Restriction Fragment Length Polymorphism (RFLP)

It is a brief description of restriction fragment length polymorphism enzymes.

1. Restriction enzymes RFLP Hafiz.M.Zeeshan.Raza COMSATS University Islamabad, Sahiwal Campus , Punjab, Pakistan [email protected]

2. Restriction enzymes 1962: “molecular scissors” discovered in in bacteria These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction ; while host DNA is protected by a modification enzyme (a methylase) that modifies the prokaryotic DNA and blocks cleavage. 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially

3. Methylation

4. Restriction enzymes Restriction enzymes are endonucleases (Endo (inside), nuclease(cuts nucleic acid), which catalyze the cleavage of the phosphodiester bonds within both strands of DNA. They require Mg +2 for activity and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the point of cleavage.

5. Restriction enzymes The distinguishing feature of restriction enzymes is that they only cut at very specific sequences of bases. This specific DNA sequence is called recognition sequence.

6. Restriction enzymes A restriction enzyme requires a specific double stranded recognition sequence of nucleotides to cut DNA. Recognition sites are usually 4 to 8 base pairs in length. Cleavage occurs within or near the site.

7. Restriction enzymes The type of bacteria in which the enzyme is found The order in which the restriction enzyme was identified and isolated.

8. Many recognition sequences are palindromic. For example , 5’ GAATTC 3’ 3’ CTTAAG 5’ palindromic: read the same in the opposite direction e.g: Level leveL

9. Sticky and Blunt end cutters Not all restriction endonucleases cut symmetrically and leave blunt ends.  Many endonucleases cleave the DNA backbones in positions that are not directly opposite each other or can make staggered cuts, which produce single stranded “sticky-ends” DNA from different sources can be spliced easily because of these sticky-end overhangs.

10. Example of RE that produce sticky end cutters:

11. Some restriction enzymes cut DNA at opposite base. They leave blunt ended DNA fragments These are called blunt end cutters Hae III

12. Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence. All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5'-phosphates . Types Of Restriction Enzymes

13. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements, as summarised below: Type I enzymes cleave at sites remote from recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase activities. Type II enzymes cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction) enzymes independent of methylase. Type III enzymes cleave at sites a short distance from recognition site; require ATP (but do not hydrolyse it); S-adenosyl-L-methionine stimulates reaction but is not required; exist as part of a complex with a modification methylase. Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA.

14. Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another.

15. An  RFLP probe  is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes. Polymorphisms are inherited differences found among the individuals in more than 1% of normal population.

16. Applications of RFLP: RFLPs can be used in many different settings to accomplish different objectives. 1- RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. (i.e. it has forensic applications). 2- RFLPs can be used determine the disease status of an individual. (e.g. it can be used in the detection of mutations particularly known muations) 3- RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans.

17. Southern Blotting

18. Southern blot hybridization is one of the most commonly used molecular techniques to detect specific DNA sequences using labeled probes. Four steps: DNA extraction Electrophoresis to separate Transfer to membrane Use labeled probes, which will hybridize to specific sequence, to identify sequence of interest

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