DNA Sequencing

DNA sequencing … ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…

DNA sequencing Determination of nucleotide sequence Two similar methods: 1. Maxam and Gilbert method 2. Sanger method They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined

Maxam and Gilbert Method The single stranded DNA fragment to be sequenced is end- labeled by treatment with alkaline phosphatase to remove the 5’phosphate It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base 1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified

Frederick Sanger Discovered DNA sequencing by chain termination method Nobel Prize 1 (1958) –Complete amino acid sequence of insulin Nobel Prize 2 (1980) –For DNA sequencing

Sanger Method DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system Incorporation of dideoxynucleotides into growing strand terminates synthesis Synthesized strand sizes are determined for each dideoxynucleotide rxn by using gel or capillary electrophoresis

Dideoxynucleotide no hydroxyl group at 3’ end prevents strand extension CH2 O OPPP 5’ 3’ BASE

Dideoxy nucleotides Incorporation of a dideoxynucleotide to growing DNA strand terminates its further extension Are added in small proportion –dATPddATP –dGTPddGTP –dCTPddCTP –dTTPddTTP

Chain Termination

Chain Terminator Basics Target Template-Primer Extend ddA ddG ddC ddT Labeled Terminators ddA AddC ACddG ACGddT TGCA dN : ddN 100 : 1

CCGTAC 3’ 5’ 3’ primer dNTP ddATP GGCA ddTTP GGCAT ddCTP GGCG ddGTP G GGCATG A T C G

All Possible Terminations

Polyacrylamide Gel Electrophoresis Separates fragments based on size

Electrophoresis

DNA Sequencing – vectors + = DNA Shake DNA fragments Vector Circular genome (bacterium, plasmid) Known location (restriction site)

Different types of vectors VECTORSize of insert Plasmid 2,000-10,000 Can control the size Cosmid40,000 BAC (Bacterial Artificial Chromosome) 70, ,000 YAC (Yeast Artificial Chromosome) > 300,000 Not used much recently

DNA Sequencing – gel electrophoresis 1.Start at primer (restriction site) 2.Grow DNA chain 3.Include dideoxynucleoside (modified a, c, g, t) 4.Stops reaction at all possible points 5.Separate products with length, using gel electrophoresis

Template ssDNA vectors –M13 –pUC PCR dsDNA (+/- PCR)

Primers Universal primers parallel –cheap, reliable, easy, fast, parallel –BULK sequencing Custom primers –expensive, slow, one-at-a-time –ADAPTABLE

Extension Chemistry Polymerase –Sequenase –Thermostable (Cycle Sequencing) Terminators –Dye labels (“Big Dye”) spectrally different, high fluorescence –ddA,C,G,T with primer labels

Separation Gel Electrophoresis Capillary Electrophoresis –suited to automation rapid (2 hrs vs 12 hrs) re-usable simple temperature control 96 well format

Sequencing Strategies Ordered –Divide and Conquer Random Sequence –Brute Force

DNA Sequencing 5.17)

Sequencing of DNA by the Sanger method