A Restriction Fragment Length Polymorphism (or RFLP, often pronounced as "rif-lip") is a variation in the DNA sequence of a genome which can be detected by a laboratory technique known as gel electrophoresis. 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 Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known In classical genetics the genome of a Diploid Organism including Eukarya refers to a full set of chromosomes or genes in a Gamete, thereby Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing. Genome projects are Scientific endeavours that ultimately aim to determine the complete Genome sequence of an Organism (be it an Animal, a A genetic disorder is a condition caused by abnormalities in Genes or Chromosomes While some diseases such as Cancer, are due to genetic abnormalities acquired Genetic testing allows the genetic Diagnosis of vulnerabilities to inherited Diseases, and can also be used to determine a person's Ancestry. A maternity or paternity identification test is conducted to establish whether a person is the biological Parent of another person
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Analysis technique
The basic technique for detecting RFLPs involves the fragmentation of genomic 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. A restriction enzyme (or restriction Endonuclease) is an Enzyme that cuts double-stranded DNA at specific recognition Nucleotide Endonucleases are Enzymes that cleave the Phosphodiester bond within a Polynucleotide chain in contrast to Exonucleases which cleave Phosphodiester The recognition sequence, also referred to as Recognition site, of any DNA-binding protein motif that exhibits binding specificity refers to the DNA sequence In Molecular biology, two Nucleotides on opposite complementary DNA or RNA strands that are connected via Hydrogen bonds are called A restriction digest is a procedure used in Molecular biology to prepare DNA for analysis or other processing 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. Agarose Gel electrophoresis is a method used in Biochemistry and Molecular biology to separate DNA, or RNA molecules by size A Southern blot is a method routinely used in Molecular biology to check for the presence of a DNA sequence in a DNA sample Hybridization of the membrane to a labeled DNA probe then determines the size of the fragments which are complementary to the probe. Hybridization is the process discovered by Alexander Rich, of combining complementary single-stranded Nucleic acids into a single Molecule. In Molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length (usually 100-1000 bases long which is used to detect in DNA In Molecular biology, complementarity is a property of double-stranded Nucleic acids such as DNA and RNA as well as DNARNA duplexes An RFLP occurs when the size of a detected fragment varies between individuals. Each fragment size is considered an allele, and can be used in genetic analysis. An allele (ˈæliːl (UK /əˈliːl/ (US (from the Greek αλληλος allelos, meaning each other) is one member of a pair or series of different forms Genetics (from Ancient Greek grc-Latn genetikos, “genitive” and that from grc-Latn genesis, “origin” a discipline of Biology, is
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. In biology mutations are changes to the Nucleotide sequence of the Genetic material of an organism 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). A Variable Number Tandem Repeats (or VNTR) is a location in a Genome where a short Nucleotide sequence is organized as a Tandem repeat. 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. Genetic Insertion is the addition of one or more Nucleotide Base pairs into a genetic sequence In Genetics, a deletion (also called gene deletion, deficiency, or deletion mutation) is a Mutation (a genetic aberration In Genetics, a chromosome translocation is a Chromosome abnormality caused by rearrangement of parts between nonhomologous Chromosomes. An inversion is a Chromosome rearrangement in which a segment of a chromosome is reversed end to end
Applications
Analysis of RFLP variation in genomes was 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). Genetic linkage occurs when particular genetic loci or Alleles for genes are inherited jointly 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 carriers of the mutant gene. A genetic carrier (or just carrier) is a person or other organism that has inherited a genetic trait or Mutation, but who does not display that
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. A maternity or paternity identification test is conducted to establish whether a person is the biological Parent of another person Genetic diversity is a level of Biodiversity that refers to the total number of genetic characteristics in the genetic makeup of a species
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. An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e A limited version of the RFLP method that used oligonucleotide probes was reported in 1985[1]. Oligomer Restriction (abbreviated OR) is a procedure to detect an altered DNA sequence in a Genome. 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 Human Genome Project (HGP was an international Scientific research project with a primary goal to determine the sequence of chemical base pairs which make up DNA A single nucleotide polymorphism ( SNP, pronounced snip) is a DNA sequence variation occurring when a single Nucleotide - A, T Genotyping provides a measurement of the genetic variation between members of a species 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. A national DNA database is a Government database of DNA profiles which can be used by law enforcement agencies to identify suspects of crimes The Combined DNA Index System (CODIS is a DNA database funded by the United States Federal Bureau of Investigation (FBI
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. Molecular biology is the study of Biology at a molecular level 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. Temperature Gradient Gel Electrophoresis ( TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE are forms of Electrophoresis where there is a temperature
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 by analyzed by Allele specific oligonucleotide (ASO) probes, a process that can often be done by a simple Dot blot. An Allele Specific Oligonucleotide (or ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA A Dot blot (or Slot blot) is a technique in Molecular biology used to detect Biomolecules It represents a simplification of the Northern blot
References
- ^ Saiki, RK; Scharf S, Faloona F, Mullis KB, Erlich HA, Arnheim N (Dec 20 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230 (4732): 1350-4. Science is the Academic journal of the American Association for the Advancement of Science and is considered one of the world's most prestigious Scientific
External links
- RFLP description at the National Institutes of Health. "NIH" redirects here For other meanings of NIH see NIH (disambiguation.
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