DNA Sequencing

1. 0 DNA Sequencing Kabi R. Neupane, Ph.D. Leeward Community College ABE Workshop 2006

2. 0 What to Sequence? • The RT-PCR product has been inserted into the pCR4-TOPO vector • Our goal • -Sequence the insert DNA

3. 0 We Supplied Template DNA: Your plasmid Primer DNA: T3 or T7

4. 0 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. 0 • DNA Sequencing exploits the DNA polymerase activity for deciphering DNA sequence • Modern DNA sequence use PCR technology in sequencing DNA Polymerase Action T3 Primer ATTAACCC TCACTAAAGG DNA Polymerase GACTAGTCCT GCAGGTTTAA AGGAATTCGC CCTT

6. 0 One primer PCR Reaction DNA Polymerase Primer dATP dGTP dCTP dGTP Nucleotides NEW STRAND Template DNA

7. 0 Dideoxy Nucleotides • Lack an -OH group at the 3-carbon position • Cannot add another nucleoside at that position • Prevent further DNA synthesis

8. 0 Dideoxy nucleotides • Incorporation of a dideoxynucleotide to growing DNA strand terminates its further extension • Are added in small proportion • dATP ddATP • dGTP ddGTP • dCTP ddCTP • dTTP ddTTP

9. Use of Fluorescent Dyes Flurophores

10. Flurophores

11. 0 Chain Termination

12. 0 All Possible Terminations

13. 0 Polyacrylamide Gel Electrophoresis Separates fragments based on size

14. 0

15. 0 DNA Sequence Files

16. 0 Good or Bad?

17. 0 Do not forget the other strand GGG ATATCACTCA GCATAATTGTTAAGTGACC T7 Primer

18. GenBank The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. 0 http://www.ncbi.nlm.nih.gov/

19. Involves fragment assembly using computer algorithms 0 Shotgun Sequencing 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 productsPlasmidSS TemplatesCosmid 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:  _________________________________________