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

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:  _________________________________________