1. Genome Analysis Determine locus & sequence of all the organism’s genes More than 100 genomes have been analysed including humans in the Human Genome Project Genome = total DNA of an organism

2. DNA Sequencing Chromosome Physical and Genetic Mapping

3. Techniques used Restriction digestion Ligation (place DNA fragments into plasmids) Gene cloning (to create clone library)

4. Step 1 : Cutting Up Chromosomes Fragment ≈ 150 000 - 1 000 000 base pairs 1 3 2 5 4 6 Human chromosome ≈ 50-280 million base pairs Restriction enzymes

5. Step 2: Creating a Clone Library vector Each large fragment is inserted into a separate vector (yeast or bacterial plasmid) using DNA ligation. Cells cloned to produce a clone library (each fragment in a separate culture). cells Plasmids placed in cells

6. Step 3: Mapping the Fragments Tagged fragments multiplied by PCR AA A EEE C F F F B B D DNA marker A Fragment 1 A Gel electrophoresis  low resolution map of the chromosome Made by analysing overlaps

7. Do you remember the statigraphic columns we studied in year 10? We used a similar strategy to sequence the different layers. Where have you seen this sort of sequencing before?

8. Mapping Fragments can be compared to map the genes Process repeated to make smaller fragments Small fragments mapped to determine base sequence

9. Step 4: Creating a Sub-Clone Library Large fragments cut into smaller pieces (1500-5000 bp) and process repeated  sub-clone library. 1 a b c d e f e e e e e

10. Step 5: Sequencing the Fragments Small fragments sequenced to find the exact order of the bases

11. 2 3 4 5 6 Step 6: Mapping the Chromosome fragment sequence assembled on the chromosome map

12. EXTENSION

13. Human Genome Project (HGP) Begun in 1990 Sequence 2m of DNA 3Gb (3 billion base pairs)

14. Human Genome Project $6 billion 1000 scientists 50 countries

15. HGP Goals Identify all coding genes Determine all base pair sequences Store information in databases. Improve tools for data analysis. Technologies for private sector Ethical, legal, and social issues

16. Some Benefits of the HGP  Understand genetic disorders such as Huntington’s disease, cystic fibrosis, the most common form of skin cancer, and breast cancer.  Development of a new therapeutic drugs.  Design of new molecules to specifically block metabolic pathways that lead to disease.  Development of gene therapy procedures for all genetically determined diseases.

17. Other species Yeast, E.coli, various crop foods To evaluate their potential for use by humans.

18. Some Definitions genome genome = entire DNA content of the cell gene gene = segment of genome transcribed into RNA genome analysis genome analysis = determining the exact base sequence in an organism's genome to determine position of each gene on the genome mtDNA mtDNA = mitochondrial DNA

19. Understanding the function of genes and other parts of the genome is known as functional genomics. Functional Genomics

20. Comparison of genomes from different species  understanding of: how species have evolved (using mtDNA) the function of genes the function of non coding regions of the DNA Comparative Genomics 2 gene clusters control the position of developing structures in the embryo - the same genes (or homologous ones) are found is all animals, including humans. Gene cluster 1: (anterior)Gene cluster 2: (posterior)

21. Knockout Gene Mouse Knock-out mice are used extensively to determine the function of a specific gene. Photo: Lexicon Genetics Inc Photo: Maggie Bartlett, NHGRI A knockout mouse model of obesity (left), compared with a normal mouse (right). In these mice, a single gene is disabled, leaving other genes unaffected. Such mice provide an ideal way to determine a gene’s function. Right: Laboratory mouse in which a gene affecting hair growth has been knocked out, left, next to a normal lab mouse.

22. Chromosome (physical) Mapping Single, whole chromosomes are isolated and probes are used to create low resolution maps of genes and markers. Useful in analyzing observable physical traits associated with chromosomal abnormalities (i.e. translocations, inversions, and deletions). DNA probes mixed with the chromosome to see what part of the chromosome it binds to.

23. Closely linked markers are less likely to be separated during chromosome rearrangement. The more closely linked genes are, the more likely they are to be inherited together Genetic Linkage Maps Children MotherFather Gene or Polymorphism Linkage distance Gene or Polymorphism A 1% crossover frequency = approx. 10Mb

24. Contiguous Fragment Maps Contiguous fragment maps (contigs) are assembled by overlapping fragments and arranging them so their markers overlap STS markers YACs Contig Map