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Genome Analysis

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. Chromosome. Physical and Genetic Mapping. DNA Sequencing. Techniques used. Restriction digestion

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Genome Analysis

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  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. Chromosome Physical and Genetic Mapping DNA Sequencing

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

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

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

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

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

  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 b e a d c f e Large fragments cut into smaller pieces (1500-5000 bp) and process repeated  sub-clone library. 1 e e e e

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

  11. Step 6: Mapping the Chromosome 4 6 3 5 2 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 = entire DNA content of the cell • gene = segment of genome transcribed into RNA • genome analysis = determining the exact base sequence in an organism's genome to determine position of each gene on the genome • mtDNA = mitochondrial DNA

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

  20. Comparative Genomics Gene cluster 1: (anterior) Gene cluster 2: (posterior) 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 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.

  21. Knockout Gene Mouse • Knock-out mice are used extensively to determine the function of a specific gene. Right: Laboratory mouse in which a gene affecting hair growth has been knocked out, left, next to a normal lab mouse. 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. Photo: Lexicon Genetics Inc

  22. DNA probes mixed with the chromosome to see what part of the chromosome it binds to. 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).

  23. Gene or Polymorphism Genetic Linkage Maps Father Mother Gene or Polymorphism Linkage distance A 1% crossover frequency = approx. 10Mb Children • 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

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

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