SIEW KONG WENG

INTRODUCTION

1091102863

Practical 6 & 7

Restriction enzymes are DNA-cutting enzymes found in bacteria. It binds specifically to and cleaves double-stranded DNA at specific sites within or adjacent to a particular sequence known as the recognition sequence. They have the ability to cut within the molecule, thus they are also called restriction endonucleases. Restriction Fragment Length Polymorphism (RFLP) is a technique use to difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination. It uses restriction enzymes (RE) to cut DNA at specific 4-6 bp recognition sites. Sample DNA is digested with one or more REs and resulting fragments are separated according to molecular size using gel electrophoresis. The RFLP analyses mostly are used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.). Polyacrylamide gel electrophoresis is a powerful electrophoretic technique to separate macromolecules according to size and charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. Polymerisation of acrylamide monomers into long chains by cross-linking with bis-acrylamide and initiated by free radicals in ammonium persulfate and stabilized by TEMED. The pore size is determined by %acrylamide concentrations and bis-acrylamide powder used in creating a gel will affect the pore size. Precaution steps must take when creating this type of gel because acrylamide is a potent neurotoxin in its liquid and powdered form.

METHODOLOGY Lab 6 Restriction Enzyme Reagents and Equipment: Restriction enzymes: DpnII, AvaII, 10X buffer pUC18 DNA digested by MspI, Sterile deionised distilled water, 1.5ml microcentrifuge tubes, Micropippettes and pipette tips, Microcentrifuge, Waterbath (37C), Floater PCR product, 1. The 1.5ml microcentrifuge tube was labeled. 2. The 10x buffer of RE was thawed. 3. PCR product was centrifuged at 10,000 rpm for 1 minute. 4. One tube for BglII were set up as shown in the table below. Reagents BglII Digestion 10X buffer 2.0 l PCR Product Sample 5.0 l Restriction Enzyme I 1.0 l Restriction Enzyme II 10X BSA Deionised distilled water 12.0 l Total 20.0 l Note: Keep enzymes cold. Always keep restriction enzymes on ice! Minimize the time you have restriction enzymes on ice. Use the micropipette designated for restriction enzymes only and always use a fresh micropipette tip when removing enzyme from the stock tube.
1

SIEW KONG WENG

5. 6.

1091102863

Practical 6 & 7

The master mix was prepared according to number of samples. The master mix was aliquot into 1.5ml microcentrifuge tube and 10l of PCR product was added into each tube respectively. 7. The master mix was mixed thoroughly on ice and was spin briefly. 8. The reaction mixture was incubated at 37C for 3 hours. 9. The samples were run on a 3% Nusieve agarose gel at 100 V for one hour after incubation. Lab 7 PAGE Reagents and Equipment: 40% Acrylamide/Bis, 37.5:1 (2.6%C), 10% ammonium persulfate (APS), freshly prepared, TEMED, 5 X and 1 X TBE buffer, 6 X gel loading dye, DNA marker, Ethidium bromide solution, 0.5g/ml, BIO-RAD Power PAC 300, BIO-RAD Mini-protean 3 cell [spacer plate, short plate, casting frame, casting stand, combs, buffer dam, electrode assembly, clamping frame, mini tank and lid], Micropipettes and tips, Beakers, Syringe, UV transilluminator, Digital camera, Samples Gel cassette sandwich preparation: Note: Ensure the casting stand, casting frames and glass plates are clean and dry before setting up the casting stand assembly. 1. The Casting Frame was placed upright with the pressure cams in the open position and facing on a flat surface. 2. A Spacer plate of the desired gel thickness and a Short Plate were placed on top of it (see Figure 4a) 3. The Spacer Plate was oriented so that the labeling is up. Two glass plates were slided into the Casting Frame, keeping the Short Plate facing the front of the frame (side with pressure cams) (see Figure 4b). Note: Ensure both plates are flush on a level surface and labeling on the Spacer Plate is oriented correctly. Leaking may occur if the plates are misaligned or oriented incorrectly. 4. When the glass was up in place, the pressure cams were engaged to secure the glass cassette sandwich in the Casting Frame (see Figure 4c). Both plates were checked to ensure that they were flushed at the bottom. 5. The spring loaded lever was engaged and the gel cassette assembly was placed on the gray casting stand gasket. The horizontal ribs of the back of the Casting Frame were flushed against the face of the Casting Stand and the glass plates were perpendicular to the level surface. The Spacer Place was pushed down against the gray rubber gasket by the lever (see Figure 4d).

SIEW KONG WENG

1091102863

Practical 6 & 7

Casting the 10% Polyacrylamide Gel: Caution: Acrylamide can cause cancer and heritable genetic damage if through contact with skin or inhalation. Avoid direct contact with acrylamide and wear protective clothing when handling. BIS is also harmful if swallowed and direct contact with skin. Be careful when handling the chemical. TEMED is neurotoxic and extremely flammable. 6. The casting solution was prepared by combining all reagents as follow: Reagents Volume (ml) Distilled water 2.75 5 X TBE buffer 1 40% Acrylamide/Bis 1.25 TOTAL 5 50 l of fresh 10 % ammonium persulfate and 2.5 l of TEMED were added to the mixture just before casting the gel 8. The casting solution was poured between the glass plates using pipette. The solution was poured slowly and gently to prevent bubble formation. Continued to pour until the top of the short plate was reached. 9. The comb was inserted into the assembled gel sandwich. 10. The gel was allowed to polymerize for 45 minutes to 1 hour. 11. The comb was removed gently and rinsed the wells thoroughly with distilled water or running buffer. Loading the Samples: 12. The Gel Cassette Assemblies was removed from the Casting Stand. The cams of the Casting Frames was rotated inward to release the Gel Cassette Sandwich (see Figure 5a). 13. A Gel Cassette Sandwich was placed into the slots at the bottom of each side of the Electrode Assembly. Be sure the Short Plate of the Gel Cassette Sandwich was faced inward toward the notches of the U-shaped gaskets (see Figure 5b). 14. The Gel Cassette Sandwich was lifted into place against the green gaskets and slided into the Clamping Frame (see Figure 5c).

7.

SIEW KONG WENG

1091102863

Practical 6 & 7

15. Inner Chamber was pressed down to insure a proper seal of the short plate against the notch on the U-shaped gasket. (see Figure 5d). Short plate was aligned with notch n gasket. Note: Gently pressing the top of the Electrode Assembly while closing the Clamping Frame cams forces the top of the Short Plate on each Gel Cassette Sandwich to seat against the rubber gasket properly and prevents leaking. 16. Inner Chamber Assembly was loaded into the Mini Tank. The inner chamber was filled with ~125 ml of 1 X TBE buffer until the level reached the halfway between the tops of the taller and shorter glass plates of the gel Cassettes. Note: Do not overfill the Inner Chamber Assembly. Excess buffer will cause the siphoning of buffer into the lower chamber which can result in buffer loss and interruption of electrophoresis. 17. ~200 ml of 1X TBE buffer was added to the Mini Tank (lower buffer chamber). 18. The samples were loaded into the well using pipette and tips. 19. The gel was now ready to run.

Running the Gel: 20. The lid was placed on top of the chamber to enclose the whole Mini-PROTEAN 3 cell, with the anode and cathode properly matching the central cooling core. 21. The electrical was attached leads to the BIO-RAD Power PAC 300, with proper polarity. 22. Power was applied and begun electrophoresis at 100 V for 1 hour. Visualization of DNA separation: 23.After electrophoresis, the gel was transferred into a container with ethidium bromide solution for 30 minutes. Then, the gel was transferred into another container with 1 X TBE buffer for 5 minutes. The gel was viewed over the UV transilluminator and the gel was photographed.
4

SIEW KONG WENG

RESULT AND DISCUSSION

1091102863

Practical 6 & 7

Lane 1: PCR product digested with HindIII Lane 2: PCR product digested with BamHI Lane 3: PCR product digested with MspI Lane 4: PCR product digested with BglII Lane 5: PCR product digested with EcoRI Lane 6: PCR product digested with EcorI and BamHI Lane 7: Marker 100bp (NEB Brand)

The picture above is the result of gel electrophoresis experiment. The sample label number 4 is the band produce from Bgl II restriction enzyme. It shows that it have about 1517 base pair by comparing with the maker 100bp (NBE Brand). This gave Bgl II special specificity properties that can recognize and cleave at distinct site.

QUESTIONS AND ANSWER Lab 6 Restriction Enzyme 1. Define 1 unit of restriction enzyme activiy. 1 unit of restriction enzyme activity is the amount needed to completely digest 1g of a linear lambda phage double stranded DNA in a 50 l reaction in 60 minutes. 2. Why should we store the restriction enzyme at -20C? To prevent degradation and denaturation of the enzyme under high temperature and keep restriction enzyme in active state. 3. Why must we not to make up restriction digests with the restriction enzyme composing more than 1/10 of the final volume? The enzyme activity might be inhibited by the high levels of glycerols in more than 1/10 of final volume of restriction enzyme. 4. What do AvaII and DpnII stand for? What are their recognition sites? AvaII stands for Anabaena variabilis Recognition site: 5'-G / G A C C-3' 3'-C C T G / G-5' DpnII stands for Streptococcus pneumoniae Recognition site:
5

SIEW KONG WENG

5'-N / GATCN-3' 3'-NCTAG / N-5'

1091102863

Practical 6 & 7

Name two other isoschizomers of DpnII. BfuCI and Sau3AI. Recognize site /GATC. 6. List out the features of RE. RE is an enzyme that recognizes and cut double stranded DNA only at specific nucleotide sequences. 7. How do organisms protect its DNA from cleavage of its own restriction endonucleases? Organisms DNA are protected from cleavage of its own restriction endonucleases by pairing it to methylate and hemi methylated the DNA. After methylation, DNA sites are protected from most RE. 8. What are the applications of RE digestion? Recombinant technology, gene cloning, studies of genetic polymorphisms and mutation.
5.

Lab 7 PAGE 1. Compare between agarose gel and polyacylamide gel (what are the advantage and disadvantage over one another) Agarose Horizontal Separate both large and small molecule, but better for large molecules No Can be reheated and poured. Nontoxic in powder form Nontoxic in solid form Mostly for running DNA molecule Polyacrylamide Vertical Separate both large and small molecule, but better for small molecules Yes Cannot be reheated and poured. Toxic in powder form Nontoxic in solid form Mostly for running DNA or proteins molecule Bigger gaps than polyacrylamide gel Smaller gaps than agarose gel Gel easily breaks where wells are. Gel easily rips. Advantages: Advantages: Agarose gel is not neurotoxin High degree of resolving power Can re-pour the gel Can separate molecules displaying 1 to 6 base pairs Only fewer ingredients needed to mix. effectively Disadvantages : Disadvantages : Low resolution power, small Acrylamide monomer is differences between band sizes cannot see neurotoxin so it is difficult to handle clearly More expensive than agarose gel 2. DNA molecules above a certain size (30-50 kb) migrate to a similar extent and so cannot readily be resolved by either agarose or polyacrylamide gel electrophoresis. How to resolve the above mentioned very long DNA? Use pulse field gel electrophoresis (PFGE) because it can resolve megabasesized of DNA fragments. 3. What are the APS and TEMED for? Ammonium Persulfate (APS) and TEMED will accelerate the polymerization process of gel by catalyze the polymerization of acrylamide solutions into gel matrices.
6

SIEW KONG WENG

1091102863

Practical 6 & 7

4. Why must we prevent the gel from exposure to air during polymerization? The presence of excess oxygen in the air will inhibit the polymerization elongation process and prevent acrylamide polymerization. Thus, it can lead to shorter average chain length. 5. Why is it important to use the same batch of electrophoresis buffer in both of the reservoirs and in the gel? Difference batch of buffer might have difference ionic strength or pH that will influences the production of the buffer front and will distort migration of DNA. 6. Describe non-denaturing (native) PAGE and denaturing PAGE. Non-denaturing (native) PAGE is an electrophoresis process which runs without SDS. The electrophoretic mobility of the protein is depends on the proteins charge and hydrodynamic size. For denaturing PAGE, denaturing agents - SDS is needed to unwind the DNA or RNA strands. Mobility is based on the molecular mass of the molecules and not depends on charges.

CONCLUSION Restriction enzymes (RE) recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. Application of RE are widely used in gene cloning and protein expression experiments. It also used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs). Polyacrylamide gel electrophoresis is powerful electrophoretic techniques use to separate macromolecules on the basis of molecular weight and charge. It is mostly use in Sequencing application, detection of mutation/variation for example Single Stranded Conformational Polymorphism (SSCP) which can be used to study Single Nucleotide Polymorphism (SNP)