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DNA Sequencing

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.

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DNA Sequencing

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

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

  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 • dATP ddATP • dGTP ddGTP • dCTP ddCTP • dTTP ddTTP

  9. Chain Termination

  10. All Possible Terminations

  11. Polyacrylamide Gel Electrophoresis Separates fragments based on size

  12. DNA Sequence Files

  13. Good or Bad?

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

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

  16. Involves fragment assembly using computer algorithms Shotgun Sequencing Contigs

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