Download - DNA Sequencing and Identification of Encoded Genes Kimberley Chinen June 30, 2006 ABE Workshop
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DNA Sequencing and Identification of Encoded Genes
Kimberley ChinenJune 30, 2006ABE Workshop
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Purpose
• To determine the order of nucleotides in a segment of DNA
• To verify the identity of the encoded gene– PDI2– GFP
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Template Preparation
Isolation of Total RNA from Arabidopsis thaliana
Grind and lyse Arabidopsis leavesAdsorption of total RNA to silica
membraneRemoval of contaminantsQuantify RNA
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RT-PCR
• Reverse Transcription
mRNA dsDNA
DNA transcript of
RNA
DNA-RNA hybrid
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RT-PCR
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Group 2 RT PCR Result
100b + WT 2SC GFP5er –RT H2O P+
100bp + WT 2A1 2A2 –RT H2O P+
Group 1 RT PCR Result
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Group 4 RT PCR Result
100bp + WT 2A1 2A2 –RT H2O P+
Group 3 RT PCR Result
100b + WT 2SC GFP5er –RT H2O P+
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MW Hen pdi2 p H pBIN GFP Tu Tu pdi2 BIN 35S pla 1 6
Gr 1 Gr2 Gr3 Gr4
Gel Purified RT_PCRproducts for use as inserts in TOPO cloning reactions
RT-PCR Results
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Molecular Cloning
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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
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DNA SEQUENCING
1.PCR
2.DNA Sequencing in polyacrylamide gel
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DNA Samples Sent in for Sequencing
Tube # Primer Gene Source Group
1 T3 PDI2 Pos Control 1
2 T7 PDI2
3 T3 GFP
24 T7 GFP
5 T3 PDI2
6 T7 PDI2
7 T3 PDI2 Henry's Control
38 T7 PDI2
9 T3 PDI2 Pos Control
10 T7 PDI2
11 T3 PDI2 Pos Control
412 T7 PDI2
13 T3 PDI2 Henry's Control
14 T7 PDI2
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1.PCR – 4.5 hours• We Provided
• DNA Template
• T3 or T7 Primer
• They added
• dideoxynucleotides – fluorescently labeled
• DNA polymerase
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T3 and T7 Priming Sites
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Deoxynucleotides vs. Dideoxynucleotides
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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.
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GREENWOOD MOLECULAR BIOLOGY FACILITY
Applied Biosystems 377XL DNA Sequencer at UHM’s
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GREENWOOD MOLECULAR BIOLOGY FACILITY AT UHM
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Chromatogram from ChromasPro
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Tube 7 ResultsGTCGCAGTTTAACGAATTCGCCCTTACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTNAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACNACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAANGNAGTTAGATTATACACCCAGGGACCAATCTTCTTTTTNCCCATGGCACAATTGGTTCTTTAGNCCTTCCCCCCGGGAAGGGNTGGGGCCCCCCTCACCCAAAAAAAAAACNTCNGAAAACCCTGAANACNAAACTCCCGGANTAANTCNTTTTCCCCCGCCCANCAATTTTANCCAAAAAAAACCCTNTTTCTTAANTCCCTGGGNAAAGGGGNAATTCCGGCNCCTAAATTAANTTN
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Cut at the beginning of PCR Product for T3 Primed Sequences
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Cut off left and right sequencesTube 7 Right Left Cut Off
ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTNAGCTGATCGTGCT
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ATNGATTAGCGGCCGCGAATTCGCCCTTGCCACGGAGCTTAAGGAAGATGGTGTTGTTTTGGCTAAGATTGATGCGACGGAGGAGAATGAGTTAGCTCAGGAGTATCGTGTTCAGGGCTTTCCGACGCTTCTTTTCTTCGTTGATGGTGAGCACAAGCCTTACACTGGAGGAAGGACTAAAGAAACAATTGTGACATGGGTGAAGAANAAGATTGGTCCTGGTGTGTATAATCTAACTACATTAGATGATGCTGAGAAAGTGTTGACTTCTGGGAACAAAGTTGTTTTGGGATACTTGAACTCCTTGGTGGGTGTTGAGCACGATCAGCTTAATGCTGCTTCCAAAGCTGAAGACGATGTGAACTTTTATCAAACAGTGAATCCTGATGTTGCAAAGATGTTTCACCTTGATCCGGAGTCTAAAAGGCCTGCTCTTGTCCTAGTTAAGAAGGAAGAGGAGAAAATTAGCCATTTTGATGGAGAATTTGTTAAGTCTGCTCTAGTTAGTTTTGTGTCTGCCAACAAGCTTGCTTTGGTCTCTGTTTTCCCAGAAAGACTGCTCCGGAAATTTTTGAGAGTGCAATCAAGAAACAGTTGTTGTTGTTTGTAACCAAAAATGAATCTGAAAANGTTCTTACNGAATTTCAAGAAGCAGCGAAATCATTCAAAGGAAAGCTCATCTTTTGTATCTGTGGATCTGGATAATGANGATTATNGGGAAGCCNGTCCCCTGAAAACCTTTGGGGGTGTCCTGGNAATGGGCCCNAAAACTTATTGGGCTACCCCNGGGGNAATGAAAAAACCCTAAAAAAANACCTTTTTTCCAATGGGGGAAAATNCCCNTCCCNAANAAAAAATTAAAAANANTTTNGGGGAAGGAATTTCCCGGAAACAACAAAGTTNAAANCCCNTTNTNTAAATCCNAAANCCCCNTTCCCTGAAAAAAAACCNATCANAAATNTTGNAAAAAAANGGGTTGGGANAAAAANTTTTAAANAAAATTNNTTCTGGGAANAATTTTAAAAAATGGCNTTTCCCNGGGCCTNCCCCCCCCGGGGGGAANGGGGGAAATTTNTTTAAACN
Tube 8 Results
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Reverse Complement
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Tube 8 Reverse ComplementNGTTTAAANAAATTTCCCCCNTTCCCCCCGGGGGGGGNAGGCCCNGGGAAANGCCATTTTTTAAAATTNTTCCCAGAANNAATTTTNTTTAAAANTTTTTNTCCCAACCCNTTTTTTTNCAANATTTNTGATNGGTTTTTTTTCAGGGAANGGGGNTTTNGGATTTANANAANGGGNTTTNAACTTTGTTGTTTCCGGGAAATTCCTTCCCCNAAANTNTTTTTAATTTTTTNTTNGGGANGGGNATTTTCCCCCATTGGAAAAAAGGTNTTTTTTTAGGGTTTTTTCATTNCCCCNGGGGTAGCCCAATAAGTTTTNGGGCCCATTNCCAGGACACCCCCAAAGGTTTTCAGGGGACNGGCTTCCCNATAATCNTCATTATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCAAGGGCGAATTCGCGGCCGCTAATCNAT
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Cut at the end of PCR Product for T7 Primed Sequences
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Cut at the ends of PCR Product for T7 Primed Sequences
Example: Tube 8 Reverse Complement with right and left ends cut
TATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCA
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Finding where the sequences overlap
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Finding where the sequences overlapTube 7ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTNAGCTGATCGTGCT
Tube 8 TATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCA
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Putting the Two Partial Sequences Together
ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCA
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BLAST Search for the combined sequence in tubes 7 and 8
http://ncbi.nih.gov/BLAST/
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Sequences with Significant Alignments
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Sequence Alignments
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Arabidopsis thaliana At5g60640 Gene Information
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Translation
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Full Gene Sequence
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Blast search results for combined sequences from tubes 3 and 4
GFP
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GFP Sequence Alignment Results
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Summary
• Arabidopsis thaliana
• Total RNA
• cDNA
• tDNA
• DNA Sequencing
• Identification of Genes– PDI2– GFP