DNA SEQUENCING:

FIRST GENERATION

Presented by:

Alizza Rose Ferrer ;

Nicci Rosietess Madarcos;
Gael Sastre;
Lejanni Tuiza
What is DNA Sequencing?

sequencing methods
for determining the
order of the
nucleotide bases -
adenine, guanine,
cytosine, and
thymine - in a
molecule of DNA
Applications:
a. diagnostic
Applications:
b. biotechnology
Applications:
c. forensic biology
Applications:
d. biological systematics
 How it started?
DISCOVERY: What accounts for the delay?
DNA double helix (1953)

(a) Chemical Properties of different DNA are

similar.

15 years! (b) Chain Length: DNA > proteins

(c) Existence of only four bases

First Experimental (d) No known specific DNAases

Determination of a DNA
Sequence (1968)
Early RNA sequencing
Saccharomyces cerevisiae tRNA: first nucleic acid
molecule sequenced by Holley, et al in 1965
RNAases already developed = protein sequencing
models for the structure deduced by assuming base pairing

Discovery of Type II restriction

enzymes by Hamilton Smith, et al
in 1970
Sequencing Methods:
First Generation

(1) Chain Terminator Method: Sanger and Coulson (1977)

First Generation
(1) Chain Terminator Method: Sanger and Coulson (1977)
1. Denaturation: +NaOH

2. Isolated ssDNA solution mixed

with: (a) Primer, (b) DNA
polymerase, (c) dNTPs, (d) ddNTPs
First Generation

(1) Chain Terminator Method: Sanger and Coulson (1977)

First Generation
(1) Chain Terminator Method: Sanger and Coulson (1977)
1. Denaturation: +NaOH

2. Isolated ssDNA solution mixed

with: (a) Primer, (b) DNA
polymerase, (c) dNTPs, (d) ddNTPs

3. Repeat process with other

ddNTPs

4. Gel electrophoresis

5. X-ray autoradiography
First Generation

(1) Chain Terminator Method: Sanger and Coulson (1977)

G
C
A
T
G
T
A
A
T
First Generation
(2) Maxam and Gilbert Method: Maxam and Gilbert (1977)

1. Obtain ssDNA

2. Add a 32P to 5 end

3. Cleave at specific nucleotides by

adding chemical reagents

Guanine: dimethyl sulfate + piperidine

Guanine + Adenine: dimethyl sulfate +
piperidine in Formic acid
Thymine + Cytosine: hydrazine + piperidine
Cytosine: hydrazine + piperidine in 1.5M NaCl

4. Electrophoresis thru acrylamide

gels
First Generation
(2) Maxam and Gilbert Method: Maxam and Gilbert (1977)

A
T
G
C
A
T
C
First Generation
(3) Automated Sanger Sequencing: Caltech (1986)
First Generation
(3) Automated Sanger Sequencing: Caltech (1986)
First Generation

THANK YOU!!
References:
Guzvic, M. 2013. The History of DNA Sequencing. J Med Biochem. (32)
301-312.
Heather, H.M and Chain, B. 2015. The Sequence of Sequencers: The
History of Sequencing DNA. Genomics (107) 1-8.
http://dx/doi.org/10.1016/j.ygeno.2015.11.003
Karger, B.L. and Guttman, A. 2009. DNA Sequencing by Capillary
Electophoresis. Electrophoresis. 30(Suppl 1): S-196-S202. doi:
10.1002/elps.200900218.
Maxam, A.M., and Gilbert, W. 1976. A new method for sequencing DNA.
Proc. Natl. Acad. Sci. Vol. 72, No. 2.pp. 560-564.
Sanger, F., Nicklen, S. and Coulson, A. 1977. DNA Sequencing with chain
terminating inhibitors. Proc. Natl. Acad. Sci. Vol. 74, No. 12. pp. 5463-
5467.