Curtin University of Technology School of Biomedical Sciences MOLECULAR GENETICS 331

1. Curtin University of TechnologySchool of Biomedical SciencesMOLECULAR GENETICS 331 Lecture 12 “Genome Analysis 1”

2. Genomics • Structural genomics • Characterising physical nature of whole genomes. • Functional genomics • Characterising the proteome and overall patterns of gene expression.

3. Gemonics Web SitesPublic • NCBI • National Centre for Biotechnology Information • USA • www.ncbi.nlm.nih.gov • Tigr • Institute for Genomic Research • USA • www.tigr.org • Sanger Centre • UK • www.sanger.ac.uk/

4. GenomicsPrivate • Celera Science • Www.celera.com/celerascience/ • The Microsoft of biotechnology • CEO • Craig Ventor • The Bill Gates of genomics

5. Bacteria Complete Genomes Taxonomy / List 28 Aquifex aeolicus NC_000918 1551335 bp Jul 25 1997 Bacillus subtilis NC_000964 4214814 bp Nov 20 1997 Borrelia burgdorferi NC_001318 910724 bp Dec 17 1997 Campylobacter jejuni NC_002163 1641481 bp Feb 10 2000 Chlamydia muridarum NC_002178 1069412 bp Mar 1 2000 Chlamydia trachomatis NC_000117 1042519 bp May 20 1998 Chlamydophila pneumoniae NC_000922 1230230 bp Dec 1 199 NC_002491 1228267 bp Jul 17 2000 Chlamydophila pneumoniae AR39 NC_002179 1229858 bp Mar 1 2000 Deinococcus radiodurans NC_001263 2648638 bp Nov 8 199 NC_001264 412348 bp Nov 2 1999 Escherichia coli NC_000913 4639221 bp Oct 13 1998 Haemophilus influenzae Rd NC_000907 1830138 bp Jul 25 1995 Helicobacter pylori 26695 NC_000915 1667867 bp Aug 6 1997 Helicobacter pylori J99 NC_000921 1643831 bp Jan 12 1999 Mycobacterium tuberculosis NC_000962 4411529 bp Jun 11 1998 Mycoplasma genitalium NC_000908 580074 bp Oct 30 1995 Mycoplasma pneumoniae NC_000912 816394 bp Nov 15 1996 Neisseria meningitidis MC58 NC_002183 2272351 bp Feb 25 2000 Neisseria meningitidis Z2491 NC_002203 2184406 bp Mar 30 2000 Rickettsia prowazekii NC_000963 1111523 bp Nov 12 1998 Synechocystis PCC6803 NC_000911 3573470 bp Mar 21 1997 Thermotoga maritima NC_000853 1860725 bp Jun 1 1999 Treponema pallidum NC_000919 1138011 bp Mar 6 1998 Ureaplasma urealyticum NC_002162 751719 bp Jan 10 2000 Vibrio cholerae NC_002505 2961149 bp Jun 14 2000 NC_002506 1072315 bp Jun 14 2000 Xylella fastidiosa NC_002488 2679306 bp Jun 2 2000 Revised August 4, 2000

6. Archaea Complete Genomes Taxonomy / List 6 Aeropyrum pernix NC_000854 1669695 bp Jun 19 1999 Archaeoglobus fulgidus NC_000917 2178400 bp Dec 17 1997 Methanobacterium thermoautotrophicum NC_000916 1751377 bp Nov 17 1997 Methanococcus jannaschii NC_000909 1664970 bp Jan 30 1998 Pyrococcus abyssi NC_000868 1765118 bp Jun 12 1999 Pyrococcus horikoshii NC_000961 1738505 bp Jun 19 1998 Revised August 4, 2000

7. Eukaryotae Genomes Taxonomy / List [ 5] Arabidopsis thaliana chromosomes: II, IV, [ 6] Caenorhabditis elegans chromosomes: I, II, III, IV, V, X, [ 5] Drosophila melanogaster chromosomes: 1, 2, 3, 4, Y, [24] Homo sapiens chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, Y, [36] Leishmania major chromosomes: 1, [21] Mus musculus chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, X, Y, [12] Oryza sativa chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, [14] Plasmodium falciparum chromosomes: 2, 3, [16] Saccharomyces cerevisiae chromosomes: I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, [10] Zea mays chromosomes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

8. DNA Sequencing • Standard method • Dideoxy chain terminator method • Fred Sanger 1981

9. The structure of 2’, 3’-dideoxy nucleotides

11. DNA Sequence ladder

12. Old method used incorporation of radio labelled nucleotides. • New automated methods use fluorescent labelled dideoxy nucleotides

15. Printout from an automatic sequencer that uses fluorescent dyes

16. Problem • DNA sequencing can only read up to 1000 bases.

17. Size Matters

18. Sequencing a genome is like putting together a jig saw puzzle, where you don’t have a picture.

19. Two Approaches • Hierarchial approach • Find out what the picture is • Fit pieces based on where they are in to picture • Random “shot gun” approach. • Just try and join the bits together.

20. Hierarchical Approach

21. Cut individual chromosomes into large fragments

22. Clone chromosomal fragments into Bacterial Artificial Chromosome (BAC) vectors RE map and order BAC clones

23. Break individual BAC clones into smaller fragments subclone into M13 for sequencing

24. Once sequence of individual BAC clone is complete. Join up all BAC sequences  complete chromosome.

25. Hierarchical Approach • Advantages • Know where each sequence • More conventional approach • Disadvantages • A huge amount of work mapping and aligning BAC clones • Ie, producing the jig saw “picture”

26. Shot Gun Approach

27. Clone random 2 kbp and 10 kbp fragment into plasmid vector

28. Sequence 500 bp of the ends of the 2 kbp and 10 kbp fragments 2 kbp 10 kbp

29. Align 2 kbp sequences Use 10 kbp sequences as reference

30. Random approach • Take genomic DNA • Randomly fragment • Sequence individual fragments • Use a computer to look for overlapping sequence • Assemble into contigs • Clone gaps

31. Advantage • No need to waste time mapping • Cut straight to the chase • More readily automated • Disadvantage • Sequence some areas may times • Have gaps (non-sequenced regions) that need to be filled • Problem if lots of repetitive DNA • Where does it fit