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RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
RFLP (pronounced either as “riflip”) is a technique that is used in field of molecular biology to determine the variation in sequence of DNA of different biological sample. The restriction fragment (RF) refers to the fragments produced by reaction of restriction enzyme, length means length of nucleotide and polymorphism refers to different morphologies of DNA from different individuals. It was first inexpensive technique that was used for DNA profiling. DNAs from different individuals rarely have exactly the same array of restriction site and distance between these sites. Therefor the population is said to be polymorphic having many forms for these restriction fragments patterns. The comparison is particularly of restriction enzyme cleavage profiles based on presence of polymorphism in sequence of DNA in relation to other sequences. It involves detection of fragments produced by digestion process using a gel electrophoresis southern blot analysis technique which separate fragments based on their molecular size along with a marker.
Restriction fragment length polymorphism (RFLP) in molecular biology is a technique which display the variation that are present in a DNA sequence which are homologous in nature. The principle of RFLP technique basically involves comparing the restriction digestion profiles of different samples of DNA that are isolated from different individuals. The variation in homologous DNA is detected by presence of different lengths of DNA sequences after its digestion with restriction endonuclease. The RFLP makes use of DNA probe for their detection. The fragments generated are visualized using molecular probe for hybridisation. The principal basis of RFLP approach is to detect few polymorphism that are present in the sequence related to probe and a probe allows selection of small fractions of DNA sequence used to study polymorphism. The DNA sequence to be used as a probe is either obtained as cDNA (complementary DNA) which is derived from mRNA from metabolically active tissue or may be as genomic DNA. After obtaining fragments agarose gel electrophoresis is carried out. RFLP molecular markers are used for estimating the size of fragments. The length of the fragments varies between different samples or individuals.
For RFLP analysis
- The initial step involves extraction and purification of DNA sample from different individuals, who all are subjects of study.
- Then with help of restriction enzymes the samples of DNA are digested, each sample separately. (Same set of restriction enzymes are used for all samples).
- Each DNA sample that is digested is then subjected to gel electrophoresis in different lane on same gel slab due to which the fragments of DNA separate out according to their size on gel slab.
- These separated fragments of DNA are then transferred to a nitrocellulose paper (or membrane) from the gel by using the technique known as southern blotting.
- Then these transferred southern blots on nitrocellulose membrane are hybridized with molecular probe that are radioactively labelled.
- After this the hybridized blots are exposed to X-ray film due to which an autoradiogram is developed and the fragments that are fully or partially homologous to probe are visualized on the autoradiogram obtained.
A diagram showing all the steps involved in RFLP analysis:
During electrophoresis for RFLP analysis the digested fragments of DNA are separated on agarose gel. These fragments under a high volt electric current tend to move on gel and the rate of movement is directly related to size. Heavier the fragment, closer it is to the site of loading and the lighter the fragment run further on the gel and in this way the fragments separation of DNA samples takes place.
After electrophoresis, southern blot analysis is done under which the separated fragments are transferred onto nitrocellulose paper and are hybridised with probe. These transferred bands of DNA are referred as southern blot. For this the fragments separated on gel are further denatured with help of alkali solution to form single strand of separated DNA fragment. Then the nitrocellulose paper is placed on the gel after which a stack of paper is placed on the top of nitrocellulose paper onto which a weight of approximately 500g is placed and this assembly is left undisturbed for few hours to allow the buffer to be drawn up by filter paper wick and passes to nitrocellulose paper from the gel due to this all single stranded (ss) DNA carried with the buffer tends to bind with nitrocellulose sheet. The transferred ssDNA is fixed onto nitrocellulose paper by baking it at 80 ÌŠC for few hours and then hybridization of probe to the fixed ssDNA is done after which the X-ray film is exposed to hybridized membrane which leads to generation of autoradiogram for analysis of fragments.
- In case of genetic counselling. For example: if a couple is expecting a child and just to know whether their child or children which they are going to have will be free of any kind of genetic disease or not, RFLP analysis can be done. for this the pedigree analysis is done and if the couple is homozygous for allele responsible for genetic disorder then it is expected that all their children will carry the disorder and if they have heterozygous allele then their children can be either heterozygous or homozygous (in 2:1 ratio).
When couple is homozygous when couple is heterozygous
- For genetic mapping:
- In case of cystic fibrosis to know whether the person is carrying any cystic fibrosis(CF) allele and if yes then how many, to check this RFLP analysis is carried out and since to have CF the alleles should be homozygous because cystic fibrosis is a recessive disease.
- To determine disease status of an individual, like in case of Huntington’s chorea to locate the RFLP marker linked to the disease which is located on the chromosome 4.
- To check presence of sickle cell anaemia. For this restriction enzyme is used to cut extracted DNA (eg. DdeI) and a probe used is fragment of ß-globin coding sequence after which gel analysis followed by hybridisation is done.
- Used for paternity case analysis. For example to check whether the suspected person is father of the child or not. In this case the DNA sample is extracted from WBC of suspected father and also from mother and child and then is subjected to RFLP analysis and then if the bands obtained on the gel of both suspected father and the child is at same position then he will be confirmed to be father and if both the bands obtained are at different positions then the suspected scenario will be proved wrong.
- Used in field of agriculture as RFLP is a direct method for selecting desirable genes such as disease resistance.
- Also used in forensics science to determine the source of the sample collected from the crime scene. Also genetic fingerprinting with help of RFLP technique helps to find out the criminal and is used in forensics.
- From books:
- Genetic analysis of genes and genomes by Daniel L. Hartl and Elizabeth W. Jones, 7th edition.
- Principles of gene manipulation and genomics by S.B. Primrose and R.M. Twyman, 7th edition.
- Form internet: