Recombinant DNA Technology RESTRICTION ENDONUCLEASES, CLONING AND TRANSFORMATION

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1 Recombinant DNA Technology RESTRICTION ENDONUCLEASES, CLONING AND TRANSFORMATION

2 What are restriction enzymes? Restriction endonuclease = Restriction enzymes Endo (inside), nuclease (cuts nucleic acid) Molecular scissors that cut double stranded DNA molecules at specific points. Found naturally in a wide variety of prokaryotes The specific DNA sequence is called recognition sequence An important tool for manipulating DNA. Arbor and Dussoix in 1962 discovered that certain bacteria contain Endonucleases which have the ability to cleave DNA. In 1970 Smith and colleagues purified and characterized the cleavage site of a Restriction Enzyme. Werner Arbor, Hamilton Smith and Daniel Nathans shared the 1978 Nobel prize for Medicine and Physiology for their discovery of Restriction Enzymes.

3 BIOLOGICAL ROLE Most bacteria use Restriction Enzymes as a defense against bacteriophages. Restriction enzymes prevent the replication of the phage by cleaving its DNA at specific sites. The host DNA is protected by Methylases which add methyl groups to adenine or cytosine bases within the recognition site thereby modifying the site and protecting the DNA.

4 restriction enzymes Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1

5 restriction enzymes Recognition sites have symmetry (palindromic) Able was I, ere, I saw Elba Bam H1 site: 5 -GGATCC-3 3 -CCTAGG-5 CIVIC, Madam

6 Protection of Self-DNA Bacteria protect their self DNA from restriction digestion by methylation of its recognition site. Methylation is adding a methyl group (CH 3 ) to DNA. Restriction enzymes are classified based on recognition sequence and methylation pattern.

7 TYPES OF RESTRICTION ENZYMES Cleavage site Location of methylase Examples Type I Random Around 1000bp away from recognition site Endonuclease and methylase located on a single protein molecule EcoK I EcoA I CfrA I Type II Specific Within the recognition site Endonuclease and methylase are separate entities EcoR I BamH I Hind III Type III Random bp away from recognition site Endonuclease and methylase located on a single protein molecule EcoP I Hinf III EcoP15 I

8 Few Restriction Enzymes Enzyme Target sequence Organism from which derived (cut at *) 5' -->3' Bam HI Bacillus amyloliquefaciens G* G A T C C Eco RI Escherichia coli RY 13 G* A A T T C Hind III Haemophilus inflenzae Rd A* A G C T T Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii C T G C A * G Sma I Serratia marcescens C C C * G G G Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G G

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10 Mechanism of Action of Restriction Enzymes Restriction endonuclease scan the length of the DNA, binds to the DNA molecule when it recognizes a specific sequence and makes one cut in each of the sugar phosphate backbones of the double helix by hydrolyzing the phoshpho-diester bond. Specifically, the bond between the 3 O atom and the P atom is broken. 3 OH and 5 PO 4 3- is produced. Mg 2+ is required for the catalytic activity of the enzyme.

11 Structure of EcoR V endonuclease Consists of two subunits dimers related by two fold rotational symmetry. Binds to the matching symmetry of the DNA molecule at the restriction site and produces a kink at the site.

12 Hydrogen bonding interactions between EcoRv and its DNA substrate

13 Palindrome, Restriction Enzyme, Sticky Ends CIVIC, Madam Sticky Ends (Cohesive Ends) GAATTC G AATTC G AATTC GAATTC EcoRI Get An Apple To The Class G AATTC G AATTC

14 USES FOR RESTRICTION ENZYMES RFLP analysis (Restriction Fragment Length Polymorphism) Genotyping DNA sequencing Mapping of DNA DNA libraries Transformation Large scale analysis of gene chips

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16 Agarose gel electrophoretogram of restriction digests. Digest of Agrobacterium radiobacter plasmid pagk84 digested with: A. BamHI, B. B. PstI, C. C. BglII, D. D. HaeIII, E. E. HincII, F. F. SacI, G. G. XbaI, H. H. HpaI. I. Lane I contains l phage DNA digested with HindIII as standards bp 9416 bp 6557 bp 4361 bp 2322 bp 2027 bp

17 Restriction Mapping of DNA CK A Restriction enzymes B A+B M A B 10 kb A B A + B 8 kb 2 kb 7 kb 3 kb 5 kb 3 kb 2 kb Juang RH (2004) BCbasics

18 DNAs with different genotypes

19 The Specific Cutting and Ligation of DNA GAATTC GAATTC CTTAAG CTTAAG EcoRI G AATTC G AATTC CTTAA G CTTAA G G CTTAA EcoRI sticky end G AATTC CTTAA G DNA Ligase G AATTC CTTAA G AATTC G EcoRI sticky end

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21 Uses Restriction enzymes are most widely used in recombinant DNA technology.

22 Transformation and Cloning 1-Transformation

23 Transformation Remember that a gene is a piece of DNA. It provides the instructions (codes) for a protein that gives an organism a particular trait. Genetic transformation This is the uptake of DNA from the environment. Which can be nduced to happen in a laboratory situation, inserting the DNA you want the microbe to express. Uptake of new DNA by microorganism will provide cell with new characteristics. In the laboratory situation From a population of 1000 or more treated cells only one or two may pick up foreign DNA. Those which do are said to be transformed. Bacterium E. coli and the yeast S. cerivisiae are widely used as recipients for foreign DNA. Both are unicellular and quick growing. Ideal for large-scale production of proteins such as industrial enzymes.

24 E. coli advantages 1. Several different types of plasmid can be used to introduce foreign DNA. 2. Foreign DNA can account for up to 60% of its total protein production. 3. Fast growing. 4. Easy to transform. 5. Easy to manipulate. E. coli disadvantages 1. Does not carry out post-translational modifications to proteins. 2. These modifications are necessary for protein function in, for example, human cells. 3. Bacterium occurs naturally in the intestines of humans and under certain circumstances can cause disease.

25 S. cerivisiae advantages 1. Rarely a human pathogen. 2. Genes organised, expressed and controlled in ways that are similar to human genes. 3. Carries out post-translational modifications, eg addition of sugar residues, which are a common feature of human proteins. S. cerivisiae disadvantages 1. Difficult to transform. 2. Yields less protein than bacteria. 3. Plasmids easily lost from yeast

26 Transformation and Cloning 2-CLONING

27 Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

28 CLONING 1-CLONING PLASMIDS OR VECTORS

29 Cloning vectors In gene cloning, once recombinant DNA (rdna) has been constructed it is introduced into a host. In the host, rdna has to be: maintained replicated passed from one generation to another. This is achieved by introducing rdna into a cell on a DNA vehicle called a cloning vector, commonly known as plasmid cloning vectors. PLASMID Extra-chromosomal DNA found in bacteria. Loops of double-stranded DNA. Some of them present in multiple copies. Independently replicate inside bacteria.

30 CLONING VECTORS

31 Page 106 The puc18 cloning vector.

32 CLONING VECTORS

33 Features of plasmids PLASMID VECTORS ARE KB AND CONTAIN: A selectable marker usually an antibiotic resistance gene: required for maintenance of plasmid in the cell advantageous for bacteria to keep the plasmid (can grow in presence of antibiotic). Under the selective conditions, only cells that contain plasmids with selectable marker can survive Commonly used selectable markers are ampicillin, neomycin and chloramphenicol. Origin of replication (Ori) is a DNA segment recognized by the cellular DNAreplication enzymes. Without replication origin, DNA cannot be replicated in the cell. Many cloning vectors contain a multiple cloning site or polylinker (MCS): a DNA segment with several unique sites for restriction endonucleases located next to each other Restriction sites of the polylinker are not present anywhere else in the plasmid. Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

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35 Target Genes Carried by Plasmid to the transformants Restriction Enzyme Target Gene Recombination Target Genes Recombination ( DNA ligase) PCR + Restriction Enzyme Chromosomal DNA Transformation Host Cells

36 STEPS BACTERIAL TRANSFORMATION A-Incubation of antibiotic-sensitive bacterial cell with calcium chloride treatment to disrupt the cell wall. b-treated bacterial cell incubated with plasmid and then bacteria plated out onto antibiotic containing agar plates. C- Only those bacteria that have taken up plasmid will be able to grow on agar plus antibiotic. D-Transformed bacteria can then be isolated and grown in bulk with appropriate antibiotic. Bacteria multiply to produce genetically identical offspring clones.

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38 THANKS