Transcript Dideoxy nucleoside Triphosphate

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 plasmid
Primer 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
• DNA Sequencing exploits the DNA polymerase activity
for deciphering DNA sequence
• Modern DNA sequence use PCR technology in
sequencing
T3 Primer
ATTAACCC TCACTAAAGG
GACTAGTCCT GCAGGTTTAA
DNA Polymerase
AGGAATTCGC CCTT
One primer PCR Reaction
DNA Polymerase
Primer
dATP
dGTP
dCTP
dGTP
Nucleotides
Template DNA
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
– dGTP
– dCTP
– dTTP
ddATP
ddGTP
ddCTP
ddTTP
Use of Fluorescent Dyes
Flurophores
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
GGG ATATCACTCA GCATAATTGTTAAGTGACC
T7 Primer
• 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 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: [email protected]
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.
TEMPLATE
PRIMER
PCR products
20ng/100 bp
3.2 pmole
Plasmid
0.5-1.0 μg
3.2 pmole
SS Templates
0.25-0.5 μg
0.8 pmole
Cosmid
0.5-1.0 μg
25pmole
<><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><><>
SAMPLE NAME:
1. _______________
2. _______________
3. _______________
4. _______________
5. _______________
6. _______________
7. _______________
8. _______________
9. _______________
10. _______________
11. _______________
12. _______________
13. _______________
14. _______________
15. _______________
16. _______________
17. _______________
18. _______________
19. _______________
20. _______________
21. _______________
22. _______________
23. _______________
24. _______________
25. _______________
26. _______________
27. _______________
28. _______________
29. _______________
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: _________________________________________