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DNA Restriction Anaysis

DNA Restriction Anaysis. DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus. Model of DNA DNA is Comprised of Four Base Pairs. Deoxyribonucleic Acid (DNA). O. O. P. O. Base. O. O. CH 2. Sugar. O. O. P. O. Base. O. O. CH 2. Sugar. OH. DNA Schematic.

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DNA Restriction Anaysis

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  1. DNA Restriction Anaysis

  2. DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus

  3. Model of DNADNA is Comprised of Four Base Pairs

  4. Deoxyribonucleic Acid(DNA)

  5. O O P O Base O O CH2 Sugar O O P O Base O O CH2 Sugar OH DNASchematic 5’ phosphate 3’ hydroxyl

  6. DNARestriction Enzymes • Evolved by bacteria to protect against viral DNA infection • Endonucleases = cleave within DNA strands • Over 3,000 known enzymes

  7. Enzyme Site Recognition Restriction site Palindrone • Each enzyme digests (cuts) DNA at a specific sequence = restriction site • Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Fragment 2 Fragment 1

  8. 5 vs 3 Prime Overhang Enzyme cuts • Generates 5 prime overhang

  9. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

  10. The DNA DigestionReaction • Restriction Buffer provides optimal conditions • NaCI provides the correct ionic strength • Tris-HCI provides the proper pH • Mg2+ is an enzyme co-factor

  11. DNA DigestionTemperature Why incubate at 37°C? • Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? • Too hot = enzyme may be denatured (killed) • Too cool = enzyme activity lowered, requiring longer digestion time

  12. AgaroseElectrophoresisLoading • Electrical current carries negatively-charged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel Power Supply

  13. AgaroseElectrophoresisRunning • Agarose gel sieves DNA fragments according to size • Small fragments move farther than large fragments Gel running Power Supply

  14. Analysis of Stained Gel Determine restriction fragment sizes • Create standard curve using DNA marker • Measure distance traveled by restriction fragments • Determine size of DNA fragments • Identify the related samples

  15. Molecular Weight Determination Fingerprinting Standard Curve: Semi-log Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0

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