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Introduction to Bioinformatics

Introduction to Bioinformatics. Molecular Biology Tools. Cut and Paste. Restriction enzyme Protein cuts double-stranded DNA molecules at a specific string of nucleotides called restriction site Example Eco RI 5'-GAATTC-3'

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Introduction to Bioinformatics

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  1. Introduction to Bioinformatics Molecular Biology Tools

  2. Cut and Paste • Restriction enzyme • Protein cuts double-stranded DNA molecules at a specific string of nucleotides called restriction site • Example EcoRI • 5'-GAATTC-3' • Restriction site occurs randomly in DNA sequences only once every 4nbase pairs  once every 4,096 base pairs for this one • Sticky ends and blunt ends • Sticky ends (as shown by the red lines) • Blunt ends (some enzymes cut it straight) • E.g., AluI cuts 5’-AGCT-3’  5’-AG CT-3’ • A sticky end can paste to a complementary sticky end

  3. Cut and Paste • What’s special about the site? • More examples • 5'-GATC-3' • 5'-GCGGCCGC-3' • They are palindromes! • Useful for • Restriction mapping • Isolation and experimental manipulation of individual genes for the very first time

  4. Sorting • Gel electrophoresis • Molecular biology tool separating fragments of different size from each other • Load DNA (or RNA or protein) fragments into wells at one end of gel • Apply electric field across the gel, DNA (and RNA) with its negatively charged phosphate backbone is drawn toward the positively charged electrode • Small molecules move easier through the gel than larger ones  larger molecules closer to the wells • Molecules are sorted on the basis of their size! • Roberts and Sharp will tell us how cutting and sorting work …

  5. Copy • Large quantity of a specific DNA fragment required for analysis • Cloning  not fast enough • Quicker way? • PCR (Kary Mullis, 1993 Nobel Prize, Chemistry) • Polymerase Chain Reaction • 3 steps/cycle • Denaturalization of double-stranded sequence • Annealing of primers • Extension by heat-stable polymerase • # doubled every cycle  grows exponentially! • Animation 4 – 5 minutes per cycle

  6. DNA Sequencing • Determining the linear order of nucleotides in the DNA molecule • Sanger • As shown • Animation • Shotgun • Will be discussed

  7. Chip and Parallel Processing • GeneChip ® • Animation • Microarray • Animation

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