DNA Separation Techniques

1. Lysis

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   • Cell lyses with Sodium dodecyl sulfate (detergent that disrupt membrane and denature proteins)
   1. Purification

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      • 2 possible ways to do it. Protein precipitation: A high salt content solution is added to precipitate the protein due to the hydrophobic effect. Protein will be grouped together and after centrifuging, the supernatant will be the purified DNA. Phenol/Chloroform: 3 layers will be form with DNA in the top layer.
      1. Test of Purity

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         • A measure of how pure the supernatant is. DNA/RNA absorb UV light best at 260nm while protein does best at 280nm. A ratio (A260/A280) greater than 1.8 indicates that DNA/RNA sample is pure.
      2. Precipitation

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         • Add organic solvent as DNA is a ionic compound (phosphate backbone is negatively charged) DNA will precipitate after centrifuge. Alternatively, just cool it down to extreme cold temperatures.

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• Used to separate DNA or other molecules. The bromophenolblue is used as a dye to "smear" plus glycerol makes it denser. The process takes about 45 minutes. Used ethidium or gel red and irradiated with UV light to see glow. In alternative, use a camera.

1. Factors

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   • Affected by size shape and charge. Smaller molecules travel further Compact molecules travel further Negative charge travel further.

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• Exponential amplification of a defined sequence of DNA. Visualized on the agarose gel. Oligonucleotides provide the specificity for the reaction.

1. Properties

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   • a) Low pH: At low pH the purine (2 ringed, A,G) bases are released. Used in chemical sequencing of DNA b)High pH: DNA is resistant and RNA will hydrolyse due to the extra OH side. c) DNA is also very long and weak, quite susceptible to shearing. d) DNA with higher G/C content is stronger than the one with A/T due to the extra hydrogen bonds

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• Enzymes degrade nucleic acids with chemical specificity. 1. DNase or Rnase 2.DNase: Single stranded, Double stranded or both. 3. Either act on the end or in the strand itself.

1. Exonucleases

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   • Depending on the nuclease, it will chew off from one end and it will cleave one nucleoside at a time.
2. Endonucleases

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   • Cleave from within. Depends on the enzyme how to cut it will determine how it cleave
   1. Restriction Enzymes

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      • Type II enzymes that act on unique  genetic sequence, remember, read from 5' to 3'.  Some recognise 4 base pairs other recognise 6. Arrows indicate cleavage sites. Needs to be palindromic.
      1. Applications

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         • With usage of DNA ligase. We cans splice DNA into plasmids and put it into bacteria as recombinant bacteria.

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• Arranged in chromatin bacteria as a nucleosome core and it did it in a spiral. In a eukaryotic cell it arranges in chromatids while bacteria it arranged in loops.