Biology 340 Molecular Biology
Lecture 12
Genomics
Reading: Chap. 8 pp. 274-281
Positional Cloning: Use of recombinant DNA techniques to locate the gene
responsible for a genetic disease based on its chromosomal location.
Four steps to positional cloning:
(1) Identify cloned segment of DNA near gene of interest.
(2) Isolation of a contiguous stretch of DNA and construction of a physical map
of DNA in the region.
(3) Locate the possible site of the gene of interest.
(4) Detect the change in DNA sequence between mutant and wild type versions
of the gene.
1. Approaches to identify chromosomal region near gene:
RFLPs: Restriction fragment length polymorphisms: difference in length of a
restriction fragments in DNA of individual with disease vs. individuals without
disease.
FISH: Fluorescence in situ hybridization. (Radioactive tag=in situ hybridization)
Use of tagged probe to locate region of gene on chromosome.
Somatic cell hybrids: When human cells and rodent cells are fused, most human
chromosomes are lost. To map genes to human chromosomes, a series of lines
are constructed representing different subsets of human chromosomes. DNA is
isolated from the lines and digested for gel electrophoresis. Southern
hybridization of the DNA to a RFLP or tentative gene probe can localize a gene
to a particular chromosome.
2. Isolation of a region of DNA in a relevant location:
Chromosome walking: Isolation of a series of overlapping genomic clones
representing a region of a particular chromosome. Fig. 8-24. Starting clone is
used to isolate a series of genomic clones. Restriction mapping identifies
regions of overlap. To continue the search in one direction or the other a probe
derived from one end of a genomic clone is used for rescreening. It is important
to chose probes that lack repetitive DNA elements.
Chromosome jumping: Technique developed by Francis Collins (director of the
human genome project) that allows a series of overlapping clones to be obtained
while skipping over some repetitive regions of the genome. Involves ligation of
the two distant ends of a genomic clone to derive a probe.
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Sequence tagged sites: cDNAs that are derived from unique regions of the
genome. These represent parts of individual genes that haven't been completely
mapped out yet. These can form major landmarks along stretches of DNA. Long
stretches of contiguous DNA can be assembled from overlapping BACs
(bacterial artificial chromosomes) or YACs (yeast artificial chromosomes).
3. Locate the possible site of the gene of interest.
Use physical markers along the DNA to locate the region of the gene. Physical
markers can include known genes and RFLPs.
4. Detect the change in DNA sequence between mutant and wild type
versions of the gene.
Above approaches may not be able to distinguish between several genes in
close proximity to determine which is responsible for a genetic disease. To
detect difference:
Use Southern analysis on mutant vs. normal DNA: may observe difference in
size of hybridizing fragments.
Northern analysis may uncover differences in mRNA expression (in sizes
produced or tissues in which it is found) between mutant and normal gene.
Point mutation detection methods (Fig. 8-28) for large or small fragments.
Method for analyzing small fragments=SSCP, single-stranded conformation
polymorphisms.
5. Bioinformatics tools for genome analysis.
NCBI Web page for human genome data and analysis.
Tutorial for bioinformatics.
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