Rapd and rflp

RAPA AND RFLP

1. RAPD AND RFLP U.DEEPALAKSHMI 18PY04 IIM.SCMICROBIOLOGY

2. Molecular markers: The variant DNA fragment that gives some information about genes of interest is called molecular marker/genetic marker .

3. Types of molecular marker: 1.PCR based marker. RAPD. 2.Hybridization based markers. RFLP. AFLP. SNP.  

4. Restriction fragment length polymorphism: The variation in the restriction DNA fragment lengths between individuals of a species is called restriction fragment length polymorphism. This is a best laboratory technique to analyze and compare DNAs of two or more individuals of a species. It is extensively used in genome analysis.

5. RFLP method involves the following steps: Sample collection. Isolation DNA. Restriction digestion. Electrophoresis. Blotting of DNA. Making genomic DNA probes. Nucleic acid hybridization. Autoradipgraphy .

6. sampe collection: Tissues or cells of individuals are collected to extract their DNA. The samples are collected separately. Isolation of DNA : The DNA was isolated from the tissues or cells. restricton digestion: Genomic DNA of each sample is cut with a restriction enzymes separately to generate variable lengths of DNA fragments.restricion enzymes such as EcoRI .Hind III, PstI are of much use for RFLP. This restriction digest is divided into two halves one half is used for DNA DETECTION and other is used for PROBE MAKING.

7. Electrophoresis: The digested genomic DNA of all the samples are loaded into separate wells in agarose or polyacrylamide gel and are subjected to electrophoresis. The DNA fragments get separated according to their size .use of molecular weight markers in one lane is likely to know the molecular weight of separated DNA fragments. Blotting of DNA : As in southern blotting the DNA is transferred to a nitrocellulose filter by using a blotting setup. The nitrocellulose filter is dried in between filter discs before performing hybridization.

8. Making genomic DNA probes : One half genomic DNA digest from each sample is electrophoresed to separate the DNA fragments. Fragments of 0.5-2.0 kb are extracted from the gel and cloned in Puc21 vector to construct rDNAs . These rDNA are amplified by introducing them into bacterial host and the amplified rDNAare isolated from the bacteria . the target DNA fragments are excised from the rDNA by using the same restriction enzyme that was Used for cutting the DNA. They are purified by electrophoresis.

9. These DNA fragments are radiolabelled with p35 isotope incorporated nucleotide by nick translation or random primer extension or end labeling. As a result, genomic probes are formed Nucleic acid hybridization: DNA blotted nitrocellulose membrane is kept immersed in a hybridization solution containing genomic DNA probes to bring out hybridization. After hybridization unbound probes are washed out of the membrane.

10. Autoradiography: Images of radioactive probes are captured on an X-ray film using autoradiography. The autoradiogram shows DNA bands in distinct lanes each of which is characteristics of an individual. Variation in the lengths of DNA fragments between individuals as shown by the lanes in the autoradiogram is called RFLP.

11. Application: preparation of genetic map. Mapping of trait. Phylogenetic analysis. DNA fingerprinting.

12. Random amplified polymorphic DNA : The RAPD is a PCR based method to detect variations between individuals of a species by selective amplification of some polymorphic sequences in their genome. this method was developed by J.G.K.WILLIAMS in 1991. only least number of DNA fragments are considered for RAPD analysis. RAPD are of much use to construct genetic maps.

13. Steps involved in RAPD analysis: 1.Sample cells or tissues are collected from individuals to be distinguished from one another. 2.Genomic DNA of each and every sample is isolated by using a standard procedure. 3.each genomic DNA is separately treated with Taq polymerase, a primer , Datp,dttp,dctp,dgtp.the primer is 9-10 bp 4. all these reaction mixture are subjected to repeated cycles.

14. denaturation,primer annealing, polymerization upto 35-40 cycles. Denaturation : temperature is maintained at 940 c for 1 mins Annealing :360c for 2 mins Polymerization : 720c for 1.5 mins Repeated cycle of denaturation, annealing, polymerization leads to amplification of polymorphic sequences in the genome. 5.As a result of PCR amplification each reaction tube contains RAPD fragments. Ethidium bromide is added to it. 6.The RAPDs thus obtained from the samples are separately loaded into wells of an AGE or PAGE and molecular weight markers are added on the well.

15. 7.The loaded DNAs are electrophoresed to separate the DNA fragments based on their size. 8.The electrophoresed gel is examined under a UV light illuminator to detect light bands and photographed. If a DNA band of molecular weight 1Kb distinguish an individual from others it is considered as RAPD marker of that individual. An individual may differ from others in one or more RAPD markers.

16. Advantages: There is no need for species specific probes in RAPD analysis. RAPD is quick method. RAPD analysis can be performed in crude DNA samples also. RAPD requires only small amount of DNA samples. It does not require radioactive probes and hybridization.

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