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Restriction Digestion and its Applications

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Description of Restriction Digestion and its applications like Cloning, Southern Blot, RFLP, PCR-RFLP and DNA Fingerprinting.

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Restriction Digestion and its Applications

  1. 1. 1 RESTRICTION DIGESTION AND ITS APPLICATIONS Endonucleases cleave internal phosphodiester bonds in polynucleotides, while exonucleases cleave polynucleotides from their ends. Restriction endonucleases are group of enzymes that cleave DNA at or near a specific sequence. Evolutionarily, their role is to protect bacteria from invading foreign DNAs and that is why they are endowed with exquisite sequence specificity. There are 4 types of restriction endonucleases:  Type I enzymes are complex, multisubunit enzymes that cut DNA at random sites far from their recognition sequences.  Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for routine DNA analysis and gene cloning.  Type III enzymes also cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests.  Type IV enzymes recognize modified DNA, usually, methylated DNA Restriction endonucleases are named based on the bacterial strain from which they are isolated. Example: EcoRI is named after Escherichia coli RY13 strain and was the first (I) restriction endonuclease to be isolated from this strain. Digestion of the DNA by a restriction endonuclease can lead to generation of digested products either with flushed ends (blunt ends) or with overhangs (sticky ends). Because of the ability of Type II enzymes cut DNA at defined positions close to or within their recognition sequences, they have applications in various molecular biology techniques, as detailed below: 1. MOLECULAR CLONING: Molecular cloning refers to the isolation of a DNA sequence from any species (often a gene), and its insertion into a vector for propagation, without alteration of the original DNA sequence. Plasmids are the most commonly used vectors are can accept up to 10 kilobase pairs of foreign DNA.
  2. 2. 2 The human diploid genome is composed of 6 billion base pairs and isolation of a specific gene to be inserted into a vector is a laborious process. Hence, the source of the gene to be inserted into the vector can be (i) a gene library, in which the clones containing the gene of interest have already been identified or (ii) complementary DNA (cDNA) obtained from mRNA of a cell expressing the gene in large quantities. The cDNA can be amplified using a polymerase chain reaction with primers specific for the gene of interest and the amplicon obtained can be used for cloning. Both the plasmid and the DNA to be inserted are digested by the same restriction enzyme(s). Usually, restriction enzymes that produce sticky ends are used. This will lead to generation of 5’ and 3’ overhangs in the plasmid and the insert, which are complementary to each other, as shown in the diagram:
  3. 3. 3 The insert and the plasmid can then be ligated using DNA ligase in the presence of ATP, to generate the recombinant plasmid. Note in the above diagram that the plasmid also contains various other sequences: a. Origin: Refers to the sequence for origin of replication, which is necessary for multiplication of plasmid in the host organism. b. Plasmid also contains one or more markers that enable us to differentiate between the host organisms in which the plasmid is present, compared to those which do not have the plasmid. Example: Presence of an antibiotic resistance gene enables bacteria containing the plasmid to grow in a growth medium containing that particular antibiotic. c. MCS or Multiple Cloning Site, aka polylinker is a feature of engineered plasmids and contains multiple restriction sites close to each other, and thus provide versatility in the DNA fragments that can be inserted. Once the recombinant plasmid has been made after the ligation of restriction-digested plasmid and insert, the recombinant plasmid is introduced into a bacteria. The process by which foreign DNA is introduced into a cell is known as transformation. A bacterial cell can be transformed by any of the following methods: 1. Incubating the bacterial cells under cold conditions in presence of a divalent cation (eg.Ca++ ) along with the plasmid, followed by a heat shock. The divalent cations generate coordination complexes with the negatively charged DNA molecules, facilitating their entry into the cell. 2. Electroporation in which the cells are briefly shocked with an electric pulse. This leads to transient pore formation in the plasma membrane, through which the plasmid can enter the cell; the pore is later repaired by various repair mechanisms. The bacteria that is transformed are then grown in the presence of appropriate marker, so as to select the colonies containing the plasmid. These colonies are then used to isolate the plasmid in a procedure that is analogous to genomic DNA isolation along with a few modifications. APPLICATIONS OF MOLECULAR CLONING: 1. Production of recombinant proteins: Recombinant vector that contains the coding sequence of a particular protein of interest under suitable promoter can be introduced into appropriate prokaryotic or eukaryotic vector to express proteins in larger amounts. Examples of recombinant proteins include various drugs like insulin, erythropoietin, clotting factors etc. and vaccines like hepatitis B vaccine which contains HBsAg (Hepatitis B surface Antigen). 2. Production of DNA library: DNA library is a collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study. There are basically two kinds of libraries: genomic DNA and cDNA libraries. 3. Site directed mutagenesis: The sequence of a protein can be altered at specific amino acid residues by mutating the specific codons in the coding sequence which can be then cloned and expressed. This is helpful in understanding the pathogenesis of genetic diseases, mapping the active site amino acids of enzymes etc. 4. Gene therapy: Preparation of lentiviral/adenoviral vectors with the correct insert coding for the protein to be expressed involves molecular cloning. 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. After a DNA segment has been digested using a restriction
  4. 4. 4 enzyme, the resulting fragments can be examined agarose gel electrophoresis, which separates the fragments of DNA generated according to their size. One common method for constructing a restriction map involves digesting the unknown DNA sample in three ways. Here, two portions of the DNA sample are individually digested with different restriction enzymes, and a third portion of the DNA sample is double-digested with both restriction enzymes at the same time. Next, each digestion sample is separated using gel electrophoresis, and the sizes of the DNA fragments are recorded. The total length of the fragments in each digestion will be equal. However, because the length of each individual DNA fragment depends upon the positions of its restriction sites, each restriction site can be mapped according to the lengths of the fragments. The final representation of the DNA segment that shows the positions of the restriction sites is called a restriction map. 2. RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP): RFLP refers to the presence of DNA fragments of different lengths, after digestion of homologous DNA samples in question with specific restriction endonucleases. In RFLP analysis, the DNA fragment is digested by one or more restriction endonucleases; depending on the presence or absence of the restriction sites for the specific restriction endonucleases, the nucleotide changes in the DNA can be profiled. Let us see an example: The genetic defect in sickle cell anemia is a point mutation (transversion) where T is substituted for A in the coding region of beta hemoglobin gene. This substitution results in a change in the codon from GAG (glutamic acid) to GUG (valine). The figure below shows the restriction site of MstII corresponding to 6th position of beta globin:
  5. 5. 5 Beta globin gene has 3 restriction sites for MstII. One of these restriction sites is lost in sickle cell anemia. Thus the restriction pattern will be different in a DNA sample from a normal individual, compared to those with sickle cell trait or sickle cell disease. This can be detected using: 1. Sequence specific probes against these fragments using a Southern Blot. In Southern Blot:  DNA is digested with a set of restriction enzymes  The fragments are separated based on their molecular weight (size) using gel electrophoresis  The DNA is transferred to a polymer membrane (usually nitrocellulose or nylon) from the gel. This process is known as blotting. Blotting helps to prevent the diffusion of DNA which can occur in a gel. Moreover, blots are less fragile, compared to a gel.  Sequence specific labelled nucleotide oligomers (also known as probes) are incubated with the blot. The probes are radiolabelled or fluorescent labelled. The probes will bind only to their complementary DNA sequence in the blot (hybridization).  The DNA fragment of interest can be detected by exposing the blot to an X-ray film (in case of radiolabelled probes) or by examining under a fluorescence microscope (in case of fluorescent labelled probes).
  6. 6. 6 The below diagram shows MstII restriction pattern of beta globin gene in sickle cell trait, sickle cell disease and a normal individual, when detected using Southern blotting: 1. PCR-RFLP: In PCR-RFLP, PCR is performed to amplify a particular region of DNA and the PCR products are subjected to digestion using restriction endonucleases. Since the sequence of interest in the DNA is amplified to signifance amounts, the products can be visualised under UV light after an agarose gel electrophoresis, thus avoiding the radioactive hazards that are involved in Southern blotting. The diagram given in the next page explains the principle of PCR-RFLP in diagnosis of sickle cell anemia Note that RFLP techniques are NOT frequently used for genotyping at present. The preferred method is DNA sequencing. Can you think why?
  7. 7. 7 3. DNA FINGERPRINTING: In DNA fingerprinting, genomic DNA is subjected to digestion by a specific set of restriction endonucleases. Humans share a genome sequence similarity of 99.9%. This means DNA sequence of two individuals differs in 1 in every 1000 nucleotides. Some of these sites that differ may be restriction sites. So the restriction pattern of DNA from two individuals generally differ. This is another example of RFLP. DNA fingerprinting was used to establish a link between a biological evidence and a suspect in a criminal investigation. A DNA sample taken from a crime scene is compared with a DNA sample from a suspect. If the two DNA profiles are a match, then the evidence came from that suspect. Conversely, if the two DNA profiles do not match, then the evidence cannot have come from the suspect. Since RFLPs are inherited, DNA fingerprinting were also used to establish paternity. The procedure is almost same as the RFLP analysis using Southern Blot which we discussed earlier, except for the fact that multiple enzymes are used here, and different samples are compared. Nowadays, RFLP has been replaced by analysis of STR (short tandem repeats) in criminalistics. PROTOCOL FOR RESTRICTION DIGESTION OF PLASMID: You have been provided with the following: 1. 4 µg of plasmid DNA 2. Restriction enzymes A and B (20 Units each) 3. Buffer for restriction enzyme 4. Nuclease free water 5. 1% Agarose gel (containing ethidium bromide) 6. Gel loading dye 7. DNA ladder (molecular weight marker) 8. PCR tubes (0.2 mL)
  8. 8. 8 Make the reaction mixtures as shown below: Sl No Component Tube A Tube B Tube C 1. Plasmid 1 µg 1 µg 1 µg 2. Nuclease free water Upto 50 µL Upto 50 µL Upto 50 µL 3. Buffer 5 µL 5 µL 5 µL 4. Enzyme A 10 Units - 10 Units 5. Enzyme B - 10 Units 10 Units 1. Mix components by pipetting the reaction mixture up and down. Follow with a quick spin-down in a microcentrifuge. 2. Incubate at 37 ⁰C for 15 minutes. 3. Add 10 µL of gel loading dye to each tube. 4. Also, prepare tube D by adding 1 µg DNA to 1 µL gel loading dye. 5. Load the contents of the tube into the agarose gel, along with molecular weight marker. 6. Perform agarose gel electrophoresis (80 V for 60 minutes) and visualise your gel under uv transilluminator/ gel documentation system. 7. Based on your findings, create a restriction map of the plasmid. PRECAUTIONS: 1. Wear gloves while performing all the steps. 2. Restriction enzyme should be added as the last component of the reaction mixture 3. Restriction enzymes should be always kept on ice 4. Use UV safety measures while visualising the gel.

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