Introduction to Bioinformatics Molecular Biology Tools.
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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 EcoRI –5'-GAATTC-3' –Restriction site occurs randomly in DNA sequences only once every 4 n base 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
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
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 …how cutting and sorting work
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! –AnimationAnimation 4 – 5 minutes per cycle