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DNA Sequencing
Kabi R. Neupane, Ph.D.
Leeward Community College
ABE Workshop 2006
What to Sequence?
• The RT-PCR product has been inserted into the pCR4-TOPO vector
• Our goal-Sequence the insert DNA
We Supplied
Template DNA: Your plasmidPrimer DNA: T3 or T7
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
DNA Polymerase Action
ATTAACCC TCACTAAAGG
T3 Primer
GACTAGTCCT GCAGGTTTAA AGGAATTCGC CCTTDNA Polymerase
• DNA Sequencing exploits the DNA polymerase activity for deciphering DNA sequence
• Modern DNA sequence use PCR technology in sequencing
One primer PCR Reaction
Template DNA
Primer
DNA Polymerase
dATPdGTPdCTPdGTP
Nucleotides
NEW STRAND
Dideoxy Nucleotides
• Lack an -OH group at the 3-carbon position
• Cannot add another nucleoside at that position
• Prevent further DNA synthesis
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
Use of Fluorescent DyesFlurophores
Flurophores
Chain Termination
All Possible Terminations
Polyacrylamide Gel Electrophoresis
Separates fragments based on size
DNA Sequence Files
Good or Bad?
Do not forget the other strand
T7 Primer
GGG ATATCACTCA GCATAATTGTTAAGTGACC
• GenBank• The Basic Local
Alignment Search Tool (BLAST) finds regions of local similarity between sequences.
http://www.ncbi.nlm.nih.gov/
Shotgun Sequencing • Involves fragment assembly using computer algorithms
Contigs
GREENWOOD MOLECULAR BIOLOGY FACILITYUNIVERSITY OF HAWAII AT MANOA
3050 Maile Way, Gilmore Hall 411, Honolulu, HI 96822Phone: (808) 956-6718 Fax: (808) 956-9589 E-mail: [email protected]
DNA SEQUENCING FORM
PRIMARY INVESTIGATOR: _________________________________ DATE: _________________YOUR NAME: ____________________________________________ DEPARTMENT: __________ADDRESS: _____________________________________________________________________
_____________________________________________________________________PHONE: _______________ FAX: ______________ E-MAIL: _____________________________PURCHASE ORDER/REQUISITION NUMBER: __________________________________________BILLING ADDRESS: _______________________________________________________________
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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 CosmidTEMPLATE 20ng/100 bp 0.5-1.0 μg 0.25-0.5 μg 0.5-1.0 μgPRIMER 3.2 pmole 3.2 pmole 0.8 pmole 25pmole
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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: _________________________________________
