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

A A A

E E E

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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