5. Manual DNA sequencing
1- DNA preparation:
- High quality
- At least two microgram DNA
- The more the merrier
2- Single strand DNA is ready to go, double strand DNA needs denaturation:
- Heat and annealing with the primer (on ice-water)
- Using one micro of 1N NaOH and neutralize with HCl
Plasmid Phagemid
M13
3- Labeling, DNA polymerase, little dNTPs, Radioactive dATP (S35, P33, P32)
4- Prepare 4 tubes, each contain dNTP plus one of the ddNTP, label the tubes
AGC or T. Transfer one fourth of the mixture from (3) to the A tube, one
fourth to the G tube, one fourth to the C tube and one fourth to the T tube
6. A tube
AGCT
A
DNA polymerase
5- Stop the reaction in the four tubes using loading buffer containing
formamide, heat up and load the four reaction in adjacent four lanes on
polyacrylamide gel that contain 50% urea
6- Run the gel at high voltage, dry, expose to X-ray film and develop the film
Have a nice reading
7. 1-DNA amount and quality. Columns, impurities
2-Double Strand DNA, full denaturation, excess primer
3-Single stand DNA, too many steps in preparation
4-PCR products, fast re-annealing of the fragment, excess primer, fast
annealing
A G C T
5-GC compression: Secondary structure in the
newly synthesized strand
Gel temperature
Nucleotides analogues, 7-diaza
8. A G C T
6-Secondary structure in the template
Extension temperature (40 T7 polymerase)
Chasing with dNTP and terminal transferase
7-gel problems:
Smiling
Gel compression at the top
A G C T
Aluminum plates
Gradient gel
Gradient buffer
9. - Four reaction in four separate tubes
- Very low amount of DNA (0.1 picomole)
1picomole of double strand in ng = size of DNA x 0.66
- Linear increase of the newly synthesized DNA
-Less sensitivity to impurities
-Full denaturation of DNA
-Less secondary structure in the template DNA
