genomic technologies cit581n michael kane, ph.d. lecture 1: sequencing technology and dna microarray...

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