ Saiki, R.; Scharf, S; Faloona, F; Mullis, K.; Horn, G.; Erlich, H.;
Arnheim, N (1985). "Enzymatic amplification of beta-globin genomic
sequences and restriction site analysis for diagnosis of sickle cell
anemia". Science. 230 (4732): 1350–1354.
doi:10.1126/science.2999980. ISSN 0036-8075.
Molecular genetics: key methods of study
Restriction fragment length polymor