UNIT 5 Recombinant DNA Technology. Objectives Define Recombinant DNA technology Define Recombinant DNA technology Outline steps involved in creating recombinant

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  • UNIT 5Recombinant DNA Technology

  • ObjectivesDefine Recombinant DNA technologyOutline steps involved in creating recombinant DNA Define type II restriction enzymes and their use in recombinant DNA technologyExplain restriction mapping and construct restriction mapsState the properties of cloning vectors and reproduce genetic map of common cloning vectorsOutline the steps involved in cloning prokaryotic DNA into a plasmid cloning vectorOutline the steps involved in cloning eukaryotic DNA into a plasmid cloning vectorUnderstand the methods of transformation and the steps involved in the selection of transformantsExplain the concept of gene libraries and methods of screening a gene library

  • Recombinant DNA technologya body of techniques for cutting and splicing together different pieces of DNA. When segments of foreign DNA are transferred into another cell or organism, the protein for which they code may be produced along with substances coded for by the native genetic material of the cell or organism. These cells become "factories" for the production of the protein coded for by the inserted DNA

  • Restriction EnzymesThe major tools of recombinant DNA technology are restriction enzymes (nucleases)first discovered in the late 1960s They work by cutting up the foreign DNA, a process called restriction. Most restriction enzymes are very specific recognizing short, specific nucleotide sequences in DNA molecules and cutting at specific points within these sequences. There are hundreds of restriction enzymes and more than 150 different recognition sequences.

  • Restriction enzymesNucleases are further described by addition of the prefix "endo" or "exo" to the name:

    endonuclease applies to sequence specific nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of the molecule.

    exonucleases function by removing nucleotides from the ends of the molecule

  • Types of Restriction EnzymesType I Recognise specific sequencesbut then track along DNA (~1000-5000 bases) before cutting one of the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA

    Type II Recognise a specific target sequence in DNA, and then break the DNA (both strands), within the recognition site

    Type III Intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site

  • Restriction EndonucleasesThe first endonucleases discovered was from Escherichia coli EcoRI

    A restriction endonuclease functions by "scanning" the length of a DNA molecule.

    Once it encounters its particular specific sequence (recognition site), it will bond to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones of the double helix.

    Once the cuts have been made, the DNA molecule will break into fragments.

  • Restriction EndonucleasesThe positions of these two cuts, both in relation to each other, and to the recognition sequence itself, are determined by the identity of the restriction endonuclease

    The length of the recognition site for different enzymes can be four, five, six, eight or more nucleotide pairs long.

    Restriction endonucleases that cleave recognition sites of four and six nucleotide pairs are used for most of the protocols for molecular cloning since these restriction sites are common in DNA.

  • Restriction Endonucleases

    Different endonucleases yield different sets of cuts, but one endonuclease will always cut a particular base sequence the same way.

    Some restriction endonucleases digest DNA leaving 5-phosphate extensions(protruding ends, sticky ends)

    Some leave 3-hydroxyl extensions

    Some cut the backbone of both strands within a recognition site to produce blunt-ended (flush-ended) DNA molecules.

  • Use of Restriction Enzymes in recombinant DNA Technology

  • How Restriction Enzymes are namedgenerally have names that reflect their originThe first letter of the name (capitalized ) comes from the genus of the bacteria the second two letters (lower case) come from the species Roman numerals following the nuclease names indicate the order in which the enzymes were isolated from that strain of bacteria. For example EcoRI comes from Escherichia coli RY13

  • Type II Restriction EnzymesMany Type II restriction endonucleases recognise PALINDROMIC sequencesSymmetrical sequences which read in the same order of nucleotide bases on each strand of DNA (always read 5' to 3')

    For example, EcoRI recognises the sequence 5'-G A A T T C-3'3'-C T T A A G-5 '

    The high specificity for their recognition site means that DNA (target sequence and cloning vector) will always be cut reproducibly into defined fragments (important for molecular cloning)

  • Note: Only one strand of the target DNA is shown

    Enzyme Organism from which derived Target sequence (cut at *) 5' -3' Bam HI Bacillus amyloliquefaciens G* G A T C C Bgl II Bacillus globigii A* G A T C T Eco RI Escherichia coli RY 13 G* A A T T C Eco RII Escherichia coli R245 * C C A/T G G Hae III Haemophilus aegyptius G G * C C Hind III Haemophilus inflenzae Rd A* A G C T T Hpa I Haemophilus parainflenzae G T T * A A C Kpn I Klebsiella pneumoniae G G T A C * C Pst I Providencia stuartii C T G C A * G Sma I Serratia marcescens C C C * G G G SstI Streptomyces stanford G A G C T * C Sal I Streptomyces albus G G * T C G A C Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G G

  • Frequency of cuttingBecause of their restriction site specificity, the restriction endonucleases cut DNA into fragments whose average length is determined by the number of base pairs in the restriction site and to a lesser extent by the ratio of bases in the DNA. For DNA that has equal amounts of all four bases, each base has a probabilty of 1/4 at any particular position in the DNA sense strand.

  • Frequency of cuttingBecause of their restriction site specificity, the restriction endonucleases cut DNA into fragments whose average length is determined by the number of base pairs in the restriction site (and to a lesser extent by the ratio of bases in the DNA). For DNA that has equal amounts of all four bases, each base has a probability of 1/4 at any particular position in the DNA strand.

  • Frequency of cuttingFor a restriction site of 4 base pairs, the probability of random occurrence of that sequence is (1/4)(1/4)(1/4)(1/4) = 1/256.

    For 6 base pairs, the probability is 1/4,096, and for 8 base pairs it is 1/65,536.

  • Frequency of cuttingThus, a restriction endonuclease with a 6 base pair restriction site would generate fragments whose average length is 4,096 base pairs.

    Such fragments are large enough to contain a complete gene (provided that the are no cut sites within the gene for the restriction endonuclease that is used).

  • Frequency of cuttingEffect of base composition: For DNA whose base composition differs from 50% GT it is necessary to calculate the probability of a site as the product of the probabilities of each of its components. For example, if a DNA is 66.7% GC (2/3 of its base pairs are GC) and one assumes random orientation of the base pairs, A and T will each have probabilities of 1/6 and G and C will have probabilities of 1/3 each.

  • Frequency of cuttingEffect of base composition contd. : Thus the probability of GAATTC would be:(1/3)(1/6)(1/6)(1/6)(1/6)1/3) = 1/11,664as opposed to 1/4096 when all four bases are present in equal amounts.

    Thus, the average fragment length generated by Eco RI would be longer in a DNA with a higher GC content.

  • Steps in creating recombinant DNAThe DNA (insert, cloned DNA, target DNA, foreign DNA) from a donor organism is extractedThen DNA is enzymatically cleaved (cut, digested) and joined (ligated) to another DNA entity (cloning vector) to form a new recombined DNA molecule (cloning vector-insert DNA construct, DNA construct).

  • Steps in creating recombinant DNA

    The cloning vector-insert DNA construct is transferred into and maintained within a host cell, a process called transformation.

    The host cell that takes up the DNA construct (transformed cells) are identified (by screening) and selected (separated, isolated) from those that do not.

  • Transformation

    Many cells lack the ability to take up DNA from their surroundings and are hence not competent

    The cell membranes of such cells have to be made porous to make them competent.

    Competence can be achieved by treatment of cells with low temperature and calcium chloride.

    Heat shock treatment during transformation closes these pores.

  • Transformation

    Transformation is an inefficient process (1 cell in 1000). Furthermore a few cells will be transformed by recircularized plasmid DNA

    others by nonplasmid DNA

    while a few by plasmid-insert DNA construct.

  • Desirable features of cells used in Transformation exercises

    To ensure that the plasmid-insert DNA construct is perpetuated in its original form the E. coli cells used:

    must lack REs

    must also be incapable of homologous recombination.

  • Screening of TransformantsAfter transformation, it is necessary to identify the cells that contain the plasmid-cloned DNA constructs

    This procedure is called screening

    The screening method used will depend on the plasmid system used in the transformation process.

  • Screening when a pBR322 system is used In a pBR322 system in which the target sequence was inserted into the BamH1 site, the identification is ac