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
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
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
