( RESTRICTION DIGESTION | INTRODUCTION Endonucleases are DNases that recognizes specific oligonucleotide sequence, make double stranded cleavages and generate unique, equal molar fragments of DNA molecules. By the nature of controllable, predictable, infrequent and site specific cleavage of DNA, restriction endonucleases provided to be extremely useful as a tool in dissecting, analyzing and reconfiguring genetic information at molecular level. Three distinct types of restriction modification system have been characterized onthe basis of subunit composition, cofactor requirements and type of DNA cleavage. Bacteria remain a source of restrictionendonuclease. More than 600 restriction enzymes with more than 100 specificity have been reported, but similar enzymes are rare in eukaryotes. PRINCIPLE Restriction enzyme hydrolyses the backbone of DNA between deoxyribose and phosphate groups. This leaves a phosphate group on the 5' ends and a hydroxyl group on the 3' ends of both strands. The restriction enzymes most used in molecular biology lab cuts within the recognition sites and generate two different types of ends. The 5' or 3' overhangs generated by enzymes that cut asymmetrically are called sticky or cohesive ends , because they will Teadily stick or anneal with their complementary sequences by base pairing E.g. Bam HI. Some enzymes cutat precisely opposite sites 4in two strands of DNA and generate blunt ends without overhangs called bluntends. E.g. Haell. Every restriction enzyme has unique target sites for digestion. Lambda DNAhas multiple restriction sites for both EcoRI and Hindlll which result into several fragments of varying sizes. 1 Enzyme | Source Recognition | Cut Fragment | sizes Sequence (bp) EcoRI | Escherichia | 5'GAATTC | 5'G AATTC3’ | 21226, 7421, coli 3'CTTAAG | 3'CTTAA GS! | 5go4' | 5643, 4878, 3530 | Hind Ill | Haemophilus | 5'AAGCTT | 5'A AGCTT-3' | 23130, 9416, influenzae 3'TTCGAA | 3'TTCGA AS’ | 6557, 4361, 2322, 2027, 564, 125 Applications: Restriction enzymes are powerful too! used to: Map DNA molecules Analyze population polymorphisms Rearrange DNA molecules Prepare molecular probes and Create mutants s of molecular geneticsMATERIALS REQUIRED BUT NOT PROVIDED 1. Micropipettes and tips 2. Distilled water 3. Incubator 4. Electrophoresis unit with a power pack 5. UVTransilluminator. PROCEDURE All the micropipettes should be well calibrated and the pipetting should be accurate to get optimum results. 1. Take the restriction digestion tool from -20°C and keep it in icebox, gently thaw to liquefy the crystallized samples. 2. Briefly spin all the tubes for few seconds to settle down all the droplets sticking at the top or sides. 3. A single or double digestion of the DNA molecule is set up depending upon the requirements or conveniences. Single digestion 1. Digestion of a DNA with a single enzyme (Eco RI or Hind Ill separately) is known as single digestion. 2. Take a new sterile 0.5 ml Microfuge tube provided.3. Add the following reagents in the order listed, to the tube1 for 20 ul reaction Distilled water - 13ul Restriction buffer 10X - 2ul DNA : 3yl Enzyme (EcoRI) - 2ul 20pl 4. Add the following reagents in the order listed, to the tube 2 for 20 ul reaction Distilled water - 13ul Restriction buffer 10X - 2ul DNA : 3ul Enzyme (Hind III) - 2ul 20ul Double Digestion 1. Digestion of a DNA with two enzymes (Eco RI and Hind Ill) is known as double digestion. 2. Take a new sterile 0.5 ml Microfuge tube provided.3. Add the following reagents in the order listed to the tube for 201 reaction Distilled water - Vl Restriction buffer 10X = - 2ul DNA - 3yl Hindll : 2ul EcoRI - 2ul 4. Gently mix well by repeat pipetting or tapping. Briefly spin for few seconds and incubate the reaction mix at 37°Cfor 1 hour 5. After 1 hour incubation, place the tubes at room temperature for 10 minutes 6. Run the samples on 1% Agarose gel electrophoresis, observe the bands under UV transilluminator. Preparation of 1% Agarose gel electrophoresis 1.Prepare 1% Agarose solution(0.5g/50ml) in the buffer provided (0.5X TEB). 2.Boil the Agarose until itis completely dissolved and make sure no obvious particle of Agarose remain in the suspension.10. 11 12. 13. 14. When the gel temperature is around 40°C, add 4 ul of ethidium bromide and mix properly. Seal the gel-casting tray on two sides; place the comb on the gel tray in appropriate place. Pour the agarose mixture in to the tray-containing comb. Allow the agarose to solidify in the tray. Then remove the seal from the two sides of the tray without disturbing the gel. Then keep the gel tray in tank containing 0.5-X TEB buffer with the wells in the cathode (Negative) side. The buffer level in the tank should be maintained above the gel tray. Gently lift the comb to avoid damage of the wells, the gel is now ready for loading. Connect the cords between the electrophoresis tank and the power pack before loading the samples. To prepare samples for electrophoresis, add 5ul of gel loading dye in to the sample and mix well by pipetting. Load 15yl of the sample in to the well. For control (undigested) aliquot 3 ul of Lambda DNA (from the vial provided) in to a microfuge tube, with that add 3! of gel loading dye, mix it and load (61) to the nearby well. After loading, switch on the power pack and adjust the voltage to 50 V or 100V. Continue the electrophoresis until the dye reaches to 1/3” of the gel or above. Observe the bands under UV transilluminator.(sBueysare ON) sBueuseaQ sebpy wioyupy Areyuswe;dwo5 ays uonjuBosay ays UoWUBOI9y SOseajInuopuy” Uoljoi4soy PUA 3UNIg, Sesvejonuopuy von sjajsey ,puy Ay213S)Meaox- 3/0 Baa Restriction Digestion Teaching kt RESULT AND INTERPRETATION The given sample DNA has 7 and 5-restriction site for Hing \ll and Eco RI respectively. When a digestion of DNA is set up with one enzyme either Hind Ill or Eco RI (i.e. single digestion) 7 or 5 bands were observed. Double digestion of DNAwith both the enzymes Hind Il and Eco RI results in 11 bands. Whereas in control a single band was observed