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