Sanger

Sequencing
A Brief Introduction &
Explanation
DNA
 Blueprint of life
 Chemical building blocks= Nucleotides
 Nucleotides

i. Phosphate
ii. Sugar
iii. Nitrogen Base (A, T, G, C)
 Sequence of base=biological
instruction.
Structure
History of Sequencing
 Watson & Crick identified DNA double-helix
structure.
 Walter Fieserr= RNA sequencing of
Bacteriophage MS2
 Maxam & Gilbert developed chemical
degradation method
 Sanger developed di-deoxy chain termination
method
 Automation in DNA sequencing by Applied
Biosystems
Maxam-Gilbert Method

 Denature DNA to
get ssDNA

 32
P as 5’ phosphate

 Cleavage using
chemicals

 Electrophoresis

 Radiography
Why Obsolete?
 Time Consuming

 Quite Problematic (Too many steps)

 200-300 bases of DNA every few days

 Working with radioactive material

 More complicated than Sanger

Sanger Sequencing
 Usingthird form of a Ribose Sugar
(Dideoxyribose)

 Ribose has OH group on 2’ and 3’ carbon

 Deoxyribose has O removed from 2’

carbon.

 Dideoxyribose has O removed from 2’ and

3’carbons.
 ddNTPs are labeled with fluorescent dyes
 Fragments readable through laser light in capillary
gels
Conclusion
 15-20% of DNA molecules need to contain the mutation to
be reliably detected by Sanger. (Rohlin et al., 2009)

 Sanger is being replaced by NGS (Mu et al., 2016)

 NGS technologies have lowered the cost and effort of DNA

sequencing

 NGS employs massively parallel sequencing of millions of

DNA fragment simultaneously

 However, Sanger is the gold standard for short fragments

(Totomoch et al., 2017) & NGS is the future
References
Rohlin, A., Wernersson, J., Engwall, Y., Wiklund, L., Björk, J., & Nordling,
M. (2009). Parallel sequencing used in detection of mosaic mutations:
Comparison with four diagnostic DNA screening techniques. Human
Mutation, 30(6), 1012-1020. doi:10.1002/humu.20980

Mu, W., Lu, H., Chen, J., Li, S., & Elliott, A. M. (2016). Sanger
Confirmation Is Required to Achieve Optimal Sensitivity and Specificity
in Next-Generation Sequencing Panel Testing. The Journal of Molecular
Diagnostics, 18(6), 923-932. doi:10.1016/j.jmoldx.2016.07.006

Totomoch-Serra, A., Marquez, M. F., & Cervantes-Barragán, D. E.

(2017). Sanger sequencing as a first-line approach for molecular
diagnosis of Andersen-Tawil syndrome. F1000Research, 6, 1016.
doi:10.12688/f1000research.11610.1