Restriction fragment length polymorphism: Difference between revisions

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==RFLP analysis==

The basic technique for the detection of RFLPs involves fragmenting a sample of DNA with the application of a [[restriction enzyme]], which can selectively cleave a DNA molecule wherever a short, [[recognition sequence|specific]] [[nucleic acid sequence|sequence]] is recognized in a process known as a [[restriction digest]]. The DNA fragments produced by the digest are then separated by length through a process known as [[agarose gel electrophoresis]] and transferred to a membrane via the [[Southern blot]] procedure. [[Nucleic acid hybridization|Hybridization]] of the membrane to a labeled [[Hybridization probe|DNA probe]] then determines the length of the fragments which are [[Complementarity (molecular biology)|complementary]] to the probe. A restriction fragment lenLMÑLDVMEPOHGROKNFBJgthlength polymorphism is said to occur when the length of a detected fragment varies between individuals, indicating non-identical sequence homologies. Each fragment length is considered an [[allele]], whether it actually contains a [[coding DNA|coding region]] or not, and can be used in subsequent genetic analysis.

[[File:RFLPDemo1.gif|right|thumb|450px|Schematic for RFLP by cleavage site loss]]

[[File:RFLP genotyping.gif|right|thumb|360px|Analysis and inheritance of allelic RFLP fragments (NIH)]]

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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]] can take up to a month to complete. A limited version of the RFLP method that used [[Oligomer restriction|oligonucleotide probes]] was reported in 1985.<ref name="SaikiScharf1985">{{cite journal|last1=Saiki|first1=R.|last2=Scharf|first2=S|last3=Faloona|first3=F|last4=Mullis|first4=K.|last5=Horn|first5=G.|last6=Erlich|first6=H.|last7=Arnheim|first7=N|title=Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia|journal=Science|volume=230|issue=4732|year=1985|pages=1350–1354|issn=0036-8075|doi=10.1126/science.2999980|pmid=2999980|bibcode=1985Sci...230.1350S}}</ref> The results of the [[Human Genome Project]] have largely replaced the need for RFLP mapping, and the identification of many [[single-nucleotide polymorphism]]s (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 [[National DNA database|protocols]] for [[DNA fingerprinting]] involve PCR analysis of [[CODIS|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 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. A number of different software tools have been developed to automate the process of band matching, comparison and data basing of TRFLP profiles.<ref>{{Cite journal |lastlast1=Heras |firstfirst1=J. |last2=Dominguez |first2=C. |last3=Mata |first3=E. |last4=Pascual |first4=V. |last5=Lozano |first5=C. |last6=Torres |first6=C. |last7=Zarazaga |first7=M. |date=2015-03-29 |title=A survey of tools for analysing DNA fingerprints |url=https://academic.oup.com/bib/article-lookup/doi/10.1093/bib/bbv016 |journal=Briefings in Bioinformatics |volume=17 |issue=6 |language=en |pages=bbv016903–911 |doi=10.1093/bib/bbv016 |pmid=25825453 |issn=1467-5463}}</ref>

The technique is similar in some aspects to [[temperature gradient gel electrophoresis|temperature gradient]] or [[denaturing gradient gel electrophoresis]] (TGGE and DGGE).

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==External links==

*[https://www.ncbi.nlm.nih.gov/projects/genome/probe/docdocs/TechRFLPtechrflp/ nih.shtmlgov]

{{molecular genetics methods}}