1. DNA Sequencing Kabi R. Neupane, Ph.D. Leeward Community College ABE Workshop 2006

2. What to Sequence? The RT-PCR product has been inserted into the pCR4-TOPO vector Our goal -Sequence the insert DNA

3. We Supplied Template DNA: Your plasmid Primer DNA: T3 or T7

4. Frederick Sanger Discovered DNA sequencing by chain termination method Nobel Prize 1 (1958) –Complete amino acid sequence of insulin Nobel Prize 2 (1980) –For DNA sequencing

5. DNA Polymerase Action ATTAACCC TCACTAAAGG T3 Primer GACTAGTCCT GCAGGTTTAA AGGAATTCGC CCTT DNA Polymerase DNA Sequencing exploits the DNA polymerase activity for deciphering DNA sequence Modern DNA sequence use PCR technology in sequencing

6. One primer PCR Reaction Template DNA Primer DNA Polymerase dATP dGTP dCTP dGTP Nucleotides NEW STRAND

7. Dideoxy Nucleotides Lack an -OH group at the 3-carbon position Cannot add another nucleoside at that position Prevent further DNA synthesis

8. Dideoxy nucleotides Incorporation of a dideoxynucleotide to growing DNA strand terminates its further extension Are added in small proportion –dATPddATP –dGTPddGTP –dCTPddCTP –dTTPddTTP

9. Use of Fluorescent Dyes Flurophores

11. Chain Termination

12. All Possible Terminations

13. Polyacrylamide Gel Electrophoresis Separates fragments based on size

15. DNA Sequence Files

16. Good or Bad?

17. Do not forget the other strand T7 Primer GGG ATATCACTCA GCATAATTGTTAAGTGACC

18. GenBank The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. http://www.ncbi.nlm.nih.gov/

19. Shotgun Sequencing Involves fragment assembly using computer algorithms Contigs

20. GREENWOOD MOLECULAR BIOLOGY FACILITY UNIVERSITY OF HAWAII AT MANOA 3050 Maile Way, Gilmore Hall 411, Honolulu, HI 96822 Phone: (808) 956-6718 Fax: (808) 956-9589 E-mail: biotech@hawaii.edu DNA SEQUENCING FORM PRIMARY INVESTIGATOR: _________________________________ DATE: _________________ YOUR NAME: ____________________________________________ DEPARTMENT: __________ ADDRESS: _____________________________________________________________________ _____________________________________________________________________ PHONE: _______________ FAX: ______________ E-MAIL: _____________________________ PURCHASE ORDER/REQUISITION NUMBER: __________________________________________ BILLING ADDRESS: _______________________________________________________________ <><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><> Templates and primers should be supplied in ultrapure deionized water. Do not use Tris or other buffers. Please supply 12 µl of sample per reaction (template + one primer) in 0.5mL thin-walled PCR microcentrifuge tubes. Pre-reacted samples should be provided dry in 1.5mL centrifuge tubes. Please do not attach tags to tubes. Samples may not be run simultaneously. PCR products Plasmid SS Templates Cosmid TEMPLATE 20ng/100 bp 0.5-1.0 μg 0.25-0.5 μg 0.5-1.0 μg PRIMER 3.2 pmole 3.2 pmole 0.8 pmole 25pmole <><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><> SAMPLE NAME: 1. _______________ 11. _______________ 21. _______________ 2. _______________ 12. _______________ 22. _______________ 3. _______________ 13. _______________ 23. _______________ 4. _______________ 14. _______________ 24. _______________ 5. _______________ 15. _______________ 25. _______________ 6. _______________ 16. _______________ 26. _______________ 7. _______________ 17. _______________ 27. _______________ 8. _______________ 18. _______________ 28. _______________ 9. _______________ 19. _______________ 29. _______________ 10. _______________ 20. _______________ 30. _______________ SPECIAL INSTRUCTIONS: __________________________________________________________ DATA DELIVERY: 3.5’’ disk or ZIP disk (must provide) ______ FTP _______ E-mail attachment _______ Electrophoregram print-out ($2.00 per sample) _________ Type of Computer used: MAC _______ PC _________ SIGNATURE: _________________________________________