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Methods in DNA Sequencing

Methods in DNA Sequencing. Big Picture. Large-scale sequencing requires DNA to be broken into fragments Cutting (with enzymes) Shearing (with mechanical forces) DNA is duplicated into a vector Individually sequenced Assembled electronically Shotgun sequencing. Brief Bio Background.

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Methods in DNA Sequencing

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  1. Methods in DNA Sequencing

  2. Big Picture • Large-scale sequencing requires DNA to be broken into fragments • Cutting (with enzymes) • Shearing (with mechanical forces) • DNA is duplicated into a vector • Individually sequenced • Assembled electronically • Shotgun sequencing

  3. Brief Bio Background • Nucleotides • Components in DNA, consists of 3 portions: • Nitrogenous base (Adenine, Guanine, etc.) • Sugar • Phosphate Primer -Strand of nucleic acid -Serves as starting point for DNA replication

  4. Brief Bio Background • Polymerase • DNA polymerase can add free nucleotides • No known DNA polymerase is able to begin a new chain • Ligase • Links together DNA fragments

  5. DNA Extraction/Prep • Break open cells • grinding • lysine • Remove cellular proteins and lipids • Detergent • Precipitate DNA • alcohol • DNA is insoluble in alcohol • Add Primer

  6. Chain Termination • Sanger Method • Uses a DNA template, primer, polymerase, and fluorescent nucleotides • DNA sample fragments separated into four lanes • One for each nucleotide (A, T, G, C) • DNA bands are then visualized by UV light, • Positions of the different bands used to read the DNA sequence

  7. Sample: Chain Termination Output

  8. Dye Sequencing • Four different labels • Each of the four nucleotide chains has a different dye • Individual dyes fluoresce at unique wavelengths • Vast majority of sequencing projects • easier • cheaper

  9. Sample: Dye Sequencing Output

  10. Sequencing by Ligation • Ligase identifies the nucleotide • instead of polymerase • doesn’t create a second strand • Ligase joins probe sequences • produces a fluorescence. • Based on the fluorescence one can infer the identity of the nucleotide

  11. 2-Base Encoding • Construct library of Probes • Small fragments representing two bases • Combination results in sixteen unique probes • Each fluoresces at a different wavelength • Sequencing Reaction • 2-base encoding is based on sequencing by ligation • Decoding Data • Remember each color indicates two bases • Need to know one of the bases in the sequence

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