Restriction enzyme digestion
- 2. INTRODUCTION Gene manipulation requires enzymes to cut DNA molecules. The restriction endonucleases cut at the interior part of DNA. These enzymes are found in bacteria and in vivo are involved in recognition and destruction of foreign DNA. Invading phage DNA for instance will be restricted by such enzymes. The bacteria protect their own DNA by modification process.
- 3. The essential feature of restriction endonucleases is that these enzymes recognize a particular sequence of bases. Type II restriction enzymes cut the DNA within the recognized sequences. Each enzyme has its own characteristic recognition sequence and it may be 4 to 7 bases long with dyad symmetry. With the availability of nearly 50 restriction enzymes commercially, it is now possible to construct the physical map of genes after digestion of the DNA with different restriction enzymes and subsequent of DNA fragments on agarose gel.
- 4. PRINCIPLE Restriction enzyme EcoRI(isolated from Escherichia coli) that recognized and cleaves the sequence 5”-GAATTC-3” TO generate cohesive or sticky ends similarly, HindIII isolated from Haemophilus influenza and cleave the sequence 5”- AAGCTT-3” to generate cohesive or sticky ends. EcoRI G|AATTC CTTAA|G HindIII A|AGCTT TTCGA|A *Recognition sequence and cutting sites are shown by “|”
- 5. TYPES OF RESTRICTION ENZYME Three types of restriction enzyme are there. TYPE I TYPE II TYPE III
- 6. TYPE I RE They are complex enzymes which act as endonuclease and methylase function. They require ATP,Mg+2. They are single, multi-functional enzyme which recognizes 15bp in length and cleavage site is 1000bp. They show specificity for recognition but not for cleaveage. They produce heterogeneous fragments. Not used in gene cloning techniques. Eg. EcoK,EcoB
- 7. TYPE II RE Most common RE used in gene cloning. They are simple enzyme having single polypeptide. They have separate methylase and endonuclease activity. The recognition and cutting site is the same one. They generally recognize six nucleotide.some also recognize 4,5,or 8bp too. Eg.PvuI,PvuII,EcoRI
- 8. Cutting site of RE Type II Cleave DNA to generate different “ends” Staggered cut 5’ extension 3’ extension Blunt end
- 9. TPYE III There are two subunits. One is for recognition and modification another for nuclease activity. They require ATP,Mg. The problem is that the recognition sites are assymetric non pallindromic. The single strand ends produce by TYPEIII differ from each other and cannot recombine at random. They lack ATPase activity since cleaved products are not uniformed they are not useful in gene cloning. Eg.HgaI,MboII
- 10. Restriction Enzymes Recognition sites Generally 4, 6, or 8 bp in length Most sites are palindromic OTTO / HANNAH / REGAL LAGER A MAN A PLAN A CANAL PANAMA For REases - sequence reads the same in a 5’--- >3’ direction on each strand
- 11. NOMENCLATURE OF RE Nomenclature EcoRI E = Escherichia genus name co = coli species name R = strain RY12 strain or serotype I = Roman numeral one = first enzyme HinDIII Haemophilus influenza serotype d 3rd enzyme
- 12. MATERIALS REQUIRED Plant DNA, Restriction Buffer (10x) Sodium acetate, Ethanol, Sterile double distilled water, Restriction enzymes, microfuge tubes, micropipette (0-20 μl), micro centrifuge, DNA marker, marking pen, bucket with crushed ice, pipette tips, agarose gel electrophoresis unit and its chemicals
- 13. CHEMICALS Recipes (μl) 1 2 3 4 5 Distilled Water 16 16 14 14 14 10x buffer 2 2 2 2 2 Marker DNA 2 - - - - Sample DNA - 2 2 2 2 - - 2 - - Hind III - - - 2 - Bam HI - - - - 2 Total volume (μl) 20 20 20 20 20
- 14. PROTOCOL/PROCEDURE 1. Label microfuge tubes 1 to 5 and arrange them open in a rack. 2. Bring all reactants on ice. DNA samples stored frozen are thawed quickly and brought on ice. 3. Prepare the following reaction mixes by carefully pipetting into the bottom of microfuge tubes. 4. Fasten the cap on each tube. Mix contents of tubes carefully. Centrifuge all the microfuge tubes for 2 second to settle the contents at the bottom of tubes. 5. Incubate 30OC in water bath for 60 minutes or longer if necessary.
- 15. 6. To the digested reaction mixture, add 2 μl of 3 M sodium acetate and 50 μl absolute ethanol for precipitation. Keep it at -20OC for 20 minutes. 7. Recover the DNA by centrifugation for 15 minutes. 8. Wash the pellets twice with 70 % ethanol. Remove supernatant. 9. Air dry the pellets. 10. Dissolve the pellets in 20 μl TE buffer and store at -40OC 11. The digested DNA fragments can be separated through electrophoresis.
- 16. INFORMATION REGARDING RE Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific sequences. There are hundreds of different restriction enzyme , allowing scientists to target a wide variety of recognition sequences. For a list of many commonly used restriction enzyme .
- 17. Restriction enzyme digestion is a commonly used technique for molecular cloning such as in cloning by either PCR or restriction enzyme digest. It is also used to quickly check the identity of a plasmid by diagnostic digest.
- 18. PRECAUTION Make sure that the restriction enzyme does not exceed more than 10% of the total reaction volume, otherwise the glycerol and the EDTA in the enzyme storage buffer may inhibit digestion process.
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