Bioinformatics Lecture By: Ms AQSAD RASHDA Genome Sequencing

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

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

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

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

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

PCR Process: 20-30 CYCLES Denaturation 92-96°C Annealing 40-55°C Extension 70-72°C 20-30 CYCLES

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

Cloning of DNA

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.

Difference b/w deoxynucleotides and Dideoxy nucleotides

DNA Sequencing