Restriction mapping

Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.

1. Restriction mapping By: Minali Singh

2. What is restriction mapping? It is a process of generating a map of a DNA molecule either linear or circular( a plasmid),indicating where the sites for certain restriction enzymes are using the data from restriction digests with those enzymes.

3. TECHNIQUES INVOLVED : USE OF RESTRICTION ENDONUCLEASES GEL ELECTROPHORESIS

4. What are restriction endonucleases (REs)? How can REs be used to identify DNA molecules?

5. Historical accounts of restriction enzymes The term restriction enzyme originated from the studies on phage lambda(bacterial virus) bacteriophage. In 1950s Salvador L uria and Giuseppe B ertani found that bacteriophage can grow well in E.coli. In 1960s Werner A rber and Matthew M esselson found that restriction is caused by enzymatic cleavage of phage DNA(type 1= EcoRl ). In 1970s Hamilton O.Smith and Thomas Kelly discovered Hind II restriction enzyme. Daniel Nathans and Kathleen showed the cleavage of DNA by restriction enzymes into fragments by using gel electrophoresis. Thus this showed that restriction enzyme can be used in mapping. In 1978 Werner A rber,Daniel N athans,Hamilton O.Smith were awarded the N obel prize for the discovery of restriction enzymes.

6. REs with 6-nucleotide recognition sites (6-cutters) are widely used in molecular biology RE Strain of origin Recognition site EcoR I E. coli (strain RY13) G A A T T C Hind III H . influenza A A G C T T Recognition sites are often palindromes

7. GAATTC CTTAAG 3’ 5’ 5 ’ 3 ’ GAATTC CTTAAG 3’ 5’ 5 ’ 3 ’ G AATTC CTTAA G 3’ 5’ 5 ’ 3 ’ EcoRI recognition site is a palindrome with an axis of symmetry EcoRI dimer binds sequence and catalyzes double-strand cleavage Products have “sticky ends”: unpaired hydrogen bonds on nitrogen bases

8. METHOD OF RESTRICTION MAPPING ISOLATION OF DNA DIGESTION WITH A SPECIFIC RESTRICTION ENZYME. EXTRACTION OF DNA AND ELECTROPHORESIS.

9. Agarose Gels To visualize the results of a restriction digest, you need to separate the different fragments of DNA, and determine their size We will do this by agarose gel electophoresis

10. Agarose Agarose is very water soluble polysaccharide Forms porous, aqueous gels after heating and cooling

11. Electrophoresis power supply - + 200 500 1000 1500 2000 3000 4000 6000

12. Gel Visualized Under UV Light

13. CONSTRUCTION OF A RESTRICTION MAP It involves successive digests with 2 individual enzymes , where we extract each fragment produced in the individual digest with either enzyme A or enzyme B and then cleave it with other enzymes. The original DNA sample is also digested by a mixture of both the enzymes to confirm the results of individual successive digests. The former and the latter are known as Reciprocal digests and Double digest respectively. Using the information we can find the over lapping regions in A and B digest and find out the sites of cleavage by A and B .This will then allow us to prepare a restriction map.

14. CLEAVAGE OF DNA BY 2 RESTRICTION ENDONUCLEASES A AND B LENGTH OF EXPERIMENTAL DNA = 5000 bp

15. TECHNIQUE OF RECIPROCAL AND DOUBLE DIGESTS

16. Restriction map in the form of linear arrangement of cleavage sites.

17. SIGNIFICANCE In molecular biology restriction maps are used as a reference to engineer plasmid or other relatively short pieces of DNA. It is used to sequence the whole molecule of DNA and run it through computer program that will find the recognition sites that are present for every restriction enzyme known.