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Recombinant dna technology tools and techniques

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Methods used to prepare and amplify recombinant DNA; identification of specific DNA, RNA and proteins, DNA sequencing

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Recombinant dna technology tools and techniques

  1. 1. Recombinant DNA Technology: Tools and Techniques R. C. Gupta M.D. (Biochemistry) Jaipur, India
  2. 2. Recombinant DNA technology is used to produce recombinant DNA Recombinant DNA is made by combining different fragments of DNA Usually, the DNA fragments are derived from different sources
  3. 3. Recombinant DNA, having unrelated genes, is also known as chimeric DNA Chimera is a mythological creature having the head of a lion, the trunk of a goat and the tail of a serpent
  4. 4. Recombinant DNA technology came into existence in the 1970s It has led to revolutionary changes not only in biochemistry but in all life sciences
  5. 5. Recombinant DNA technology has proved to be of immense value in: Medical science Agriculture Animal husbandry Industry
  6. 6. Opened the doors of recombinant DNA research The most important tools of recombinant DNA technology Restriction endonucleases Discovered by Arber, Smith and Nathans in the early1970s Arber Smith Nathans
  7. 7. Restriction endonucleases (enzymes) are found in bacteria They protect the bacteria against viral infections When a virus infects a bacterial cell, restriction enzymes split the viral DNA Thus, the virus is destroyed
  8. 8. Hundreds of restriction enzymes have been discovered so far They are named after the bacterium in which they are found e.g. Hin I, Hae III, Eco RI
  9. 9. The three-letter abbreviation is derived from the name of the bacterium e.g. Hin from Haemophilus influenzae Hae from Haemophilus aegyptius Eco from Escherichia coli
  10. 10. Sometimes, a strain designation is also included in the name e.g. R after Eco means strain R of E.coli The numerals I, II, III etc indicate the serial numbers of enzymes from the same bacterium in the order of their discovery
  11. 11. Each restriction enzyme: Recognizes a specific base sequence in double-stranded DNA Splits the DNA at this site
  12. 12. The base sequence recognized by a restriction enzyme: Is 4-8 base pairs long Is palindromic
  13. 13. A palindrome is a word or a sentence which reads the same from left to right and from right to left e.g. DAD, MADAM, RADAR etc
  14. 14. In DNA, the base sequences are read in 5’ → 3’ direction If a sequence reads the same on both the strands in 5’ → 3’ direction, it is known as a palindromic sequence 5’ GGCC 3’ 5’ TTTAAA 3’ 3’ CCGG 5’ 3’ AAATTT 5’
  15. 15. Restriction enzymes: Split both the strands of DNA Produce blunt or sticky ends Blunt ends Sticky ends
  16. 16. Blunt ends are also known as even or non-overlapping ends Sticky ends are also known as cohesive or overlapping ends
  17. 17. 5’ GG CGCC 3’ 5’ GG CGCC 3’ 3’ CC GCGG 5’ 3’ CCGC GG 5’ 5’ GG 3’ CC CGCC 3’ GCGG 5’ + 5’ GG 3’ CCGC CGCC 3’ GG 5’ + RE Blunt ends produced by a restriction enzyme (RE) Sticky ends produced by another RE RE
  18. 18. Sticky ends are more useful in recombinant DNA technology They can be easily ligated to the complementary sticky ends of another fragment of DNA by DNA ligase GGCCTCAAT AGTATCCGG
  19. 19. Two complementary sticky ends joined Restriction enzyme DNA ligase Sticky ends of two fragments joined Fragment having sticky end Fragment having sticky end Another DNA cut by the same enzyme The enzyme cuts both the strands at its restriction site A T T A A A T T
  20. 20. The site recognized by a restriction enzyme is known as its restriction site DNA fragments produced by restriction enzymes are known as restriction fragments Every DNA has got a number of restriction sites for a number of restriction enzymes
  21. 21. 5’ – – G AATTC – – 3’ 3’ – – CTTAA G – – 5’ 5’ – – G GATCC – – 3’ 3’ – – CCTAG G – – 5’ 5’ – – GG CC – – 3’ 3’ – – CC GG – – 5’ 5’ – – G AATTC – – 3’ 3’ – – CTTAA G – – 5’ 5’ – – G GATCC – – 3’ 3’ – – CCTAG G – – 5’ 5’ – – GG CC – – 3’ 3’ – – CC GG – – 5’ Eco RI Bam HI Hae III 4 4 4 + + + 5 6 6 6 5 5 Restriction sites of some enzymes
  22. 22. Recombinant DNA: Can be formed by joining restriction fragments obtained from different sources Will have a base sequence different from that of the original DNA
  23. 23. A foreign gene can be inserted into DNA by this method The gene is clipped out using a restriction enzyme It is ligated to another DNA using DNA ligase
  24. 24. 5’ – – GGATCC – – 3’ 3’ – – CCTAGG – – 5’ Foreign gene DNA having Bam HI restriction site Foreign gene having Bam HI restriction site on either side 6 Bam HI 6 Bam HI Foreign gene inserted between restriction fragments by ligating complementary sticky ends 5’ – – GGATCC 3’ – – CCTAGG GGATCC – – 3’ CCTAGG – – 5’ Foreign gene – – – – – – – – – – – – – – – – 5’ – – G GATCC – – 3’ 3’ – – CCTAG G – – 5’ 5’ – – G GATCC G GATCC – – 3’ 3’ – – CCTAG G CCTAG G – – 5’ Foreign gene – – – – – – – – – – – – – – – – DNA ligase + + + 6 5’ – – GGATCC – – – – – – – – GGATCC – – 3’ 3’ – – CCTAGG – – – – – – – – CCTAGG – – 5’
  25. 25. Amplification Amplification is often required to prepare multiple copies of the recombinant DNA Amplification can be done: Either by cloning the DNA Or by polymerase chain reaction
  26. 26. Herbert Boyer Stanley Cohen Cloning of recombinant DNA Cloningof DNA was pioneered by Herbert Boyer and StanleyCohen
  27. 27. DNA can be cloned in living cells A vector is required to transfer recombinant DNA into the cells A clone is a population of identical organisms or cells or molecules derived from the same source
  28. 28. Some useful cloning vectors are: Plasmids Bacteriophages Cosmids Bacterial artificial chromosomes Yeast artificial chromosomes
  29. 29. Plasmids are small circular double- stranded DNA molecules They are present in prokaryotes in addition to the chromosomal DNA They may replicate independently of the chromosomal DNA Plasmids
  30. 30. Plasmids contain one or more antibiotic- resistance genes These genes provide antibiotic-resistance to the bacteria Plasmids can be transferred from one bacterium to another Plasmids can accept foreign DNA frag- ments up to 10 kb in size
  31. 31. Therefore, DNA fragments up to 10 kb (kilo bases) in size can be easily inserted into plasmids, and cloned in bacteria e.g. E.coli
  32. 32. Nick the plasmid by a restriction enzyme to generate sticky ends Ligate the desired DNA fragment having complementary sticky ends to the plasmid by DNA ligase Introduce the plasmid into a bacterial cell Technique
  33. 33. Multiplication of the bacterial cell and independent replication of the plasmid produce multiple copies of the foreign DNA within a short time
  34. 34. pSC 101 is a plasmid present in E. coli This was the first vector used for cloning pSC 101 has a single restriction site, a single antibiotic-resistance gene and it replicates poorly
  35. 35. An ideal vector should replicate rapidly, should have several restriction sites for different restriction enzymes and more than one antibiotic-resistance genes
  36. 36. Some plasmids having these features have been constructed in the laboratory Examples are pBR 322, pBR328, pUC18 etc
  37. 37. Plasmids can be introduced into bacteria by exposing them to high concentration of divalent cations This increases the permeability of bacterial cell membrane so as to allow plasmids to enter the bacterial cells
  38. 38. However, plasmids may not enter all the bacterial cells Some plasmids might not have taken up the foreign DNA We have to select the bacterial cells having recombinant plasmids
  39. 39. Antibiotic-resistance genes are useful in selection of recombinant plasmids For example, plasmid pBR322 has got ampicillin- and tetracycline-resistance genes Restriction sites are present in the middle of these genes
  40. 40. Ampicillin-resistance gene Tetracycline-resistance gene pBR322
  41. 41. A foreign gene/DNA fragment can be inserted in the middle of an antibiotic-resistance gene Ampicillin-resistance gene Tetracycline-resistance gene (disrupted) Foreign DNA
  42. 42. Bacteria having recombinant plasmid would lose resistance to tetracycline But they would be resistant to ampicillin
  43. 43. We first grow the bacteria on an agar plate containing ampicillin The bacterial cells which have taken up and those which have not taken up the recombinant plasmid will grow
  44. 44. We partially transfer the bacterial colonies on a replica plate containing tetracycline Cells having the recombinant plasmid will be destroyed Thus, the colonies on the original plate having the recombinant plasmid can be identified
  45. 45. Agar plate containing ampicillin Replica plate containing tetracycline Colonies having recombinant plasmid
  46. 46. Bacteriophages are viruses that infect bacteria These viruses have a DNA genome surrounded by a protein coat Bacteriophages
  47. 47. The virus infects a bacterial cell by injecting its DNA into the bacterial cell
  48. 48. Landing Pinning DNA injectionTail contraction and penetration
  49. 49. After entering the bacterial cell, the viral DNA may enter one of the two alternate pathways: Lysogenic pathway Lytic pathway
  50. 50. In this pathway, the viral DNA gets incorporated into the bacterial genome, and becomes dormant (provirus) The proviral DNA replicates only when the bacterial cell divides Lysogenic pathway
  51. 51. In the lytic pathway, the viral DNA remains separate It replicates independently Lytic pathway
  52. 52. The proteins encoded by the viral DNA are synthesized by the bacterial cell Each DNA molecule is packaged into a protein coat forming a new virus particle When the number of viruses becomes very large, the bacterial cell ruptures
  53. 53. The provirus can break out of the bacterial DNA and enter the lytic pathway if the bacterial cell is exposed to some DNA damaging agent e.g. ultra-violet light
  54. 54. Bacteriophages can be used as vectors for cloning A portion of viral DNA, not essential for its replication and packaging, is clipped out by restriction enzymes The foreign DNA to be cloned is inserted in the viral DNA
  55. 55. The virus infects a bacterial cell It multiplies inside the bacterial cell A large number of copies of foreign DNA are formed in the bacterial cell
  56. 56. DNA is packaged into protein coat Phage DNA Restriction enzyme Foreign DNA DNA ligase Recombinant phage DNA E. Coli cell Virus attaches to E. Coli cell; injects its DNA into the cell Infected E. Coli cell Viral DNA replicates in the E. coli cell Virus Restriction fragments
  57. 57. Lambda phage and M13 phage are commonly used as cloning vectors DNA fragments up to 20 kb in size can be inserted in phage vectors, and can be cloned in E.coli Moreover, it is easier to infect E. coli with a phage than with a plasmid
  58. 58. Cosmids are hybrids of plasmids and lambda phage Lambda phage DNA possesses sticky ends on either side known as cos sites Cos sites are necessary for packaging phage DNA into the protein coat Cosmids
  59. 59. Cosmids are prepared by inserting the cos sites of phage DNA in plasmids Cosmids can infect E. coli just like plasmids DNA fragments up to 45 kb in size can be inserted in cosmids
  60. 60. Origin of replication Ampicillin-resistance gene Restriction sites Cos siteCosmid
  61. 61. BAC is a genetically engineered vector BAC is constructed from a fertility plasmid (F factor) Bacterial artificial chromosome (BAC)
  62. 62. BAC possesses chloramphenicol-resistance gene This gene helps in selecting the cells having recombinant DNA A polylinker contains several restriction sites for inserting foreign DNA
  63. 63. Origin of replication Chloramphenicol- resistance gene Restriction sites BAC
  64. 64. BAC can be used to clone DNA in bacterial cells e.g. E. coli BAC can accept foreign DNA of 100-300 kb BAC is nicked with a restriction enzyme and foreign DNA is inserted in it
  65. 65. The BAC is introduced into a bacterial cell It acts as an extra chromosome in the bacterial cell As the bacterial cell divides, the BAC is also replicated
  66. 66. Multiple copies of BAC are formed in the bacterial cells The BAC can then be isolated BACs are commonly used to clone DNA for determining its base sequence
  67. 67. YACs are formed by adding yeast telo- meres to a centromere-containing plasmid It has got an autonomous replicating sequence necessary for replication It has also got some restriction sites and markers Yeast artificial chromosome (YAC)
  68. 68. Eco RI restriction site BamHI restriction site Marker TelomereTelomere Marker Centromere Autonomous replicating sequence Ampicillin- resistance gene Yeast artificial chromosome
  69. 69. YAC can accept large fragments of foreign DNA Foreign DNA up to 3,000 kb in size can be inserted in YAC, and cloned in yeast cells
  70. 70. Polymerase chain reaction (PCR) Polymerase chain reaction is a technique for rapid ampli- fication of DNA devised by Kary Mullis in the 1980s
  71. 71. PCR is far quicker, easier and cheaper than cloning The only limitation of PCR is the size of DNA that can be amplified DNA up to 3 kb in length can be amplified by PCR
  72. 72. The DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq) in PCR Thermus aquatics is a bacterium found in hot water springs
  73. 73. T. aquaticus is used to high temperatures Its enzymes are not denatured even at 95°C Optimum temperature of its enzymes, including DNA polymerase, is 72°C
  74. 74. In PCR, DNA has to be heated to 94-95°C for separation of strands Since DNA polymerase of T. aquaticus is not destroyed at this temperature, it is an ideal enzyme for PCR DNA polymerase of T. aquaticus is usually called Taq polymerase
  75. 75. The reaction mixture for PCR contains: • The DNA to be amplified • Taq polymerase • A large quantity of primers • Deoxyribonucleotides (dATP, dGTP, dCTP & dTTP) • Buffer with Mg++ and K+
  76. 76. Primers have to be added to the reaction mixture as they are required to initiate each cycle of replication To prepare primers, we must know short flanking sequences on either side of the target DNA sequence
  77. 77. Amplification occurs in three steps: Strand separation Primer binding Primer extension (addition of deoxyribonucleotides to primer)
  78. 78. The temperature is raised to 95°C to separate the DNA strands First step The temperature is lowered to 56°C for binding of primers to DNA strands Second step The temperature is raised to 72°C for Taq polymerase to replicate the strands Third step
  79. 79. Target sequence of DNA to be amplified The two strands separate Primers hybridize with template strands Taq polymerase replicates both the strands. Two copies of target DNA are formed Target sequence 95°C for 30 sec 56°C for 30 sec 72°C for 2 to 5 min 3’ ´ 5’ 5’ ´ 3’ ´ 5’ 3’ ´ 3’ 5’ ´ 5’ 5’ 3’ 3’
  80. 80. Both the double-stranded DNA molecules are separated into single strands . Primers bind to all the four strands Taq polymerase replicates all the four strands. Four copies of target sequence are formed 72°C for 2-5 min ´ 95°C for 30 sec 56°C for 30 sec 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’5’ 3’ 5’ 3’5’ 3’ 5’ Second cycle begins
  81. 81. By repeating the cycles again and again, enormous amplification can be achieved After twenty cycles, nearly a million copies of target sequence of DNA are formed After thirty cycles, nearly a billion copies are formed
  82. 82. After preparing the reaction mixture, the only thing to be done to repeat the cycles is to change the temperature cyclically This can be done auto- matically in instruments known as thermocyclers
  83. 83. Strand separation (95°C for 30 sec) Primer binding (56°C for 30 sec) Replication (72°C for 2-5 min) Strand separation (95°C for 30 sec) Temperature cycles in thermocycler
  84. 84. Several new variants of the classical PCR have been devised: Nested PCR Multiplex PCR Reverse transcriptase-PCR Quantitative PCR
  85. 85. Amplification is done twice using two different sets of primers One pair of primers is used first to produce a DNA product This may contain unintended DNA sequences besides the intended target Nested PCR
  86. 86. The product of first PCR is then used as target sequence in the second PCR Different primers are used that bind downstream from the primers used first Nested PCR increases the specificity of DNA amplification
  87. 87. Nested PCR First primer | Second primer |
  88. 88. Multiple sets of primers are used in the same run Different target sequences are amplified in a single run Multiplex PCR
  89. 89. Multiplex PCR
  90. 90. A DNA complementary to an RNA is prepared by reverse transcription This is amplified by PCR This DNA can be used to determine the base sequence of RNA Reverse transcriptase-PCR
  91. 91. Fluorescent dyes are used to measure the amount of the amplified DNA in real time Quantitative PCR is also known as real time PCR Quantitative PCR
  92. 92. The initial PCR methods were able to amplify DNA of the size of 3 kb only With refinements in methodology, the size has increased to10 kb now
  93. 93. Applications of PCR • Study of genes • Genome mapping • Diagnosis of genetic diseases • Diagnosis of infectious diseases • DNA finger printing
  94. 94. Techniques for identification of DNA and RNA In recombinant DNA technology, we often require techniques to identify a specific: DNA RNA Protein
  95. 95. Complementary DNA (cDNA) probes are used for identification of DNA and RNA The cDNA probe hybridizes with comple- mentary DNA or RNA strand Antibodies can be used as probes to identify specific proteins
  96. 96. A technique for identification of a specific DNA was devised by Southern in1975 This technique is known as Southern blot transfer or Southern blotting
  97. 97. DNA is hydrolysed by a restriction enzyme The DNA fragments are separated by gel electrophoresis Agarose gel is used for high molecular weight compounds Polyacrylamide gel is used for low molecular weight compounds Southern blotting
  98. 98. Samples DNA ladder Gel Gel Gel Nitrocellulose sheet Nitrocellulose sheet Sponge Buffer Paper towels The fragments are transferred to a nitrocellulose sheet
  99. 99. A probe labeled with 32P is added which binds to the band of interest Unbound probe molecules are washed off The sheet is exposed to an x-ray film The band of interest becomes visible (called autoradiography)
  100. 100. Southern blot transfer Unbound probe washed off and the sheet exposed to x-ray film 32P-Labelled probe added Nitrocellulose sheet overlaidElectrophoresis Band having the probe becomes visible Probe find the complementary fragment and binds to it Fragments are transferred onto nitrocellulose sheet Fragments are separated and melted (denatured) DNA fragments produced by restriction enzymes are put on gel
  101. 101. A similar technique for identification of RNA was devised in 1977 by Alwine This was jokingly named as northern blot transfer or northern blotting In this technique, a cDNA-RNA hybrid is formed Northern blotting
  102. 102. Northern blot transfer
  103. 103. A similar technique for identification of proteins was devised later by Burnette The protein of interest is identified with the help of a labelled antibody probe This technique was named as western blot transfer or western blotting Western blotting
  104. 104. Western blot transfer
  105. 105. Techniques for determining base sequence of DNA The base sequence of DNA can be determined by: Chemical method of Maxam-Gilbert Enzymatic dideoxy method of Sanger
  106. 106. Maxam-Gilbert method is also known as selective chemical cleavage method It is based on chemical cleavage of DNA at selective sites Maxam-Gilbert method
  107. 107. The DNA to be sequenced is labeled at its 3’-end with a nucleotide having 32P Reagents that destroy/remove one specific base are used to remove bases from DNA
  108. 108. Four sets of 32P labeled DNA are treated with four different reagents These reagents remove adenine, guanine, cytosine and thymine respectively Conditions are so chosen that only one base is removed per DNA strand
  109. 109. Several strands of DNA are treated with each particular reagent The given base is removed randomly from all the possible sites where it was present Chain breaks are, then, induced at the “base-less” sites
  110. 110. The fragments produced in the four tubes are separated by electrophoresis on poly- acrylamide gel in four parallel lanes Locations of 32P-labeled fragments are identified by autoradiography
  111. 111. The smallest fragment moves the farthest from the point of application The largest fragment moves the least The other fragments are in between, depending on their size
  112. 112. Relative sizes of the fragments indicate the distance between the 32P-label (3’-end) and the destroyed base By arranging the fragments in the four lanes in the decreasing order of size, the sequence of bases from 5’-end to 3’-end can be deduced
  113. 113. 5’ ATCGATCG3’ 5’ ATCGATCGC*3’ Addition of radiolabeled C* at 3’-end 5’–TCGATCGC*3’ and 5’ATCG–TCGC*3’ 5’ATC–ATCGC*3’ and 5’ATCGATC–C*3’ 5’AT–GATCGC*3’ and 5’ATCGAT–GC*3’ 5’A–CGATCGC*3’ and 5’ATCGA–CGC*3’ TCGATCGC* and ATCG+TCGC* ATC+ATCGC*3 and ATCGATC+C* AT+GATCGC* and ATCGAT+GC* A+CGATCGC* and ATCGA+CGC* 1 2 3 4 5 6 7 8 5´ 3´A Lane G Lane C Lane T Lane A T C G A T C G Removal of T Removal of C Removal of G Removal of A Electrophoresis and autoradiography Cleavage at “base-less” site
  114. 114. Sanger used dideoxynucleo- tides to sequence DNA He had earlier devised a protein sequencing method Sanger’s dideoxy method Frederick Sanger
  115. 115. The 3’-OH group can form an ester bond with the next nucleotide There is no 3’-OH group to form an ester bond with the next nucleotide H HH OH HH CH2 Base O P ~ P ~ P — O H HH HH O P ~ P ~ P — O H 2’-Deoxyribonucleoside triphosphate 2’,3’-Dideoxyribonucleoside triphosphate O Base O 2’ 2’3’ 3’ CH2
  116. 116. Sanger’s method is based on controlled interruption of replication The DNA strand to be sequenced is used as a template for replication Replication is done in four test tubes
  117. 117. The following are added to each tube: • The template strand • 32P-Labeled primer • dATP, dGTP, dCTP and dTTP • DNA polymerase • One dideoxynucleotide (ddATP, ddGTP, ddCTP or ddTTP)
  118. 118. The dideoxynucleotide competes with the normal nucleotide If a normal nucleotide enters the growing chain, replication continues If a dideoxynucleotide enters the growing chain, replication stops Several cycles of replication are carried out
  119. 119. The relative concentrations of the dideoxynucleotide and its normal counterpart are such that the dideoxy analogue enters randomly at different sites in different cycles of replication
  120. 120. After several cycles of replication, strands of different lengths are formed Each strand ends with a dideoxy- nucleotide Strands formed in the four tubes are separated by electrophoresis Electrophoresis is done in four parallel lanes
  121. 121. The fragments are visualized by auto- radiography The pattern of bands on the autoradio- gram gives the sequence of bases This sequence is complementary to the template strand
  122. 122. 8 7 6 5 4 3 2 1 3’ 5’A Lane G Lane C Lane T Lane G C T A G C T A Electrophoresis and autoradiography 5’‒ATCGATCG 3’‒TAGCTAGC Tube 1 5’‒ATCGATCG 3’‒TAGCTAGC Tube 2 5’‒ATCGATCG 3’‒TAGCTAGC Tube 3 5’‒ATCGATCG 3’‒TAGCTAGC Tube 4 5’‒ATCGATCG 3’‒TAGCTAGC 5’‒ATCGATCG 3’‒TAGCTAGC 5’‒ATCGATCG 3’‒TAGCTAGC 5’‒ATCGATCG 3’‒TAGCTAGC dGTP dCTP 3’‒TAGCTAGC 5’■ dATP dTTP ddATP* DNA poly- merase 3’‒TAGCTAGC 5’■A* 3’‒TAGCTAGC 5’■ATCGA* + 3’‒TAGCTAGC 5’■ATCG* + 3’‒TAGCTAGC 5’■ATCGATCG* 3’‒TAGCTAGC 5’■ATC* + 3’‒TAGCTAGC 5’■ATCGATC* 3’‒TAGCTAGC 5’■AT* + 3’‒TAGCTAGC 5’■ATCGAT* dGTP dCTP 3’‒TAGCTAGC 5’■ dATP dTTP ddGTP* DNA poly- merase dGTP dCTP 3’‒TAGCTAGC 5’■ dATP dTTP ddCTP* DNA poly- merase dGTP dCTP 3’‒TAGCTAGC 5’■ dATP dTTP ddTTP* DNA poly- merase
  123. 123. Sanger’s method has also been auto- mated This has made DNA sequencing much faster