Bioinformatics Lecture By: Ms AQSAD RASHDA

1. BioinformaticsLecture 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: • Denaturation 92-96°C • Annealing 40-55°C • Extension 70-72°C • 20-30 CYCLES

8. Cloning of DNA • Isolation of donor and vector DNABoth 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