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DNA Sequencing.ppt
- 3. 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
- 4. 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
- 5. 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
- 6. 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
- 7. Dideoxynucleotide no hydroxyl group at 3’ end prevents strand extension CH2 O O PPP 5’ 3’ BASE
- 8. Dideoxy nucleotides • Incorporation of a dideoxynucleotide to growing DNA strand terminates its further extension • Are added in small proportion – dATP ddATP – dGTP ddGTP – dCTP ddCTP – dTTP ddTTP
- 10. Chain Terminator Basics Target Template-Primer Extend ddA ddG ddC ddT Labeled Terminators ddA AddC AC ddG ACG ddT TGCA dN : ddN 100 : 1
- 11. CCGTAC 3’ 5’ 5’ 3’ primer dNTP ddATP GGCA ddTTP GGCAT ddCTP GGC G ddGTP GG GGCATG A T C G
- 14. Electrophoresis
- 15. DNA Sequencing – vectors + = DNA Shake DNA fragments Vector Circular genome (bacterium, plasmid) Known location (restriction site)
- 16. Different types of vectors VECTOR Size of insert Plasmid 2,000-10,000 Can control the size Cosmid 40,000 BAC (Bacterial Artificial Chromosome) 70,000-300,000 YAC (Yeast Artificial Chromosome) > 300,000 Not used much recently
- 17. 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
- 18. Template • ssDNA vectors – M13 – pUC • PCR • dsDNA (+/- PCR)
- 19. Primers • Universal primers – cheap, reliable, easy, fast, parallel – BULK sequencing • Custom primers – expensive, slow, one-at-a-time – ADAPTABLE
- 20. Extension Chemistry • Polymerase – Sequenase – Thermostable (Cycle Sequencing) • Terminators – Dye labels (“Big Dye”) • spectrally different, high fluorescence – ddA,C,G,T with primer labels
- 21. Separation • Gel Electrophoresis • Capillary Electrophoresis – suited to automation • rapid (2 hrs vs 12 hrs) • re-usable • simple temperature control • 96 well format
- 22. Sequencing Strategies • Ordered – Divide and Conquer • Random Sequence – Brute Force
- 24. Sequencing of DNA by the Sanger method