Title: Use of molecular biology in environmental toxicology
1Use of molecular biology in environmental
toxicology
- Joe Staton
- University of South Carolina,
- Columbia
2Overview
- Classical methods
- Molecular methodology
- Applications to toxicological studies
3What are the new moleculartechnologies?
4Tracking a chosen gene
- DNA cloning and sequencing (E. coli)
- Polymerase chain reaction (PCR)
- Reverse Transcriptase PCR (RT-PCR)
- Expression (bacterial) vectors
- In situ hybridization
5Methods for finding new genes
- cDNA libraries
- Expressed sequence tags (ESTs)
- Microarrays
- Subtractive libraries
- Serial Analysis of Gene Expression
- Genome Projectshigh-throughput sequencing
- Gene chips
6Good candidate genes? Considerations
- Population marker?
- Direct effect on enzyme?
- Main link in pathway?
- Indicator of organism life history?
7Getting the data...
- Introduction to Polymerase Chain Reaction and DNA
Sequencing
8Polymerase Chain Reaction(PCR)1993 Nobel prize
in Chemistry to Kerry Mullis
9Recovering DNA
Conserved DNA Unknown DNA Conserved DNA
5-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3 3
-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5
10Recovering DNA
Conserved DNA Unknown DNA Conserved DNA
5-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3 3
-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5
GTC CAG
11Making Primers
Primer a 5-CGTCGGATGTA-3 5-CGTCGGATGTAA
GAGACTCTCACAAACGCTCCGATCGGCGT-3 3-GCAGCCTACATTCT
CTGAGAGTGTTTGCGAGGCTAGCCGCA-5
3-GCTAGCCGCA-5
Primer b
12Temperature Profile of PCR
Melting
94
? Temp increasing ( C)
72
Extension
50
Annealing (stringency)
One cycle
Time increasing?
13Mechanics 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
14Net 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
151
1
2
3
Larger Fragments
2
Smaller Fragments
3
16DNA Sequencing
- Sanger random termination method (enzymatic)
17Model 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
18Demo 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
19Demo of the autosequencer
ddATP ddGTP ddCTP ddTTP
Origin
20Demo of the autosequencer
1 2 3 4
Origin
21Actual gel image
22Tracked gel image
23Single scanned sequence
24How 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
25What if you want to measure protein, too?
26Use 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.)
27What 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
28Tracking multiple genes to whole genomes
- Prospects for the future or Gordian Knot?
29Multigene technologies
- cDNA libraries
- Expressed sequence tags (ESTs)
- Microarrays (hybridization)
- Subtractive libraries
- Serial Analysis of Gene Expression (SAGE)
- Genome Projectshigh-throughput sequencing
- Gene chips (hybridization)
30ESTs 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
31Microarray mechanics
32What if you are unsure about the genes affected?
33Subtractive 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.
34Serial 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
35Step 1 Isolating mRNA fragments
e.g., AE NlaIII
36Step 2 Add linkers to each half for reaction
37Step 3 Release from beads with TE (BsmFI)
38Step 3 Increasing amount of product
39Step 4 Purify and clone
40Sequencing clones
- Sequence many DITAGS
- 26-bp units
- 23 to 30 DITAGS per clone
- Sequence 1000-1500 clones!
- Normalize data and perform statistical analysis
41Microarrays 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
42Genome projects
- Method Brute force cloning and sequencing of
entire genome of an organism - Use whole genome microarray or gene chip
43Drawbacks
- 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)