DNA Technology

• Gene Cloning and Transfer:

  1. Isolation
  2. Insertion
  3. Transformation
  4. Identification
  5. Growth / Cloning
• Isolation - there are 3 ways to isolate a particular gene before using it to transfer another species: conversion of mRNA to cRNA by reverse transcriptase, cutting DNA at specific, palindromic recognition sequences using restriction endonuclease and the gene machine.
• Conversion of mRNA to cDNA by reverse transcriptase:

  • mRNA is bound to a DNA strand by reverse transcriptase, forming a DNA/RNA heteroduplex
  • the strands separate, and cDNA is formed by DNA polymerase
• Conversion of mRNA to cDNA is used because

  1. mRNA has no non-coding regions
  2. only 2 copies of the gene in DNA but many with mRNA
  3. know where to locate the gene
• Using restriction endonuclease:

  Restriction endonuclease cut up the DNA at recognition sequences. Some of these leave fragments with two straight edges called blunt ends. Others leave ends with uneven edges called sticky ends. If the sequence of bases on one of these uneven ends is CAGGCCTG, them the sequence on the other strand, if read in the same direction will be GTCCGGAC. DNA from one source can be joined to DNA from another source if they are cut using the same restriction endonuclease and then joined using DNA ligase.
• The Gene Machine:

  1. The protein which is required has a known sequence of amino acids
  2. The amino acids mRNA codons are looked up.
  3. The complementary DNA triplets are looked up.
  4. This sequence of nucleotides is fed into the computer.
  5. Checked for ethical and biosafety reasons.
  6. Small, single strands of oligonucleotides are assembled, joined and then fed into PCR.
• Oligonucleotides are short sequences of <20 nucleotides.
• 2. Insertion

  The addition of promotor and terminator regions to the DNA fragment:

  • needed for transcription of gene
  • for attachment both transcription factors and RNA Polymerase
• Insertion

  Addition of promoter and terminator regions to the DNA fragments:

  • Needed for transcription of gene
  • For attatchment both transcription factors and RNA polymerase
• Transformation

  • Plasmids and bacterial cells mixed together in a medium containing calcium ions
  • Changes in temperature make the bacterial cell permeable so the plasmid can enter
  • Not all bacterial cells will contain the DNA fragments
• Identification

  Marker genes

  Has the plasmid been successfully uptaken by the bacteria?

  • Antibiotic resistance

  Has the gene been successfully uptaken by the plasmid?

  Inactivation of:

  • A second antibiotic resistance gene (replica plating)
  • A fluorescent marker
  • A enzyme marker
• Growth / cloning

  Once the bacteria which have successfully been 'transformed' - ie uptaken the vector with the gene - are identified - they can be grown on appropriate media and allowed to express the gene
• In vitro

  • high error rate, since no error-correction, hence prone to contamination
  • DNA is made in the test tube, so cannot be expressed directly
  • can use DNA from different kinds of source, including degraded DNA from crime scenes, or archaeological sources
• In vivo

  • low error rate due to cellular error - correcting mechanisms
  • DNA is made in cells, so can be expressed
  • Needs intact pure DNA
• PCR

  1. DNA Polymerase and many of the 4 nucleotides are added to the thermocycler
  2. Heat to 95C, to break the hydrogen bonds
  3. Then cool to below 70C, and add primers, these anneal
  4. Heat to 72C, to bind the two strands, replicating the DNA
• Primers - short single stands of DNA which are complementary to ends of the DNA strands to be copied. Primers join to the single strands to stop the strands reannealing; and also to allow the DNA polymerase to bind and join nucleotides together.