Genome Analysis
description
Transcript of 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
DNA Sequencing
Chromosome
Physical and Genetic Mapping
Techniques used
• Restriction digestion • Ligation (place DNA fragments into
plasmids)• Gene cloning (to create clone library)
Step 1 : Cutting Up Chromosomes
Fragment ≈ 150 000 - 1 000 000 base pairs
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Human chromosome ≈ 50-280 million base pairs
Restriction enzymes
Step 2: Creating a Clone Library
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).
Plasmids placed in cells
Step 3: Mapping the Fragments Tagged fragments multiplied by PCR
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DNA marker A
Fragment 1A
Gel electrophoresis low resolution map of the chromosome
Made by analysing overlaps
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?
Mapping
• Fragments can be compared to map the genes
• Process repeated to make smaller fragments• Small fragments mapped to determine base
sequence
Step 4: Creating a Sub-Clone Library
Large fragments cut into smaller pieces (1500-5000 bp) and process repeated sub-clone library.
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Step 5: Sequencing the Fragments
Small fragments sequenced to find the exact order of the bases
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Step 6: Mapping the Chromosome
fragment sequence assembled on the chromosome map
EXTENSION
Human Genome Project (HGP)
Begun in 1990
• Sequence 2m of DNA
• 3Gb (3 billion base pairs)
Human Genome Project
$6 billion
1000 scientists
50 countries
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
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.
Other species
Yeast, E.coli, various crop foods
To evaluate their potential for use by humans.
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
Understanding the function of genes and other parts of the genome is known as functional genomics.
Functional Genomics
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)
Knockout Gene MouseKnock-out mice are used extensively to determine the function of a specific gene.
Photo: Lexicon Genetics Inc
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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.
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.
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 orPolymorphism
Linkage distance
Gene orPolymorphism
A 1% crossover frequency = approx. 10Mb
Contiguous Fragment Maps
Contiguous fragment maps (contigs) are assembled by overlapping fragments and arranging them so their markers overlap
STS markers
YACs
Contig Map