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