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Sanger sequencing 
Dr. R. D. Kulkarni, MD 
Dept. of Microbiology, SDM College of 
Medical Sciences & Hospital, Dharwad - 
580009
One of the 
greatest 
women who 
ever walked on 
earth..!
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
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
S 
P 
Base 
5 
3 
Nucleotide
Principle 
• Utililizes 2',3'-dideoxynucleotide triphosphate 
(ddNTPs) 
• Are different from dNTPs at the 3’carbon
Difference
dd NTPs 
• Specially designed 
nucleotides 
• Have a hydrogen 
atom attached to 
the 3' carbon 
rather than an OH 
group
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
ddNTPs are the terminator molecules…
ddNTPs are the terminator molecules… 
• dd ATP • dd GTP 
• dd CTP • dd TTP
• 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
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
• 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
• 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
dNTP
• ddNTP - random 
addition 
• Free permutation 
and combination
Electrophoresis
Computerized alignment
Compress all in one plain
Electropherogram
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
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
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
Acinetobacter PCR runs
99 to 100% homology with Acinetobacter
Rockefeller University, New York
Rockefeller University, New York
Rockefeller University, New York
Temple of Science …
Temple of Science …
Temple of Science …
Temple of Science …
- 130C on day 1
Not to impress that I received brief training 
here…. 
• My dear youngsters 
• Places like this should 
be your dream 
destination…!

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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..!
  • 3.
  • 4.
  • 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
  • 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
  • 14.
  • 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
  • 19.
  • 20. dNTP
  • 21. • ddNTP - random addition • Free permutation and combination
  • 22.
  • 23.
  • 24.
  • 25.
  • 28.
  • 29.
  • 30. Compress all in one plain
  • 31.
  • 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
  • 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…!