Use of molecular biology in environmental toxicology

1
Use of molecular biology in environmental
toxicology

• Joe Staton
• University of South Carolina,
• Columbia

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Overview

• Classical methods
• Molecular methodology
• Applications to toxicological studies

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What are the new moleculartechnologies?
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Tracking a chosen gene

• DNA cloning and sequencing (E. coli)
• Polymerase chain reaction (PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Expression (bacterial) vectors
• In situ hybridization

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Methods for finding new genes

• cDNA libraries
• Expressed sequence tags (ESTs)
• Microarrays
• Subtractive libraries
• Serial Analysis of Gene Expression
• Genome Projectshigh-throughput sequencing
• Gene chips

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Good candidate genes? Considerations

• Population marker?
• Direct effect on enzyme?
• Main link in pathway?
• Indicator of organism life history?

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Getting the data...

• Introduction to Polymerase Chain Reaction and DNA
  Sequencing

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Polymerase Chain Reaction(PCR)1993 Nobel prize
in Chemistry to Kerry Mullis
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Recovering DNA
Conserved DNA Unknown DNA Conserved DNA
5-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3 3
-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5
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Recovering DNA
Conserved DNA Unknown DNA Conserved DNA
5-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3 3
-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5
GTC CAG
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Making Primers
Primer a 5-CGTCGGATGTA-3 5-CGTCGGATGTAA
GAGACTCTCACAAACGCTCCGATCGGCGT-3 3-GCAGCCTACATTCT
CTGAGAGTGTTTGCGAGGCTAGCCGCA-5
3-GCTAGCCGCA-5
Primer b
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Temperature Profile of PCR
Melting
94
? Temp increasing ( C)
72
Extension
50
Annealing (stringency)
One cycle
Time increasing?
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Mechanics of PCR
Cycle 1
Melt - 94?C
Anneal - 45-60?C
Extend - 72?C
Repeat 30 times
Cycle 30
1.07x109 copies!
Primers
Synthesized DNA
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Net effect of PCR
What Goes In
What Comes Out
Total DNA PCR primers dNTPs (A,C,G,T) DNA
Polymerase Buffer, etc.
What went in 1 Billion Copies of the Amplified
Fragment
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1
1
2
3
Larger Fragments
2
Smaller Fragments
3
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DNA Sequencing

• Sanger random termination method (enzymatic)

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Model of the chemistry-- ddATP
G A T C T G G G C T A C T C G G G C G T
C G C A
A G C C C G C A
A T G A G C C C G C A
A C C C G A T G A G C C C G C A
A G A C C C G A T G A G C C C G C A
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Demo of the autosequencer
ddATP ddGTP ddCTP ddTTP
Origin
G
C
C
A
G
T
G
C
A
G
C
T
G
G
A
FINISH!
G
C
T
A
G
T
C
G
A
C
T
A
T
C
C
T
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Demo of the autosequencer
ddATP ddGTP ddCTP ddTTP
Origin
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Demo of the autosequencer
1 2 3 4
Origin
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Actual gel image
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Tracked gel image
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Single scanned sequence
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How does RT-PCR differ?

• Uses RNA as a start template
• No introns
• Can quantify amount
• Uses a retroviral enzyme to make cDNA from RNA
• PCR as usual

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What if you want to measure protein, too?
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Use antibodies

• Purified protein from subjects injected into
  vertebrate (e.g., rabbits)
• Rabbits produce antibodies
• Antibodies from purified from rabbit blood
• Used for detection of proteins (ELISA, in situ
  hybridization, etc.)

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What if protein is difficult to get?

• Get RNA for gene
• Put in a genetically engineered plasmid
  (expression vector)
• Use bacteria to generate protein in super-large
  quantities
• Inject into vertebrate host to create antibodies

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Tracking multiple genes to whole genomes

• Prospects for the future or Gordian Knot?

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Multigene technologies

• cDNA libraries
• Expressed sequence tags (ESTs)
• Microarrays (hybridization)
• Subtractive libraries
• Serial Analysis of Gene Expression (SAGE)
• Genome Projectshigh-throughput sequencing
• Gene chips (hybridization)

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ESTs and Microarrays

• Make cDNA library of tissues of interest
• Sequence all unique cDNAs and identify
• Create unique DNA fragments for each gene
• Bind cloned fragments onto a solid support (e.g.,
  nylon filter)
• Label RNA from test subject and bind to DNA array
• Use brightness of color at each spot as data

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Microarray mechanics
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What if you are unsure about the genes affected?
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Subtractive libraries

• Concept control cDNAs block tester cDNAs from
  amplification in PCR
• Use will amplify only differentially expressed
  cDNAs in a reaction, which can then be used for
  in situ hybridization, microarrays, etc.

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Serial Analysis of Gene Expression (SAGE)

• Measures amount of mRNA products in a tissue
• Compare levels in control vs. test organisms
• Relate treatment to specific genes or suites of
  genes

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Step 1 Isolating mRNA fragments
e.g., AE NlaIII
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Step 2 Add linkers to each half for reaction
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Step 3 Release from beads with TE (BsmFI)
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Step 3 Increasing amount of product
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Step 4 Purify and clone
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Sequencing clones

• Sequence many DITAGS
• 26-bp units
• 23 to 30 DITAGS per clone
• Sequence 1000-1500 clones!
• Normalize data and perform statistical analysis

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Microarrays vs. SAGE

• Chip-less
• Large post-sequencing effort
• Digital data of copy number in library

• Must have developed chip
• Large pre-sequencing effort
• Analog data of relative binding to site

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Genome projects

• Method Brute force cloning and sequencing of
  entire genome of an organism
• Use whole genome microarray or gene chip

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Drawbacks

• Expensive
• Limited organisms
• Bacteria (Escherichia coli)
• Virus (Lambda)
• Fly (Drosophila melanogaster)
• Round worm (Caenorhabditis elegans)
• Cabbage relative (Arabidopsis thaliana)
• Frog (Xenopus laevis)
• Human (Homo sapiens)