GeNei TM Restriction Digestion Teaching Kit Manual

Transcription

1 Teaching Kit Manual Cat No. New Cat No. KT KT01A KT01B Revision No.:

2 CONTENTS Page No. Objective 3 Principle 3 Kit Description 7 Materials Provided 9 Procedure 10 Observation & Interpretation 12 AGAROSE GEL ELECTROPHORESIS Introduction 15 Principle 15 Procedure 18 ORDERING INFORMATION 20 1

3 Objectives: To perform restriction digestion of λ DNA with EcoR I and Hind III enzymes. To analyze the restriction pattern by agarose gel electrophoresis. Principle: Restriction Enzyme Activity: Bacteria are under constant attack by bacteriophages. To protect themselves, many types of bacteria have developed defence mechanism in the form of enzymes called endonucleases that chop up any foreign DNA. Since these enzymes restrict the infection of bacteriophages they are termed "restriction endonucleases". These molecular scissors found in the bacterial cytoplasm can prove dangerous to the cell, so bacteria protect their own DNA by methylating the adenine or cytosine bases. The methyl groups block the binding of restriction enzymes, but not the normal reading and replication of genetic information. DNA from an attacking bacteriophage will not have these protective methyl groups and will be destroyed. Together restriction enzyme and its modification methyltransferase form a restriction modification (RM) system. Four kinds of RM systems are known. These are distinguished based on subunit composition, kinds of sequences recognised and cofactors needed for activity. Most characterised enzymes (about 93%) belong to Type II class. They comprise the commercially available restriction enzymes used for DNA analysis and other manipulations. Note: All discussions in the manual pertain to Type II Restriction enzymes 2 3

4 Each restriction enzyme has a unique name, derived from genus, species and strains of bacteria that produce them, followed by a number that refers to discovery order. e.g. EcoR I and EcoR V are both from Escherichia coli, strain R; I and V are the order in which they were discovered. Restriction enzymes are powerful tools of molecular genetics used to: Map DNA molecules Analyze population polymorphisms Rearrange DNA molecules Prepare molecular probes Create mutants The Type II restriction enzymes recognize specific DNA sequences and cleave the DNA at fixed locations at or near the recognition sites. They act as dimers, each subunit recognizing the same 5' 3' nucleotide sequence in complementary DNA strands and hence are said to recognize palindromic sequences. Before the restriction enzyme cuts at its specific recognition site on a long DNA, it binds to a site amidst a very large number of non-cognate sites. This protein is then translocated from the initial site to specific site, which occurs by "jumping or sliding". At this recognition site in presence of Mg 2+, enzyme undergoes a conformational change, which kinks the helix and cleaves the DNA producing 'blunt' or 'sticky' ends. For e.g. Hae III produced by Haemophilus aegypticus cleaves straight across the double helix when it encounters the following sequence to produce blunt ends. 5'-GGCC-3' 3'-CCGG-5' 5'-GG CC-3' 3'-CC GG-5' However, many restriction enzymes cut in an offset fashion to give sticky ends, which have protruding 5' or 3' ends with unpaired bases depending upon the restriction enzyme, for e.g. EcoR I recognizes the following sequence and cleaves each backbone between G and A base residue giving 5' protruding ends. 5'-GAATTC-3' 5'-G AATTC-3' 3'-CTTAAG-5' 3'-CTTAA G-5' Pst I recognizes the following sequence, cleaves between A and G residue to give 3' protruding ends. 5'-CTGCAG-3' 5'-CTGCA G-3' 3'-GACGTC-5' 3'-G ACGTC-5' The resulting sticky ends can base pair with any DNA molecule that has the complementary sticky ends to give a recombinant DNA molecule. 4 5

5 Any restriction endonuclease will cut only a specific base sequence, no matter what DNA molecule it is acting on. However, a given recognition sequence can be recognised by multiple enzymes called isochizomers. e.g., Sma I and Xma I. These can cleave the DNA at same or different position within the recognition sequence. Frequency of cleavage depends on the probability of occurrence of recognition sequences. Thus enzyme with longer recognition sequences cut less frequently and consequently produces large fragments than do enzymes with shorter recognition sequences. Factors affecting Restriction Enzyme Activity: Temperature: Most digestions are carried out at 37 C. However, there are a few exceptions e.g., digestion with Sma I is carried out at lower temperatures (~25 C), while with Taq I at higher temperature i.e., 65 C. Buffer Systems: Tris-HCl is the most commonly used buffering agent in incubation mixtures, which is temperature dependent. Most restriction enzymes are active in the ph range Ionic Conditions: Mg 2+ is an absolute requirement for all restriction endonucleases, but the requirement of other ions (Na + /K + ) varies with different enzymes. Methylation of DNA: Methylation of specific adenine or cytidine residues within the recognition sequence of the restriction enzyme affects the digestion of DNA. Kit description: Using this kit, students will perform restriction enzyme digestion of λ DNA with two different enzymes, EcoR I and Hind III. Lambda DNA is a linear double stranded DNA, base pairs, isolated from lambda bacteriophage. It has recognition sites for many restriction enzymes and is commonly used for molecular weight size markers in gel analysis of DNA and as a substrate in restriction enzyme activity assays. Enzymes supplied in this kit, EcoR I and Hind III have 5 and 7 recognition sequences on λ DNA. On digestion with EcoR I and Hind III, six and eight fragments of different sizes are obtained respectively. These bands are then resolved by agarose gel electrophoresis to observe the different restriction enzyme patterns. Simultaneously, a molecular weight marker λ/mlu I will also be electrophoresed to assess the various fragment sizes (Refer fig. 1). Enzyme Recognition Site on λ Fragment Size (in bp) EcoR I 21226, 26104, 31749, 39168, , 7421, 5804, 5643, 4878, 3530 Hind III 23130, 25157, 27479, 36895, 37459, 37584, , 9416, 6557, 4361, 2322, 2027, 564,

6 KT01 : Kit is designed to carry out 5 experiments. Each experiment involves restriction enzyme digestion of λ DNA with EcoR I and Hind III, followed by band pattern analysis by agarose gel electrophoresis. The kit also includes electrophoresis equipment (ETS-1) required for agarose gel electrophoresis. KT01A : KT01B : Kit is designed to carry out 5 experiments. Each experiment involves restriction enzyme digestion of λ DNA with EcoR I and Hind III and band pattern analysis by agarose gel electrophoresis. Kit is designed to carry out 20 experiments. Each experiment involves restriction enzyme digestion of λ DNA with EcoR I and Hind III and band pattern analysis by agarose gel electrophoresis. Note : Electrophoresis equipment is required for KT01A/KT01B Duration of experiment: Approximately 4 hours. 8 Materials Provided: The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival. Note: EcoR I & Hind III are supplied as single aliquot Materials Required: Equipment : Dry bath, Gel rocker (optional). Glassware : Beakers, Conical flask, Measuring cylinder, Staining tray. Reagent : Distilled water. Other Requirements: Crushed ice/genei Cooler, Tips, Micropipette. 9 Quantity Materials KT01/01A KT01B Store (5 expts.) (20 expts.) 2X Assay buffer 0.25 ml 1.0 ml -20 C λ/mlu I digest 50 µl 0.2 ml -20 C 2.5X Gel loading buffer 0.25 ml 0.25 ml -20 C Lambda DNA (substrate) 0.2 ml 0.8 ml -20 C Restriction Enzymes: EcoR I, Hind III 15 µl 60 µl -20 C Control λ DNA 0.05 ml 0.2 ml -20 C Agarose 2.5 g 10 g RT 6X Staining dye 40 ml 160 ml RT 50X TAE 20 ml 80 ml RT 1.5 ml vials 10 Nos. 50 Nos. RT

7 Note: Read the entire procedure before starting the experiment. Enzymes are temperature sensitive; hence place the vials containing enzyme on ice. Ensure thorough mixing by gently tapping the vial, after addition of buffer and substrate to the enzyme vial. Set the dry bath at 37 C prior to starting the experiment. For preparation of gel, staining and destaining, refer agarose gel electrophoresis. Add 5 µl of GLB to 50 µl of λ/mlu I and 20 µl of GLB to 200 µl of λ/mlu I digest 4. Incubate the vial at 37 C for 1 hour. 5. Meanwhile, prepare a 1% agarose gel for electrophoresis. 6. After an hour add 5 µl of gel loading buffer to vials. 7. Load the digested samples, 10 µl of Control DNA and 10 µl of marker, note down the order of loading. 8. Electrophorese the samples at V for 1-2 hours. 9. Stain the agarose gel with 1X staining dye. 10. Destain to visualize the DNA bands. Procedure: Setting up the Reaction 1. Place the vials containing restriction enzyme (EcoR I and Hind III) on ice. 2. Thaw the vials containing substrate (Lambda DNA) and assay buffer. 3. Prepare 2 different reaction mixtures using the following constituents. Reaction 1 (EcoR I digestion) λ DNA 20 µl 2X Assay Buffer 25 µl EcoR I 3 µl Reaction 2 (Hind III digestion) λ DNA 20 µl 2X Assay Buffer 25 µl Hind III 3 µl 10 11

8 Observation: Observe the band patterns obtained on digestion with EcoR I and Hind III. Compare these with molecular weight marker (λ/mlu I). (Refer fig. 1). Interpretation: Restriction patterns obtained on digestion with EcoR I and Hind III are markedly different, demonstrating the fact that each restriction enzyme recognizes and cuts only a particular base sequence unique to it. By comparing the migration distances with that of the marker, one can also determine the approximate sizes of DNA fragments Agarose Gel Electrophoresis 26,282 9,824 5,090 2,419 2,205 Lane 1 : λ/hind III Digest Lane 2 : λ/ecor I Digest Lane 3 : Control DNA Lane 4 : λ/mlu I Digest 1, Fig 1 : λ DNA digested with EcoR I & Hind III, run on 1% Agarose gel (stained with EtBr) 12 13

9 Introduction: Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their molecular weight and is an intrinsic part of almost all routine experiments carried out in molecular biology. The technique consists of 3 basic steps: Preparation of agarose gel Electrophoresis of the DNA fragments Visualization of DNA fragments Principle: Preparation of Agarose Gel: Agarose is a linear polymer extracted from seaweeds. Its basic structure is shown in the figure. HO CH2 O HO OH Fig: Basic unit structure of agarose. Purified agarose is a powder insoluble in water or buffer at room temperature but dissolves on boiling. Molten solution is then poured into a mould and allowed to solidify. As it cools, agarose undergoes polymerization i.e., sugar polymers cross-link with each other and cause the solution to gel, the density or pore size of which is determined by concentration of agarose

10 Electrophoresis of DNA fragments: Electrophoresis is a technique used to separate charged molecules. DNA is negatively charged at neutral ph and when electric field is applied across the gel, DNA migrates towards the anode. Migration of DNA through the gel is dependent upon: 1. Molecular size of DNA 2. Agarose concentration 3. Conformation of DNA 4. Applied current Matrix of agarose gel acts as a molecular sieve through which DNA fragments move on application of electric current. Higher concentration of agarose gives firmer gels, i.e., spaces between cross-linked molecules is less and hence smaller DNA fragments easily crawl through these spaces. As the length of the DNA increases, it becomes harder for the DNA to pass through the spaces, while lower concentration of agarose helps in movements of larger DNA fragments as the spaces between the cross-linked molecules is more. The progress of gel electrophoresis is monitored by observing the migration of a visible dye (tracking dye) through the gel. Two commonly used dyes are Xylene cyanol and Bromophenol blue that migrate at the same speed as double stranded DNA of size 5000 bp and 300 bp respectively. These tracking dyes are negatively charged, low molecular weight compounds that are loaded along with each sample at the start of run, when the tracking dye reaches towards the anode, run is terminated. Visualization of DNA fragments: Since DNA is not naturally coloured, it will not be visible on the gel. Hence the gel, after electrophoresis, is stained with a dye specific to the DNA. Discrete bands are observed when there is enough DNA material present to bind the dye to make it visible, otherwise the band is not detected. The gel is observed against a light background wherein DNA appears as dark coloured bands. Alternatively, an intercalating dye like Ethidium bromide is added to agarose gel and location of bands determined by examining the gel under UV light, wherein DNA fluoresces. Note: Ethidium bromide must be handled carefully as it is a mutagen and a carcinogen. Wear gloves while handling EtBr solution & gels stained with EtBr

11 Procedure: Preparation of 1% Agarose Gel 1. Prepare 1X TAE by diluting appropriate amount of 50X TAE buffer. (For one experiment, approximately 200 ml of 1X TAE is required. Make up 4 ml of 50X TAE to 200 ml with distilled water). 2. Weigh 0.5 g of agarose and add to 50 ml of 1X TAE. This gives 1% agarose gel. 3. Boil till agarose dissolves completely and a clear solution results. 4. Meanwhile place the combs of electrophoresis set such that it is approximately 2 cm away from the cathode. 5. Pour the agarose solution in the central part of tank when the temperature reaches approximately 60 C. Do not generate air bubbles. The thickness of the gel should be around 0.5 to 0.9 cm. Keep the gel undisturbed at room temperature for the agarose to solidify. 6. Pour 1X TAE buffer into the gel tank till the buffer level stands at 0.5 to 0.8 cm above the gel surface. 7. Gently lift the combs, ensuring that wells remain intact. Electrophoresis 8. Connect the power cord to the electrophoretic power supply according to the convention red: anode, black: cathode. 9. Load the samples in the wells in the desired order. 10. Set the voltage to 50 V and switch on the power supply. 11. Switch off the power when the tracking dye (bromophenol blue) from the well reaches ¾ th of the gel. This takes approximately one hour. Staining Procedure to Visualize DNA 12. Prepare 1X staining dye by diluting 6X dye (1:6) with distilled water. (Approximately 50 ml of 1X staining dye is required for one experiment. Therefore, make up 8 ml of 6X dye to 48 ml with distilled water). 13. Carefully transfer the gel (from gel tank) into a tray containing 1X staining solution. Make sure that the gel is completely immersed. 14. For uniform staining, place the tray on a rocker for approximately one hour or shake intermittently every 10 to 15 minutes. 15. Pour out the staining dye into a container. (The dye can be reused twice). Destain the gel by washing with tap water several times till the DNA is visible as a dark band against a light blue background. Note: Alternatively, Ethidium bromide can be used for visualizing DNA fragments. Add Ethidium bromide to molten agarose to a final concentration of 0.5 µg/ml (from a stock of 10 mg/ml in water), when temperature is around 50 C. Mix and cast the gel. After electrophoresis, DNA samples can be visualized under UV light, they appear fluorescent. No destaining is required in this case

12 Ordering Information Product Size Cat # 1 Pack KT01 Teaching Kit Consumables for 5 experiments & Elpho Kit (ETS 1)) 1 Pack KT01A Teaching Kit Consumables 5 experiments) 1 Pack KT01B Teaching Kit Consumables 20 experiments) Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com 20