1. DNA Sequencing Techniques

2. DNA Sequencing Methods (1977)
Alan Maxam and Walter Gilbert
Chemical protocol
Fred Sanger
Enzymatic procedures
Chain termination
Dideoxynucleotide method

3. Dideoxynucleotide Human-made molecule (Figure 5.14) dideoxynucleotide
If a dideoxynucleotide is incorporated at the end of the growing chain, DNA synthesis stops. (Figures 5.15 & 5.16)

4. Chain Termination by Dideoxynucleotide

5. Chain Termination Sequencing Procedure
Annealing a primer (17- to 24-mer) to the ss DNA
Partition into 4 tubes
Tube 1: ddATP, dNTP, DNA pol
Tube 2: ddCTP, dNTP, DNA pol
Tube 3: ddGTP, dNTP, DNA pol
Tube 4: ddTTP, dNTP, DNA pol
(--- One of dNTP is radiolabeled.)
The reactions are stopped by formamide
Poly acrylamide gel electrophoresis
Autoradiograph
Sequence determination (Figure 5.18)
About 250 to 350 bands can be resolved clearly.

6. Chain Terminator Sequencing

7. Automated DNA Sequencing
Labeling ddNTPs with four different fluorescence dyes
4-color, 1-lane detection
Run samples in one lane of polyacrylamide or polymer-filled capillary tube
Labeling with one fluorescence dye
Labeling primer
Labeling ddNTP
1-color, 4-lane detection
Detection of the fluorescence by an argon ion laser beam at the bottom of the electrophoretic matrix
Automated sequencing systems can read with high accuracy about 500 bases per run.
Under optimal conditions, one instrument can resolve about 20,000 bases per hour.

8. Automated DNA Sequencing

9. Using Bacteriophage M13 as a DNA Sequencing Vector
ss circular DNA (+ strand)
dsDNA replicative form after infection
Release of phage containing ssDNA without cell lysis
M13 as a cloning vector
dsDNA replicative form : handled as a plasmid
ssDNA : used as a template for sequencing

10. Use of bacteriophage M13 as a cloning and DNA sequencing vector

11. Sequencing of Large DNA
Primer walking
Designing serial sequencing primers based on the previous sequence data

12. PCR

13. PCR: Polymerase Chain Reaction
Amplification of a specific DNA fragment
Kary Mullis
Invented PCR in 1983
Nobel Prize in Chemistry in 1993

16. Essential Components for PCR
two primers (~20 nucleotides each)
DNA template (100 to ~35,000 bp)
Thermostable DNA polymerase
~ Taq DNA polymerase (Thermus aquatics)
dNTP
Three Steps
Denaturation
~ at 95oC for 1 min
Renaturation (annealing)
~ slowly cooled to ~55oC
Synthesis (extension)
~ raised to ~75oC (optimum temperature for Taq
DNA polymerase rxn)

21. Gene Synthesis by PCR

23. Cycle Sequencing PCR-based generation of ddNTP terminated fragment
Using single primer
 linear amplification (rather than exponential
amplification)