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Use of molecular biology in environmental toxicology

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Use of molecular biology in environmental toxicology. Joe Staton ... Cabbage relative (Arabidopsis thaliana) Frog (Xenopus laevis) Human (Homo sapiens) ... – PowerPoint PPT presentation

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Title: Use of molecular biology in environmental toxicology


1
Use of molecular biology in environmental
toxicology
  • Joe Staton
  • University of South Carolina,
  • Columbia

2
Overview
  • Classical methods
  • Molecular methodology
  • Applications to toxicological studies

3
What are the new moleculartechnologies?
4
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

5
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

6
Good candidate genes? Considerations
  • Population marker?
  • Direct effect on enzyme?
  • Main link in pathway?
  • Indicator of organism life history?

7
Getting the data...
  • Introduction to Polymerase Chain Reaction and DNA
    Sequencing

8
Polymerase Chain Reaction(PCR)1993 Nobel prize
in Chemistry to Kerry Mullis
9
Recovering DNA
Conserved DNA Unknown DNA Conserved DNA
5-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3 3
-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5
10
Recovering DNA
Conserved DNA Unknown DNA Conserved DNA
5-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3 3
-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5
GTC CAG
11
Making Primers
Primer a 5-CGTCGGATGTA-3 5-CGTCGGATGTAA
GAGACTCTCACAAACGCTCCGATCGGCGT-3 3-GCAGCCTACATTCT
CTGAGAGTGTTTGCGAGGCTAGCCGCA-5
3-GCTAGCCGCA-5
Primer b
12
Temperature Profile of PCR
Melting
94
? Temp increasing ( C)
72
Extension
50
Annealing (stringency)
One cycle
Time increasing?
13
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
14
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
15
1
1
2
3
Larger Fragments
2
Smaller Fragments
3
16
DNA Sequencing
  • Sanger random termination method (enzymatic)

17
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
18
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
19
Demo of the autosequencer
ddATP ddGTP ddCTP ddTTP
Origin
20
Demo of the autosequencer
1 2 3 4
Origin
21
Actual gel image
22
Tracked gel image
23
Single scanned sequence
24
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

25
What if you want to measure protein, too?
26
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.)

27
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

28
Tracking multiple genes to whole genomes
  • Prospects for the future or Gordian Knot?

29
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)

30
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

31
Microarray mechanics
32
What if you are unsure about the genes affected?
33
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.

34
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

35
Step 1 Isolating mRNA fragments
e.g., AE NlaIII
36
Step 2 Add linkers to each half for reaction
37
Step 3 Release from beads with TE (BsmFI)
38
Step 3 Increasing amount of product
39
Step 4 Purify and clone
40
Sequencing clones
  • Sequence many DITAGS
  • 26-bp units
  • 23 to 30 DITAGS per clone
  • Sequence 1000-1500 clones!
  • Normalize data and perform statistical analysis

41
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

42
Genome projects
  • Method Brute force cloning and sequencing of
    entire genome of an organism
  • Use whole genome microarray or gene chip

43
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)
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