1. Presentation on genome sequencing

2. Genome: the complete set of gene of an organism Genome annotation: the process by which the genes, control sequences and other interesting features are identified. First DNA completely sequenced: (5386 bp) bacteriophage ØX174 in 1975. SV40 virus: (5243 bp) in 1977. pBR322: (4363 bp) in 1978. The first chromosome sequence: chromosome III of the yeast Saccharomyces cerevisiae, in 1992, and was completed in 1996.

3. Why genome sequencing is required? GENOMICS POST GENOMICS BIOINFORMATICS ANALYSIS e.g. GENE LOCATION, CONTROL SEQUENCES FUNCTION OF UNKNOWN GENE SEQUENCE DATA CORRECT ASSAMBLY OF GENE SEQUENCES COMPUTETRIZED ASSAMBLY, EXAMINATION AND PREDICTION OF GENES LARGE AMOUNT OF DATA STORAGE

4. TWO BASIC APPROACHES FOR GENOME SEQUENCING

5. Genomic DNA Fragments Cloned into a vector Each fragment sequenced using DNA sequencing method Re-construction of genome ATGCCGTAATCGGATCAGTC

6. Clone contig method Shotgun method Isolation and sequencing of one overlapped clone from library. Hybridization with second clone Identification of second clone. Hybridization of second clone with 3 rd clone of library Randomly generated DNA fragments Cloning into vector Sequencing of each clone Check for overlapping Assembling of overlapped sequences Chromosome walking

8. The shotgun approach: the genome is randomly broken into short fragments. The resulting sequences are examined for overlaps and these are used to build up the contiguous genome sequence. Used for smaller genome. first used successfully with the bacterium Haemophilus influenzae The clone contig approach: involves a pre-sequencing phase a series of overlapping clones is identified. This contiguous series is called a contig. Each piece of cloned DNA is then sequenced, and this sequence placed at its appropriate position on the contig map in order to gradually build up the overlapping genome sequence.

9. GENOMIC/CHROMOSOMAL LIBRARY COMPLEMENTARY DNA (cDNA) LIBRARY RECOMBINENT DNA LIBRARIES

10. Genomic Library Complete digestion Mechanical shearingPartial digestion 1.Produces a large number of short DNA clones. 2.Genes containing two or more restriction sites may be cloned in two or more pieces. 1.Produces longer DNA fragments. 2.Ends are not uniform, requires enzymatic modification before fragments can be inserted into a cloning vector. 1.Cut at a less frequent restriction site and limit the amount and time the enzyme is active. 2.Results in population of large overlapping fragments. 3.Selected by agarose electrophoresis. 3 WAYS TO MAKE GENOMIC LIBRARY Set of recombinant clones that contains all the DNA present in an individual organism

12. 1.cDNA is derived from mature mRNA, does not include introns. 2.cDNA may contain less information than the coding region. 3.cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue and with time). 4.Creating a cDNA library: 1.Isolate mRNA 2.Synthesize cDNA 3.Clone cDNA 1.cDNA is derived from mature mRNA, does not include introns. 2.cDNA may contain less information than the coding region. 3.cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue and with time). 4.Creating a cDNA library: 1.Isolate mRNA 2.Synthesize cDNA 3.Clone cDNA cDNA Library:

13. Fig. 8.15, Synthesis of cDNA

14. Vectors that enable artificial chromosomes to be created and cloned into yeast. Features: 1.Yeast telomere at each end. 2.Yeast centromere sequence. 3.Selectable marker (amino acid dependence, etc.) on each arm. 4.Autonomously replicating sequence (ARS) for replication. 5.Restriction sites (for DNA ligation). 6.Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments. Vectors that enable artificial chromosomes to be created and cloned into yeast. Features: 1.Yeast telomere at each end. 2.Yeast centromere sequence. 3.Selectable marker (amino acid dependence, etc.) on each arm. 4.Autonomously replicating sequence (ARS) for replication. 5.Restriction sites (for DNA ligation). 6.Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments. Yeast artificial chromosome (YAC)

15. Vectors that enable artificial chromosomes to be created and cloned into E. coli. Features: 1.Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors. 2.Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects. 3.Like other vectors, BACs contain: 1.Origin (ori) sequence derived from an E. coli plasmid called the F factor. 2.Multiple cloning sites (restriction sites). 3.Selectable markers (antibiotic resistance). Vectors that enable artificial chromosomes to be created and cloned into E. coli. Features: 1.Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors. 2.Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects. 3.Like other vectors, BACs contain: 1.Origin (ori) sequence derived from an E. coli plasmid called the F factor. 2.Multiple cloning sites (restriction sites). 3.Selectable markers (antibiotic resistance). Bacterial artificial chromosome (BAC)