Chapter 13

Nucleic Acid Biotechnology Techniques

Restriction Endonucleases

  • recognize short DNA sequences and cleave DNA phosphodiester bonds

  • produce specific double-strand fragments containing terminal 5’-phosphate

    • common in bacteria: eliminates infectious viral DNA

    • make staggered cuts: sticky ends

    • make straight cuts: blunt ends

  • Type I

    • large, multi-subunit protein

    • ATP- and SAM-dependent

    • demethylase activity

    • cleave DNA at random sites (far from recognition sequence)

  • Type II

    • cleave DNA within or at short specific distances from recognition site

    • Not ATP- or SAM-dependent

    • smaller, less complex than Type I or Type III

    • found most used in creating recombinant DNA molecules

      • commercially available

  • Type III

    • large, multi-subunit protein

    • ATP-dependent and SAM activates

    • cleave DNA at random sites (near recognition sequence)

  • Type IV

    • target only methylated DNA

DNA Sequencing

  • Sanger Method: generation of DNA fragments (length depends on last base)

    • accomplished by controlled interruption of in vitro enzymatic synthesis DNA

  • Requirements: DNA, Primer, DNA polymerase, mix of 2’-deoxynucleoside 5’-triphosphates and 2’,3’-dideoxynucleoside 5’-triphosphates

  • DNA Sequencing Process

    • four separate sequencing reactions are carried out

      • concentration of 2’,3’-dideoxynucleoside is low so chain termination is not absolute (only occasionally)

    • four sequencing reactions are incubated for a defined time

      • allow DNA pol to copy template strand

      • generates set of DNA fragments of differing lengths

        • terminating with dideoxynucleotide

    • separate fragments and read sequence

Polymerase Chain Reaction

  • Applications: archaeology, cancer, cloning, embryology, forensics, hereditary and infectious disease, molecular phylogeny, paleontology, virology

  • developed by Kary Mullis 1983

    • 3 steps: denaturing(94ºC), annealing(Tm:5ºC 45-60ºC), extension(72ºC)

  • amplify DNA in test tube

    • regions of interests within linear DNA or complete circular plasmids

  • mix together

    • target DNA, primers, nucleotides, thermostable DNA pol

  • place mixture into thermocycler

    • melt DNA, cool separated strands, primers anneal to target, polymerase extends primers in 5’-3’ direction, after a round of elongation repeat steps

Example question:

  1. Sanger sequencing logic in the Sanger method for DNA sequencing, a small amount of a dideoxynucleotide triphosphate, ddCTP, is added to the sequencing reaction along with a larger amount of the corresponding dCTP. What result would be observed if the dCTP were omitted? If dCTP is omitted from the reaction mixture when the first G residue is encountered in the template, ddCTP is added and polymerization halts. Only one band will appear in the sequencing gel.