Sanger sequencing
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A presentation of how to sequence DNA. Basics of Sanger Sequencing. All animations are created by me. Photographs and graffiti is taken from internet.
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Sanger sequencing
- 1. Sanger sequencing Dr. R. D. Kulkarni, MD Dept. of Microbiology, SDM College of Medical Sciences & Hospital, Dharwad - 580009
- 2. One of the greatest women who ever walked on earth..!
- 5. Sanger sequencing • Frederick Sanger • British biochemist • Recipient of the Nobel Prize TWICE • 1958 - structure of proteins, Insulin • 1980 - determination of base sequences in nucleic acids
- 6. Sanger sequencing • Sequencing - Method used to sequence the stretches of the gens • Precisely write the sequence of the nucleotides as they are arranged in the stretch of the DNA
- 7. S P Base 5 3 Nucleotide
- 8. Principle • Utililizes 2',3'-dideoxynucleotide triphosphate (ddNTPs) • Are different from dNTPs at the 3’carbon
- 9. Difference
- 10. dd NTPs • Specially designed nucleotides • Have a hydrogen atom attached to the 3' carbon rather than an OH group
- 11. dd NTPs • They terminate DNA chain elongation as… – Cannot form a phosphodiester bond with the next deoxynucleotide • Each ddNTP has label for different color fluorescence
- 12. ddNTPs are the terminator molecules…
- 13. ddNTPs are the terminator molecules… • dd ATP • dd GTP • dd CTP • dd TTP
- 15. • In the actual reaction mixture of dNTPs and ddNTPs used • Proportion of the dNTPs and ddNTPs 100 : 1 • Means that 1% of the ddNTPs are fluorescence labeled terminator molecules
- 16. Technique • Gene to be sequenced subjected to standard PCR • Amplicon will have number of copies of the gene under question • Now, sequencing PCR is run using this amplicon as template • To run this reaction we add – Buffer – The template i.e. amplicon from the first PCR – Primer – BigDye – Polymerase + mixture of ddNTPs and dNTPs – Double distilled water • Amplification done
- 17. • Elongation of strand • Random addition of dNTPs and ddNTPs • When ddNTP added strand extension terminates • Being random - all possible lengths of the DNA fragments are generated having a terminal fluorescence labeled ddNTP • Product of this reaction is subjected to gel electrophoresis
- 18. • If ddNTP is added early the fragment will be short • If ddNTP is added late, fragments will be longer • Shorter fragments have faster mobility vis-à-vis the bigger fragments • Run gel electrophoresis • Image the gel • Peaks are generated using software to arrange the nucleotides based on the fragment length and the fluorescence labels • Electropherogram
- 20. dNTP
- 21. • ddNTP - random addition • Free permutation and combination
- 26. Electrophoresis
- 30. Compress all in one plain
- 32. Electropherogram
- 33. Purification of DNA before sequencing PCR • DNA amplicon purified before subjecting sequencing PCR • Purification through Sephadex gel – Filter plate – Add Sephadex powder – Add water to obtain gel – Centrifuge to remove excess water (2700 rpm x 5 mins) – Add amplicon to wells – Centrifuge to collect DNA into catch plate (having cone shaped wells) – Impurities like ddNTPs, dNTPs, enzymes etc. are caught in Sephadex – Filtrate is pure DNA - used for sequencing
- 34. Before fluorescent labels • Radio labeled ddNTPs were used before the use of fluorescent ddNTPs • Reaction was run in quadruplet. • Each of the four reactions was run with separate mixtures of dNTPs and ddNTP – ddGTP / ddCTP / ddATP and ddTTP with dNTPs • After reaction the electrophoresis was run in four different lanes and the matching was done using radiographic reading of the lanes • Final assembly using algorithms
- 35. Acinetobacter PCR at SDM • Genus level • Scola et al – France • 100% specificity • But band at 400 bp rather than at 350 bp as suggested by Scola • Discussed with Dr. Scola on Emial – it is highly specific do not bother about the size of the fragment – but get the amplicon sequenced • We got it sequenced • To our surprise – on sequencing it was 350 bp
- 37. 99 to 100% homology with Acinetobacter
- 38. Rockefeller University, New York
- 39. Rockefeller University, New York
- 40. Rockefeller University, New York
- 41. Temple of Science …
- 42. Temple of Science …
- 43. Temple of Science …
- 44. Temple of Science …
- 45. - 130C on day 1
- 46. Not to impress that I received brief training here…. • My dear youngsters • Places like this should be your dream destination…!