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Introduction to Bioinformatics: Molecular Biology Tools and Techniques

This comprehensive guide introduces essential tools used in bioinformatics and molecular biology. Key techniques include the use of restriction enzymes, which cut double-stranded DNA at specific nucleotide sequences known as restriction sites. For example, EcoRI recognizes the palindrome 5'-GAATTC-3'. The guide also explores gel electrophoresis for fragment separation based on size, cloning methods, and the Polymerase Chain Reaction (PCR) for DNA amplification. Learn about sequencing techniques, including Sanger and shotgun methods, and their applications in genomic analysis.

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Introduction to Bioinformatics: Molecular Biology Tools and Techniques

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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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