DNA Sequencing and Identification of Encoded Genes

1. DNA Sequencing and Identification of Encoded Genes Kimberley Chinen June 30, 2006 ABE Workshop

2. Purpose • To determine the order of nucleotides in a segment of DNA • To verify the identity of the encoded gene • PDI2 • GFP

3. Template Preparation Isolation of Total RNA from Arabidopsis thaliana • Grind and lyse Arabidopsis leaves • Adsorption of total RNA to silica membrane • Removal of contaminants • Quantify RNA

4. RT-PCR • Reverse Transcription mRNA DNA-RNA hybrid DNA transcript of RNA dsDNA

5. RT-PCR

6. Group 1 RT PCR Result 100bp + WT 2A1 2A2 –RT H2O P+ Group 2 RT PCR Result 100b + WT 2SC GFP5er –RT H2O P+

7. Group 3 RT PCR Result Group 4 RT PCR Result 100b + WT 2SC GFP5er –RT H2O P+ 100bp + WT 2A1 2A2 –RT H2O P+

8. RT-PCRResults Gr 1 Gr2 Gr3 Gr4 MW Hen pdi2 p H pBIN GFP Tu Tu pdi2 BIN 35S pla 1 6 Gel Purified RT_PCR products for use as inserts in TOPO cloning reactions

9. Molecular Cloning

10. Molecular Cloning • Ligate RT-PCR product into pCR4-TOPO cloning vector • Transform E. coli bacteria by heat shock • Amplify and select transformed bacteria using ampicillin treated plates • Isolate plasmid DNA • Run agarose gel electrophoresis • Extract and purify DNA

11. DNA SEQUENCING • PCR • DNA Sequencing in polyacrylamide gel

12. DNA Samples Sent in for Sequencing

13. PCR – 4.5 hours • We Provided • DNA Template • T3 or T7 Primer • They added • dideoxynucleotides – fluorescently labeled • DNA polymerase

14. T3 and T7 Priming Sites

15. Deoxynucleotides vs. Dideoxynucleotides

16. 2. DNA Sequencing of PCR Products in Polyacrylamide Gel PCR products are loaded and run in a polyacrylamide gel to separate the fragments by length in an automated DNA sequencer. A scanner in the sequencer detects the fluorescence and order of the separate bands.

17. GREENWOOD MOLECULAR BIOLOGY FACILITYApplied Biosystems 377XL DNA Sequencer at UHM’s

18. GREENWOOD MOLECULAR BIOLOGY FACILITY AT UHM

19. Chromatogram from ChromasPro

20. Tube 7 Results GTCGCAGTTTAACGAATTCGCCCTTACACCATGGTGCGTAGACCTCGAGAAGCACATCTT TAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCAT CGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAAT CCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAG GGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATT CAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTC CTTTGAATGATTTCGCTGCTTCTTGAAATTCCGTAAGAACCTTTTCAGATTCATTTTTGG TTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTC TGGTGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACT TAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAG CANGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTT GATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTNAGCTGATCGTGCTCAACAC CCACCAAGGAGTTCAAGTATCCCAAAACNACTTTGTTCCCAGAAGTCAACACTTTCTCAG CATCATCTAANGNAGTTAGATTATACACCCAGGGACCAATCTTCTTTTTNCCCATGGCAC AATTGGTTCTTTAGNCCTTCCCCCCGGGAAGGGNTGGGGCCCCCCTCACCCAAAAAAAAA ACNTCNGAAAACCCTGAANACNAAACTCCCGGANTAANTCNTTTTCCCCCGCCCANCAAT TTTANCCAAAAAAAACCCTNTTTCTTAANTCCCTGGGNAAAGGGGNAATTCCGGCNCCTA AATTAANTTN

21. Cut at the beginning of PCR Product for T3 Primed Sequences

22. Cut off left and right sequences Tube 7 Right Left Cut Off ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATC AAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGA CTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATC TGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAAT AAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTC ATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTG AAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTT GATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAG CTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCT AATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAG GTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGC TTTGGAAGCAGCATTNAGCTGATCGTGCT

23. Tube 8 Results ATNGATTAGCGGCCGCGAATTCGCCCTTGCCACGGAGCTTAAGGAAGATGGTGTTGTTTT GGCTAAGATTGATGCGACGGAGGAGAATGAGTTAGCTCAGGAGTATCGTGTTCAGGGCTT TCCGACGCTTCTTTTCTTCGTTGATGGTGAGCACAAGCCTTACACTGGAGGAAGGACTAA AGAAACAATTGTGACATGGGTGAAGAANAAGATTGGTCCTGGTGTGTATAATCTAACTAC ATTAGATGATGCTGAGAAAGTGTTGACTTCTGGGAACAAAGTTGTTTTGGGATACTTGAA CTCCTTGGTGGGTGTTGAGCACGATCAGCTTAATGCTGCTTCCAAAGCTGAAGACGATGT GAACTTTTATCAAACAGTGAATCCTGATGTTGCAAAGATGTTTCACCTTGATCCGGAGTC TAAAAGGCCTGCTCTTGTCCTAGTTAAGAAGGAAGAGGAGAAAATTAGCCATTTTGATGG AGAATTTGTTAAGTCTGCTCTAGTTAGTTTTGTGTCTGCCAACAAGCTTGCTTTGGTCTC TGTTTTCCCAGAAAGACTGCTCCGGAAATTTTTGAGAGTGCAATCAAGAAACAGTTGTTG TTGTTTGTAACCAAAAATGAATCTGAAAANGTTCTTACNGAATTTCAAGAAGCAGCGAAA TCATTCAAAGGAAAGCTCATCTTTTGTATCTGTGGATCTGGATAATGANGATTATNGGGA AGCCNGTCCCCTGAAAACCTTTGGGGGTGTCCTGGNAATGGGCCCNAAAACTTATTGGGC TACCCCNGGGGNAATGAAAAAACCCTAAAAAAANACCTTTTTTCCAATGGGGGAAAATNC CCNTCCCNAANAAAAAATTAAAAANANTTTNGGGGAAGGAATTTCCCGGAAACAACAAAG TTNAAANCCCNTTNTNTAAATCCNAAANCCCCNTTCCCTGAAAAAAAACCNATCANAAAT NTTGNAAAAAAANGGGTTGGGANAAAAANTTTTAAANAAAATTNNTTCTGGGAANAATTT TAAAAAATGGCNTTTCCCNGGGCCTNCCCCCCCCGGGGGGAANGGGGGAAATTTNTTTAA ACN

24. Reverse Complement

25. Tube 8 Reverse Complement NGTTTAAANAAATTTCCCCCNTTCCCCCCGGGGGGGGNAGGCCCNGGGAAANGCCATTTT TTAAAATTNTTCCCAGAANNAATTTTNTTTAAAANTTTTTNTCCCAACCCNTTTTTTTNC AANATTTNTGATNGGTTTTTTTTCAGGGAANGGGGNTTTNGGATTTANANAANGGGNTTT NAACTTTGTTGTTTCCGGGAAATTCCTTCCCCNAAANTNTTTTTAATTTTTTNTTNGGGA NGGGNATTTTCCCCCATTGGAAAAAAGGTNTTTTTTTAGGGTTTTTTCATTNCCCCNGGG GTAGCCCAATAAGTTTTNGGGCCCATTNCCAGGACACCCCCAAAGGTTTTCAGGGGACNG GCTTCCCNATAATCNTCATTATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAA TGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAA CAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAA ACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAAT TCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTT TTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAG TTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAG GAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCT AATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTT TCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTC GGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTA GCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCAAGGGCGAATTCGCGGCCGCTAATC NAT

26. Cut at the end of PCR Product for T7 Primed Sequences

27. Cut at the ends of PCR Product for T7 Primed Sequences Example: Tube 8 Reverse Complement with right and left ends cut TATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGA AATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTG ATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCT TGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAA TTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGT GAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTT TGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAA CAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACA CACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAG TGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGAT ACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTT CCTTAAGCTCCGTGGCA

28. Finding where the sequences overlap

29. Finding where the sequences overlap Tube 7 ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATC AAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGA CTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATC TGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAAT AAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTC ATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTG AAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTT GATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAG CTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCT AATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAG GTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGC TTTGGAAGCAGCATTNAGCTGATCGTGCT Tube 8 TATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGA AATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTG ATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCT TGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAA TTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGT GAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTT TGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAA CAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACA CACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAG TGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGAT ACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTT CCTTAAGCTCCGTGGCA

30. Putting the Two Partial Sequences Together ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATC AAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGA CTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATC TGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAAT AAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTC ATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGA AATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTG ATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCT TGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAA TTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGT GAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTT TGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAA CAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACA CACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAG TGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGAT ACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTT CCTTAAGCTCCGTGGCA

31. BLAST Search for the combined sequence in tubes 7 and 8http://ncbi.nih.gov/BLAST/

33. Sequences with Significant Alignments

34. Sequence Alignments

35. Arabidopsis thaliana At5g60640 Gene Information

36. Translation

37. Full Gene Sequence

38. Blast search results for combined sequences from tubes 3 and 4GFP

39. GFP Sequence Alignment Results

40. Summary • Arabidopsis thaliana • Total RNA • cDNA • tDNA • DNA Sequencing • Identification of Genes • PDI2 • GFP