1. Bioinformatics Lecture By: Ms AQSAD RASHDA
Genome Sequencing

2. Genome Sequencing Main steps Application of restriction enzymes
Electrophoresis to separate DNA into fragments
Southern blot technique
PCR
Cloning of DNA
Automatic DNA Sequencing

3. Restriction enzymes Sequence specific Endonucleases
Cut DNA at specific sequences
First discovered in E.choli
Protect bacteria from foriegn DNA
ReBase:Restriction enzyme data base
Eco RI
Palindromic sequences
Fingerprints of DNA

4. Electrophoresis Separates DNA strands by size Gel Charge
Speed of molecules
Distance covered by DNA fragment
distance ∞ 1/length

5. Southern blot technique
Identification of specific DNA pattern
DNA is cut with restriction enzymes
Separated by electrophoresis
DNA is denatured
Blotted on nitrocellulose membrane
Hybridized with Radio labeled probe
Radio labeled DNA detected by radiography

6. PCR Amplification of small initial amount of DNA Ingredients:
Template DNA
Polymerase enzyme
Primers
PCR Buffer
dNTPs

7. PCR Process: 20-30 CYCLES Denaturation 92-96°C Annealing 40-55°C
Extension °C
20-30 CYCLES

8. Cloning of DNA
Isolation of donor and vector DNA Both DNA digested by same endonuclease
Ends joined by Ligase
Recombinant vector inserted in the host
Replication of host Cell

9. Cloning of DNA

10. DNA Sequencing Chain termination DNA sequencing Dideoxy nucleotides
Difference b/w deoxynucleotides and Dideoxy nucleotides
Dideoxy nucleotides terminate replication process
Electrophoresis for separation of different fragments of DNA
Full content of DNA determined by reading the lengths of terminated DNA strands.

11. Difference b/w deoxynucleotides and Dideoxy nucleotides

12. DNA Sequencing