In molecular biology , restriction fragment length polymorphism ( RFLP ) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms , in order to distinguish individuals, populations, or species or to indicate the locations of For in the gene within a sequence. The term may refer to a polymorphism, as detected through different locations of restriction enzyme sites , or to a related laboratory technique by which such differences can be characterized. In RFLP analysis , a DNA sample is digested into fragments by one or more restriction enzymes ., and the resulting restriction fragments are then separated by gel electrophoresis according to their size .
Although now largely obsolete due to the emergence of inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. RFLP analysis was an important early tool in genome mapping , localization of genes for genetic disorders , determination of disease risk, and paternity testing .
RFLP analysis
The basic technique for detecting RFLPs involves fragmenting a sample of DNA with the application of a restriction enzyme , which can selectively cleave a DNA molecule where a short, specific sequence is recognized in a process known as the Restrictions Digest . The DNA fragments produced by the digest are then separated by length, 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 that are complementary to the probe.Huh . A restriction fragment length polymorphism occurs when the length of a known fragment varies between individuals, reflecting non-identical sequence homology. The length of each fragment is considered an allele , whether it actually contains a coding region or not, and can be used in subsequent genetic analysis.
Example
There are two general mechanisms by which the size of a particular restriction section can be varied. In the first schematic, a small segment of the genome is being detected by the DNA probe (thick 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 is lost by a mutation , so the probe now detects a large fused fragment running from sites 1 to 3. The second diagram shows how this fragment size variation would appear on the 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 enzymes are chosen to locate a region of the genome that contains a variable number of tandem repeat (VNTR) segments (boxes in the schematic diagram). In allele C , the VNTR has five repeats, and the probe detects a long fragment between the two restriction sites. In allele D , the VNTR contains only two repeats, so the probe detects a short fragment between the same two restriction sites. Other genetic processes, such as insertions , deletions , transfers , and inversions , can also lead to polymorphism. Short Tandem Repeats for RFLP Tests(STR) tests require much larger samples of DNA.
Application
Analysis of RFLP variation in the genome was previously an important tool in genome mapping and genetic disease analysis. If researchers were initially trying to determine the chromosomal location of a particular disease gene, they would analyze the DNA of family members with the disease, and look for RFLP alleles that show a pattern of inheritance similar to the disease ( See genetic linkage ). Once a disease gene has been localized, RFLP analysis of other families can reveal who was at risk for the disease, or who was likely to be a carrier of the mutant gene. The RFLP test is used in the identification and differentiation of organisms by analyzing unique patterns in the genome. It is also used in the identification of recombination rates at loci between restriction sites.
RFLP analysis was also the basis for early methods of genetic fingerprinting , useful in the identification of samples obtained from crime scenes, in the determination of paternity , and in the characterization of genetic diversity or breeding patterns in animal populations .
Alternative
However, the technique of RFLP analysis is slow and cumbersome. This requires a large amount of sample DNA, and the combined process of probe labeling, DNA fragmentation, electrophoresis, blotting, hybridization, washing and autoradiography can take up to a month to complete. A limited version of the RFLP method using an oligonucleotide probe was reported in 1985. [1] The results of the Human Genome Project have largely replaced the need for RFLP mapping, and many single-nucleotide polymorphisms in that project.(SNP) has been identified. (as well as the direct identification of several disease genes and mutations) has replaced the need for RFLP disease linkage analysis (see SNP genotyping). Analysis of VNTR alleles continues, but is now more commonly performed by polymerase chain reaction (PCR) methods. For example, the standard protocol for DNA fingerprinting involves PCR analysis of panels of more than a dozen VNTRs.
RFLP is still used in marker-assisted selection. Terminal restriction fragment length polymorphism (TRFLP or sometimes T-RFLP) is a technique initially developed to characterize 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 are visualized using a DNA sequencer. The results are analyzed by either counting and comparing the bands or peaks in the TRFLP profile only, or by matching bands from one or more TRFLPs to known species databases. The technique is similar to the temperature gradient or denaturation gradient gel electrophoresis (TGGE and DGGE) in some aspects. Sequence changes directly associated with RFLP can also be analyzed more rapidly by PCR. Amplification can be directed to the altered restriction site, and the products can be digested with restriction enzymes. This method is called cleaved amplified polymorphic sequence (CAPS). Alternatively, the amplified segment can be analyzed by an allele-specific oligonucleotide (ASO) probe, a procedure that can often be performed by a simple dot blot.
