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Encyclopedia > Restriction fragment length polymorphism

In molecular biology, the term restriction fragment length polymorphism (or RFLP, often pronounced "rif-lip") is used in two related contexts: as a characteristic of DNA molecules (arising from their differing nucleotide sequences) by which they may be distinguished and as the laboratory technique which uses this characteristic to compare DNA molecules. The technique is utilized in genetic fingerprinting and paternity testing. Molecular biology is the study of biology at a molecular level. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... Genetic fingerprinting or DNA testing is a technique to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. ... A paternity test is conducted to prove paternity, that is, whether a man is the biological father of another individual. ...


Method

Usually, DNA from an individual specimen is first extracted and purified. Purified DNA may be amplified by polymerase chain reaction (PCR). The DNA is then cut into restriction fragments using suitable endonucleases, which only cut the DNA molecule where there are specific DNA sequences, termed recognition sequence or restriction sites, that are recognized by the enzymes. These sequences are specific to each enzyme, and may be either four, six, eight, ten or twelve base pairs in length. The more base pairs there are in the restriction site, the more specific it is and the lower the probability that it will find a place to be cut. It could be a sticky end cut or a blunt end cut. The restriction fragments are then separated according to length by agarose gel electrophoresis. Electrophoresis separates the DNA molecules base on their molecular weight. The resulting gel may be enhanced by Southern blotting. Alternatively, fragments may be visualised by pre-treatment or post-treatment of the agarose gel, using methods such as ethidium bromide staining or silver staining respectively. The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... “PCR” redirects here. ... A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i. ... part of a DNA sequence A DNA sequence (sometimes genetic sequence) is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, The possible letters are A, C, G, and T, representing the four nucleotide subunits of a DNA strand (adenine, cytosine, guanine... The recognition sequence of any DNA-binding protein motif that exhibits binding specificity, refers to the DNA sequence (or subset thereof), to which the domain is specific. ... Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, 3 Volts/cm, stained with ethidium bromide. ... A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... Staining is a biochemical technique of adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. ...


Result

The distance between the locations cut by restriction enzymes (the restriction sites) varies between individuals, due to insertions, deletions or transversions. This causes the length of the fragments to vary, and the position of certain amplicons differs between individuals (thus polymorphism). This can be used to genetically tell individuals apart. It can also show the genetic relationship between individuals, because children inherit genetic elements from their parents. Mitochondrial DNA RFLP analyses can lead to the determination of maternal relationships. Fragments may also be used to determine relationships among and between species by comparison of the resulting haplotypes. RFLP is a technique used in marker assisted selection . Terminal Restriction Fragment Length Polymorphism (TRFLP o 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. The product of PCR or LCR; a piece of DNA that has been synthesized using amplification techniques. ... In biology, polymorphism can be defined as the occurrence in the same habitat of two or more forms of a trait in such frequencies that the rarer cannot be maintained by recurrent mutation alone. ... Mitochondrial DNA (some captions in German) Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria. ... For other uses, see Species (disambiguation). ... A haplotype, a contraction of the phrase haploid genotype, is the genetic constitution of an individual chromosome. ...


The technique 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.


  Results from FactBites:
 
Restriction fragment length polymorphism (147 words)
Restriction fragment length polymorphism (or RFLP) is a property of DNA used in molecular biology methods, for example, genetic fingerprinting or paternity testing.
The restriction fragments are then separated according to length by agarose gel electrophoresis.
The distance between the locations cut by restriction enzymes (the restriction sites) varies between individuals: so the length of the fragments varies, and the position of certain gel bands differs between individuals (thus polymorphism).
  More results at FactBites »

 
 

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