1. Genomic Technologies CIT581N Michael Kane, Ph.D. Lecture 1: Sequencing Technology and DNA Microarray Technology

2. 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.

3. A G C T 5’-AAACCAGGCCGATAAGGTACTACACGAAAAAAA-3’ dATP dCTP dTTP dGTP + ddATP 32 ddCTP 32 ddTTP 32 ddGTP 32 TTTGGTCCGGCTATTCCATGATGTGCTTTTTTT TTGGTCCGGCTATTCCATGATGTGCTTTTTTT TGGTCCGGCTATTCCATGATGTGCTTTTTTT GGTCCGGCTATTCCATGATGTGCTTTTTTT GTCCGGCTATTCCATGATGTGCTTTTTTT TCCGGCTATTCCATGATGTGCTTTTTTT CCGGCTATTCCATGATGTGCTTTTTTT CGGCTATTCCATGATGTGCTTTTTTT GGCTATTCCATGATGTGCTTTTTTT GCTATTCCATGATGTGCTTTTTTT CTATTCCATGATGTGCTTTTTTT TATTCCATGATGTGCTTTTTTT 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)

4. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html DNA Sequencing new method)

5. Phred quality score Probability that the base is called wrong Accuracy of the base call 101 in 1090% 201 in 10099% 301 in 1,00099.9% 401 in 10,00099.99% 501 in 100,00099.999% Ewing et al.1998b, Genome Research 8:186-94

6. DNA Microarray Technology: A Technical Perspective

7. 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.

8. The Key to Nucleic Acid Detection is “Sequence-Specific Affinity” CAGTAACGGTTCAGTAACGGTT 5’ 3’ GTCATTGCCAAGTCATTGCCAA 5’ 3’ Microarray-Based Assays (The Basics)

9. “PROBE” is DNA spotted (attached) to the solid substrate (non-fluorescent glass slide). “TARGET” is the fluorescence labeled cDNA representation of the mRNA and is hybridized to the probe. Microarray-Based Assays (The Basics)

10. ...GCUACGAUUGCAACGCCCGAAUGGUUACCAAAAAAAAAAA... dCTP dATP dTTP dGTP How does a DNA microarray detects gene activity? Reverse Transcription makes cDNA from gene sequence… AAAAAAAAAAAAAAAA mRNA TTTTTTTTTTTGGTAACCCCCCCATTGGGGTTGAATGTAG cDNA TTTTTTTTTTTGGTAACCCCCCCATTGGGGTTGAATGTAG

11. 2-Color System... RNA from Normal TissueRNA from Cancer or Drug Treated Tissue dCTP Reverse Transcription

12. 2-Color System... Detection

13. 2-Color Laser Scanner

14. P450 Induction Study Adult Male Sprague-Dawley rats (175-200g) were dosed daily with the following compounds (or vehicle control), and sacrificed at 1-day and 4-days of treatment. Livers were resected and flash-frozen for subsequent microsomal 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 assayed for the anticipated P450 activity.

15. -naphthoflavone (40mg/kg IP) 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-Day 1A1 Fold Change above Control

16. -naphthoflavone (40mg/kg IP) 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-Day 1A2 Methylcholanthrene-inducible P450d Fold Change above Control

17. A B C D E F G A. GAPDH B. UDP-Glucuronosyl Transferase (UGT1) C. P450d Methylcholanthrene-Inducible D. Pregnenolone 16-alpha-carbonitrile-inducible P450 E. Minoxidil Sulfotransferase F. Lactate Dehydrogenase G. Carboamyl-phosphate synthetase 1 (CPS1) -naphthoflavone (40mg/kg IP) Fold Change above Control

18. Dexamethasone (50 mg/kg IP) 3A enzyme activity is expressed as ratio of drug treated over control (measuring erythromycin to formaldehyde, nmol/min/mg protein). Day 1: 3.6 (= 3.35 / 0.94) Day 4: 7.7 (= 6.27 / 0.81) 3A 1-Day 3A Metallothionein-1 CYP 2C23 Minoxidil Sulfotransferase Fold Change above Control

19. A. P450 2C23 B. N-hydroxy-2-acetylaminofluorene sulfotransferase C. Insulin-like growth factor-  D. Metallothionein-1 E. Hydroxysteroid Sulfotransferase F. UDP-glucuronosyltransferase, phenobarbital inducible form G. UDP-glucuronosyltransferase H. Minoxidil Sulfotransferase I. Heat shock 10 kD protein 1 J. Serine protease inhibitor K. Carboamyl-phosphate synthetase 1 L. GAPDH M. Malate Dehydrogenase N. Elongation Factor 1--alpha O. Lactate Dehydrogenase P. Haptoglobin Q. Tyrosine Aminotransferase R. Vitronectin A B C D E F G H I J K L M N O P Q R Dexamethasone (50 mg/kg IP) Fold Change above Control

20. Isoniazid (100 mg/kg IP) 2E1 enzyme activity is expressed as ratio of drug treated over control (measuring p-nitrophenol to 4-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-Day4-Day Rat P450 2E1 mRNA Rat P450 2E1 Enzyme Activity 1-Day 2E1 Metallothionein-1 CYP3 Fold Change above Control

21. Isoniazid (100 mg/kg IP) A B C D E F G A. P450d Methylcholanthrene-Inducible B. Metallothionein 1 C. Hydroxysteroid Sulfotransferase A D. CYP3 E. Cyp1A2 F. Carboamyl-phosphate synthetase 1 G. Vitronectin Fold Change above Control

22. Phenobarbital (20 mg/kg IP) 1-Day 0 5 10 15 20 25 30 35 1-Day4-Day Fold Change above Control Rat P450 2B1 mRNA Rat P450 2B1 Enzyme Activity 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

23. Number of Spots Spot Size Replicates MICROARRAY SPOTTER Solid Substrate (slide) Functional Surface Chemistry DNA Probes Oligonucleotide Probes cDNA (PCR) Probes Design Synthesis Purification Clones Amplification Purification DNA Microarrays Record File (.gal file) Processing Chemistry Storage Delivery 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 Analysis Spot Finding, Background Subtraction, Signal Intensity, Normalization Sample Control Sample Test Sample RNA Isolation RT (Fluorescence) Labeling STUDY DESIGN and OBJECTIVES