Methods in DNA Sequencing Big Picture Largescale 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 • 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

Sample: Chain Termination Output

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

Sample: Dye Sequencing Output

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