Restriction fragment length polymorphism
Encyclopedia
In molecular biology
, restriction fragment length polymorphism, or RFLP (commonly pronounced “rif-lip”), is a technique that exploits variations in homologous DNA sequences. It refers to a difference
between samples of homologous DNA
molecules that come 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 (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 inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. In addition to genetic fingerprinting
, RFLP was an important tool in genome mapping, localization of genes for genetic disorder
s, determination of risk
for disease, and paternity testing
.
, 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 analyzing mixed-DNA samples. Moreover, data can be generated even when the DNA is degraded (e.g. when it is found in bone remains.)
In the third schematic, the probe and restriction enzyme are chosen to detect a region of the genome that includes a variable VNTR segment (boxes). 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.
). 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 populations.
was reported in 1985. Fortunately, the results of the 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 protocols
for 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
.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, restriction fragment length polymorphism, or RFLP (commonly pronounced “rif-lip”), is a technique that exploits variations in homologous DNA sequences. It refers to a difference
Polymorphism (biology)
Polymorphism in biology occurs when two or more clearly different phenotypes exist in the same population of a species — in other words, the occurrence of more than one form or morph...
between samples of homologous DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
molecules that come 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 (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
. Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. In addition to genetic fingerprinting
Genetic fingerprinting
DNA profiling is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier...
, RFLP was an important tool in genome mapping, localization of genes for genetic disorder
Genetic disorder
A genetic disorder is an illness caused by abnormalities in genes or chromosomes, especially a condition that is present from before birth. Most genetic disorders are quite rare and affect one person in every several thousands or millions....
s, determination of risk
Genetic testing
Genetic testing is among the newest and most sophisticated of techniques used to test for genetic disorders which involves direct examination of the DNA molecule itself. Other genetic tests include biochemical tests for such gene products as enzymes and other proteins and for microscopic...
for disease, and paternity testing
Paternity testing
Parental testing is the use of genetic fingerprinting to determine whether two individuals have a biological parent-child relationship. A paternity test establishes genetic proof as to whether a man is the biological father of an individual, and a maternity test establishes whether a woman is the...
.
Analysis technique
The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzymeRestriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
, which can recognize and cut DNA wherever a specific
Recognition sequence
The recognition sequence, sometimes also referred to as recognition site, of any DNA-binding protein motif that exhibits binding specificity, refers to the DNA sequence , to which the domain is specific...
short sequence occurs, in a process known as a restriction digest
Restriction digest
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation...
. 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
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
procedure. Hybridization of the membrane to a labeled DNA probe
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
then determines the length of the fragments which are complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
to the probe. An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
, and can be used in genetic analysis
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
.
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 analyzing mixed-DNA samples. Moreover, data can be generated even when the DNA is degraded (e.g. when it is found in bone remains.)
Examples
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 VNTR segment (boxes). 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
Genetic deletion
In genetics, a deletion is a mutation in which a part of a chromosome or a sequence of DNA is missing. Deletion is the loss of genetic material. Any number of nucleotides can be deleted, from a single base to an entire piece of chromosome...
, translocations
Chromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise separated genes, the occurrence of which is common in cancer. It is detected on...
, and inversions
Chromosomal inversion
An inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end to end. An inversion occurs when a single chromosome undergoes breakage and rearrangement within itself. Inversions are of two types: paracentric and pericentric.Paracentric inversions do not include the...
, can also lead to RFLPs.
Applications
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 linkageGenetic linkage
Genetic linkage is the tendency of certain loci or alleles to be inherited together. Genetic loci that are physically close to one another on the same chromosome tend to stay together during meiosis, and are thus genetically linked.-Background:...
). 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
Genetic carrier
A genetic carrier , is a person or other organism that has inherited a genetic trait or mutation, but who does not display that trait or show symptoms of the disease. They are, however, able to pass the gene onto their offspring, who may then express the gene...
of the mutant genes.
RFLP analysis was also the basis for early methods of Genetic fingerprinting
Genetic fingerprinting
DNA profiling is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier...
, useful in the identification of samples retrieved from crime scenes, in the determination of paternity, and in the characterization of genetic diversity
Genetic diversity
Genetic diversity, the level of biodiversity, refers to the total number of genetic characteristics in the genetic makeup of a species. It is distinguished from genetic variability, which describes the tendency of genetic characteristics to vary....
or breeding patterns in animal populations.
Alternatives
The technique for RFLP analysis is, however, slow and cumbersome. It requires a large amount of sample DNA, and the combined process of probe labeling, 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 probesOligomer restriction
Oligomer Restriction is a procedure to detect an altered DNA sequence in a genome. A labeled oligonucleotide probe is hybridized to a target DNA, and then treated with a restriction enzyme. If the probe exactly matches the target, the restriction enzyme will cleave the probe, changing its size...
was reported in 1985. Fortunately, the results of the Human Genome Project
Human Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...
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
SNP genotyping
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation...
). The analysis of VNTR alleles continues, but is now usually performed by polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) methods. For example, the standard protocols
National DNA database
A national DNA database is a government database of DNA profiles which can be used by law enforcement agencies to identify suspects of crimes....
for 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
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
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
Cleaved Amplified Polymorphic Sequence
The cleaved amplified polymorphic sequence or CAPS method is a technique in molecular biology for the analysis of genetic markers. It is an extension to the Restriction Fragment Length Polymorphism method, using polymerase chain reaction to more quickly analyse the results.Like RFLP, CAPS works...
(CAPS). Alternatively, the amplified segment can be analyzed by Allele specific oligonucleotide
Allele specific oligonucleotide
An allele-specific oligonucleotide is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay...
(ASO) probes, a process that can often be done by a simple Dot blot
Dot blot
right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate Dot-blot filters.A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods...
.
External links
- http://www.bio.davidson.edu/courses/genomics/method/RFLP.html
- http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml