Applications of Recombinant DNA Technology

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Applications of Recombinant DNA Technology
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There are many different applications of DNA
technology

• 1) Human disease gene mapping
• Identifying genes with heritable variations that
  are associated with diseases of all kinds
• 2) Prenatal and Presymptomatic testing
• Developing probes for specific genetic
  variations that have been shown to be associated
  with diseases
• 3) DNA fingerprinting for forensics and paternity
  testing
• Harnessing the wealth of genetic variations held
  in repeat sequences to identify individuals

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• 4) Mapping the Human Genome
• Combining cloning, DNA sequencing, and computing
  technologies to sequence 3 billion base pairs
• 5) Gene therapy
• Utilizing what we know about the genomes of
  bacterial and viral vectors to place specific
  genes in specific cell types
• 6) Gene expression profiling
• Creating microchips with 1000s of gene
  sequences on them to monitor gene expression
  patterns in cells and tumors

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A1 A1 A1 A2 A1 A3 A2 A2 A2 A3
A3 A3
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How do you suppose that people found these
variations?
And what were they good for? Hint Think back to
Mendel, Bateson, and Punnet but think from a
medical perspective
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• How do you suppose that people found these
  variations?
• They screened lots of people with lots of probes
  and restriction enzymes
• And what were they good for?
• Linkage analysis and finding chromosomal regions
  that segregate with disease phenotypes.

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Historical Aside How do you think people
figured out which genes were on which
chromosomes?
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• They used what information they had
• For example, sometimes they were lucky to have a
  family that had the disease because of a
  chromosomal abnormality (usually a small but
  noticeable deletion)
• Then when they found genes that were linked to
  the disease they knew they were near that
  chromosomal location
• Then they looked for other genetic variation
  that was linked to the previous locus.
• Now we have cool techniques like...
• Fluorescence in situ hybridization
• A technique involving labeling probes with
  fluorescent dyes and then hybridizing them to
  metaphase chromosomes.

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http//www.ornl.gov/hgmis/graphics/slides/images3.
html
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The Human Genome Project
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What is the Human Genome Project?

• U.S. govt. project coordinated by the Department
  of Energy and the National Institutes of Health
• Goals (1998-2003)
• Identify the approximate 100,000 genes in human
  DNA
• Determine the sequences of the 3 billion bases
  that make up human DNA
• Store this information in databases
• Develop tools for data analysis
• Address the ethical, legal, and social issues
  that arise from genome research

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Why is the Department of Energy involved?
- After atomic bombs were dropped during War War
II, Congress told DOE to conduct studies to
understand the biological and health effects of
radiation and chemical by-products of all energy
production - Best way to study these effects is
at the DNA level
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Whose genome is being sequenced?

• The first reference genome is a composite genome
  from several different people
• Generated from 10-20 primary samples taken from
  numerous anonymous donors across racial and
  ethnic groups

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Benefits of HGP Research

• Improvements in medicine
• Microbial genome research for fuel and
  environmental cleanup
• DNA forensics
• Improved agriculture and livestock
• Better understanding of evolution and human
  migration
• More accurate risk assessment

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Ethical, Legal, and Social Implications of HGP
Research

• Fairness in the use of genetic information
• Privacy and confidentiality
• Psychological impact and stigmatization
• Genetic testing
• Reproductive issues
• Education, standards, and quality control
• Commercialization
• Conceptual and philosophical implications

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For More Information...
Human Genome Project Information
Website http//www.ornl.gov/hgmis
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http//www.ornl.gov/hgmis/graphics/slides/images3.
html
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http//www.ornl.gov/hgmis/graphics/slides/3lgenoba
.jpg
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Microarray Technologies
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http//www.nhgri.nih.gov/DIR/LCG/15K/HTML/images/a
rrayer_jpeg.jpg
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The robotic setup includes 1) Glass slides
arranged in rows (about 5-10 per row) 2) The set
of cloned cDNA fragments to be put on the slides
(1000s of genes means lots of microtiter
dishes) 3) A robotic arm 4) A really good
computer 5) A really good program that directs
the process
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http//www.medsch.ucla.edu/som/humgen/cores_dna.ht
m
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http//www.medsch.ucla.edu/som/humgen/cores_dna.ht
m
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http//genome-www.stanford.edu/molecularportraits/
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From Lipshutz et al Nature Genetics Supplement
Vol 21, 1999
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From Lipshutz et al Nature Genetics Supplement
Vol 21, 1999.
The chip consists of square features (cells,
probes) about 24x24 ?m for chips we use. They
come in perfect match (PM) - mismatch (MM) pairs
(probe pairs). 20 probe pairs per gene is
typical for expression arrays. We might expect
1,000,000 molecules of probe/feature. The
sequences are typically tiled with 1 bp overlaps
rather than spaced as shown here.
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Gene expression profile of 55 samples based on
2518 genes. Red positive Green negative Black
zero
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Clustering result from 2518 genes based on 75th
percentile expression gt200 and 50 of samples
with present call, using 50 knots for normalizing
the raw data.
W well M moderate P poor
1 stage 1 3 stage 3
CC clear cell PA papillary LA large cell NE
neuroendocrine MP mucinous/papillary BA
bronchioloalveolar BD bronchial-derived ACA BM
BD/mucinous MB mucinous/MA BB BA/BD
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data stage and cluster X-square 3.1766, df
1, p-value 0.0747
data differentiation and cluster X-square
11.9501, df 2, p-value 0.0025
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N Observed Expected (O-E)2/E
(O-E)2/V cluster1 23 13 6.5
6.51 10.9 cluster2 32 4 10.5
4.03 10.9 Chisq 10.9 on 1 degrees of
freedom, p 0.00097
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N Observed Expected (O-E)2/E (O-E)2/V
stage1 35 6 11.62 2.72
8.79 stage3 20 11 5.38 5.87
8.79 Chisq 8.8 on 1 degrees of freedom, p
0.00302
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