Genomic TechnologiesCIT581N
Michael Kane, Ph.D.
Lecture 1: Sequencing Technologyand DNA Microarray Technology
Where do we get DNA sequence information?
DNA Sequencing Methods-conversion of biological/bioanalytical data into sequence information
There are automated, high-throughput sequencing centers that COMPLETELY automate (robotics and information systems) DNA sequencing, preliminary identification and publishing.
A G C T
5’-AAACCAGGCCGATAAGGTACTACACGAAAAAAA-3’
dATPdCTPdTTPdGTP
+ddATP32
ddCTP32
ddTTP32
ddGTP32 TTTGGTCCGGCTATTCCATGATGTGCTTTTTTTTTGGTCCGGCTATTCCATGATGTGCTTTTTTT
TGGTCCGGCTATTCCATGATGTGCTTTTTTTGGTCCGGCTATTCCATGATGTGCTTTTTTT
GTCCGGCTATTCCATGATGTGCTTTTTTTTCCGGCTATTCCATGATGTGCTTTTTTT
CCGGCTATTCCATGATGTGCTTTTTTTCGGCTATTCCATGATGTGCTTTTTTT
GGCTATTCCATGATGTGCTTTTTTTGCTATTCCATGATGTGCTTTTTTT
CTATTCCATGATGTGCTTTTTTTTATTCCATGATGTGCTTTTTTT
ATTCCATGATGTGCTTTTTTT
Step 1. Extend complementary sequence using “free” nucleotides with limiting amounts of radioactive “terminating” nucleotides.
Step 2. Run product out on a electrophoresis gel.
Step 3. Place gel against radiographic film, develop.
TTTTTTT
AAACCAGGCCGATAAGGTACTACACGAAAAA | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
DNA Sequencing (old method)
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
DNA Sequencing new method)
DNA Sequencing new method)
Phred quality score
Probability that the base is called wrong
Accuracy of the base call
10 1 in 10 90%
20 1 in 100 99%
30 1 in 1,000 99.9%
40 1 in 10,000 99.99%
50 1 in 100,000 99.999%
Ewing et al.1998b, Genome Research 8:186-94
DNA Microarray Technology:A Technical Perspective
From: Duggan et.al. Nature Genetics 21:10-14, 1999
Microarray-Based Assays (The Basics)
Each feature or “spot” represents a specific expressed gene (mRNA).
The fluorescent intensity of each feature correlates with the expression level of the gene (mRNA) in the samples under investigation.
The Key to Nucleic Acid Detection is “Sequence-Specific Affinity”
CAGTAACGGTT
5’
3’
GTCATTGCCAA
5’
3’
Microarray-Based Assays (The Basics)
“PROBE” is DNA spotted (attached) to the solidsubstrate (non-fluorescent glass slide).
“TARGET” is the fluorescence labeledcDNA representation of the mRNA and
is hybridized to the probe.
Microarray-Based Assays (The Basics)
...GCUACGAUUGCAACGCCCGAAUGGUUACCAAAAAAAAAAA...
dCTP
dATP
dTTP
dGTP
How does a DNA microarray detects gene activity?
Reverse Transcription makes cDNA from gene sequence…
AAAAAAAAAAAAAAAAmRNA
TTTTTTTTTTTGGTAACCCCCCC ATTGGGGTTGAATGTAG
cDNA
TTTTTTTTTTTGGTAACCCCCCC ATTGGGGTTGAATGTAG
2-Color System...
RNA from Normal Tissue RNA from Cancer or Drug Treated Tissue
dCTP dCTP
Reverse Transcription
2-Color System... Detection
2-Color Laser Scanner
P450 Induction Study
Adult Male Sprague-Dawley rats (175-200g) were dosed daily with thefollowing compounds (or vehicle control), and sacrificed at 1-day and4-days of treatment. Livers were resected and flash-frozen for subsequentmicrosomal preparation and RNA isolation.
DRUG (dose) Expected P450 Induced-naphthoflavone (40mg/kg IP) 1A1, 1A2
2) Dexamethasone (50mg/kg IP) 3A
3) Isoniazid (100 mg/kg IP) 2E1
4) Phenobarbital (20 mg/kg IP) 2B1
RNA was analyzed using the Rat P450 microarray, microsomes were assayedfor the anticipated P450 activity.
-naphthoflavone (40mg/kg IP)
0
5
10
15
20
25
30
1-Day 4-Day
Rat P450-1A1 mRNA
Rat P450-1A1 EnzymeActivity
1A1 enzyme activity is expressed as ratio of drug treated over control (ethoxyresorufin to hydroxyresorufin, nmol/min/mg protein).
Day 1: 11.4 (= 2.73 / 0.24)Day 4: 13.5 (= 2.30 / 0.17)
1A1
1-Day1A1
Fol
d C
hang
e ab
ove
Con
trol
-naphthoflavone (40mg/kg IP)
0
5
10
15
20
25
1-Day 4-Day
Rat P450-1A2 mRNA
Rat P450-1A2 EnzymeActivity
1A2 enzyme activity is expressed as ratio of drug treated over control(methoxyresorufin to hydroxyresorufin, nmol/min/mg protein).
Day 1: 4.0 (= 0.12 / 0.03)Day 4: 8.5 (= 0.17 / 0.02)
1A2
4-Day1A2
Methylcholanthrene-inducible P450d
Fol
d C
hang
e ab
ove
Con
trol
-4
-2
0
2
4
6
8
10
12
14
161-Day
4-Day
A B C D E F GA. GAPDHB. UDP-Glucuronosyl Transferase (UGT1)C. P450d Methylcholanthrene-InducibleD. Pregnenolone 16-alpha-carbonitrile-inducible P450E. Minoxidil SulfotransferaseF. Lactate DehydrogenaseG. Carboamyl-phosphate synthetase 1 (CPS1)
-naphthoflavone (40mg/kg IP)
Fol
d C
hang
e ab
ove
Con
trol
Dexamethasone (50 mg/kg IP)
3A enzyme activity is expressed as ratio of drug treated over control (measuring erythromycin toformaldehyde, nmol/min/mg protein).Day 1: 3.6 (= 3.35 / 0.94)Day 4: 7.7 (= 6.27 / 0.81)
3A
0
2
4
6
8
1-Day 4-Day
Rat P450 3A mRNA
Rat P450 3A EnzymeActivity
1-Day
3AMetallothionein-1
CYP 2C23
Minoxidil Sulfotransferase
Fol
d C
hang
e ab
ove
Con
trol
-8
-6
-4
-2
0
2
4
6
8
10
121-Day
4-Day
A. P450 2C23B. N-hydroxy-2-acetylaminofluorene sulfotransferaseC. Insulin-like growth factor-D. Metallothionein-1E. Hydroxysteroid SulfotransferaseF. UDP-glucuronosyltransferase, phenobarbital inducible formG. UDP-glucuronosyltransferaseH. Minoxidil SulfotransferaseI. Heat shock 10 kD protein 1
J. Serine protease inhibitorK. Carboamyl-phosphate synthetase 1L. GAPDHM. Malate DehydrogenaseN. Elongation Factor 1--alphaO. Lactate DehydrogenaseP. HaptoglobinQ. Tyrosine AminotransferaseR. Vitronectin
A BC D E F G H I J K L M N O P Q R
Dexamethasone (50 mg/kg IP)
Fol
d C
hang
e ab
ove
Con
trol
Isoniazid (100 mg/kg IP)
2E1 enzyme activity is expressed as ratio of drug treated over control (measuring p-nitrophenol to4-nitrocatechol, nmol/min/mg protein).Day 1: 3.4 (= 1.71 / 0.51)Day 4: 3.8 (= 1.04 / 0.27)
2E1
0
0.5
1
1.5
2
2.5
3
3.5
4
1-Day 4-Day
Rat P450 2E1 mRNA
Rat P450 2E1 EnzymeActivity
1-Day
2E1
Metallothionein-1
CYP3
Fol
d C
hang
e ab
ove
Con
trol
0
1
2
3
4
5
6
7
81-Day
4-Day
Isoniazid (100 mg/kg IP)
A B C D E F G
A. P450d Methylcholanthrene-InducibleB. Metallothionein 1C. Hydroxysteroid Sulfotransferase AD. CYP3E. Cyp1A2F. Carboamyl-phosphate synthetase 1G. Vitronectin
Fol
d C
hang
e ab
ove
Con
trol
Phenobarbital (20 mg/kg IP)
1-Day
0
5
10
15
20
25
30
35
1-Day 4-Day
Fol
d C
hang
e ab
ove
Con
trol Rat P450 2B1 mRNA
Rat P450 2B1 EnzymeActivity
2E1 enzyme activity is expressed as ratio of drug treated over control (measuring pentoxyresorufin to hydroxyresorufin, nmol/min/mg protein).Day 1: 20 (= 0.20 / 0.01)Day 4: 31 (= 0.31 / 0.07)
2B1
Number of SpotsSpot Size
Replicates
MICROARRAY SPOTTER
Solid Substrate (slide)Functional Surface Chemistry
DNA Probes
OligonucleotideProbes
cDNA (PCR)Probes
DesignSynthesis
Purification
ClonesAmplificationPurification
DNA MicroarraysRecord File(.gal file)
Processing Chemistry
StorageDelivery
Pre-Hybridization Chemistry
HYBRIDIZATION
IMAGING
Raw Data Image (.tif file)
Gene Specific Data
Biological/Bioinformatics Analysis
Time, Temp, Buffers, Wash
Resolution, Region of Interest
Raw Data AnalysisSpot Finding, Background Subtraction, Signal Intensity, Normalization
Sample
Control Sample
Test Sample
RNAIsolation
RT (Fluorescence)Labeling
STUDY DESIGNand OBJECTIVES