DNA Sequencing and Identification of Encoded Genes

Kimberley ChinenJune 30, 2006ABE Workshop

Purpose

• To determine the order of nucleotides in a segment of DNA

• To verify the identity of the encoded gene– PDI2– GFP

Template Preparation

Isolation of Total RNA from Arabidopsis thaliana

Grind and lyse Arabidopsis leavesAdsorption of total RNA to silica

membraneRemoval of contaminantsQuantify RNA

RT-PCR

• Reverse Transcription

mRNA dsDNA

DNA transcript of

RNA

DNA-RNA hybrid

RT-PCR

Group 2 RT PCR Result

100b + WT 2SC GFP5er –RT H2O P+

100bp + WT 2A1 2A2 –RT H2O P+

Group 1 RT PCR Result

Group 4 RT PCR Result

100bp + WT 2A1 2A2 –RT H2O P+

Group 3 RT PCR Result

100b + WT 2SC GFP5er –RT H2O P+

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

Molecular Cloning

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

DNA SEQUENCING

1.PCR

2.DNA Sequencing in polyacrylamide gel

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

1.PCR – 4.5 hours• We Provided

• DNA Template

• T3 or T7 Primer

• They added

• dideoxynucleotides – fluorescently labeled

• DNA polymerase

T3 and T7 Priming Sites

Deoxynucleotides vs. Dideoxynucleotides

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.

GREENWOOD MOLECULAR BIOLOGY FACILITY

Applied Biosystems 377XL DNA Sequencer at UHM’s

GREENWOOD MOLECULAR BIOLOGY FACILITY AT UHM

Chromatogram from ChromasPro

Tube 7 ResultsGTCGCAGTTTAACGAATTCGCCCTTACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTNAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACNACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAANGNAGTTAGATTATACACCCAGGGACCAATCTTCTTTTTNCCCATGGCACAATTGGTTCTTTAGNCCTTCCCCCCGGGAAGGGNTGGGGCCCCCCTCACCCAAAAAAAAAACNTCNGAAAACCCTGAANACNAAACTCCCGGANTAANTCNTTTTCCCCCGCCCANCAATTTTANCCAAAAAAAACCCTNTTTCTTAANTCCCTGGGNAAAGGGGNAATTCCGGCNCCTAAATTAANTTN

Cut at the beginning of PCR Product for T3 Primed Sequences

Cut off left and right sequencesTube 7 Right Left Cut Off

ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTNAGCTGATCGTGCT

ATNGATTAGCGGCCGCGAATTCGCCCTTGCCACGGAGCTTAAGGAAGATGGTGTTGTTTTGGCTAAGATTGATGCGACGGAGGAGAATGAGTTAGCTCAGGAGTATCGTGTTCAGGGCTTTCCGACGCTTCTTTTCTTCGTTGATGGTGAGCACAAGCCTTACACTGGAGGAAGGACTAAAGAAACAATTGTGACATGGGTGAAGAANAAGATTGGTCCTGGTGTGTATAATCTAACTACATTAGATGATGCTGAGAAAGTGTTGACTTCTGGGAACAAAGTTGTTTTGGGATACTTGAACTCCTTGGTGGGTGTTGAGCACGATCAGCTTAATGCTGCTTCCAAAGCTGAAGACGATGTGAACTTTTATCAAACAGTGAATCCTGATGTTGCAAAGATGTTTCACCTTGATCCGGAGTCTAAAAGGCCTGCTCTTGTCCTAGTTAAGAAGGAAGAGGAGAAAATTAGCCATTTTGATGGAGAATTTGTTAAGTCTGCTCTAGTTAGTTTTGTGTCTGCCAACAAGCTTGCTTTGGTCTCTGTTTTCCCAGAAAGACTGCTCCGGAAATTTTTGAGAGTGCAATCAAGAAACAGTTGTTGTTGTTTGTAACCAAAAATGAATCTGAAAANGTTCTTACNGAATTTCAAGAAGCAGCGAAATCATTCAAAGGAAAGCTCATCTTTTGTATCTGTGGATCTGGATAATGANGATTATNGGGAAGCCNGTCCCCTGAAAACCTTTGGGGGTGTCCTGGNAATGGGCCCNAAAACTTATTGGGCTACCCCNGGGGNAATGAAAAAACCCTAAAAAAANACCTTTTTTCCAATGGGGGAAAATNCCCNTCCCNAANAAAAAATTAAAAANANTTTNGGGGAAGGAATTTCCCGGAAACAACAAAGTTNAAANCCCNTTNTNTAAATCCNAAANCCCCNTTCCCTGAAAAAAAACCNATCANAAATNTTGNAAAAAAANGGGTTGGGANAAAAANTTTTAAANAAAATTNNTTCTGGGAANAATTTTAAAAAATGGCNTTTCCCNGGGCCTNCCCCCCCCGGGGGGAANGGGGGAAATTTNTTTAAACN

Tube 8 Results

Reverse Complement

Tube 8 Reverse ComplementNGTTTAAANAAATTTCCCCCNTTCCCCCCGGGGGGGGNAGGCCCNGGGAAANGCCATTTTTTAAAATTNTTCCCAGAANNAATTTTNTTTAAAANTTTTTNTCCCAACCCNTTTTTTTNCAANATTTNTGATNGGTTTTTTTTCAGGGAANGGGGNTTTNGGATTTANANAANGGGNTTTNAACTTTGTTGTTTCCGGGAAATTCCTTCCCCNAAANTNTTTTTAATTTTTTNTTNGGGANGGGNATTTTCCCCCATTGGAAAAAAGGTNTTTTTTTAGGGTTTTTTCATTNCCCCNGGGGTAGCCCAATAAGTTTTNGGGCCCATTNCCAGGACACCCCCAAAGGTTTTCAGGGGACNGGCTTCCCNATAATCNTCATTATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCAAGGGCGAATTCGCGGCCGCTAATCNAT

Cut at the end of PCR Product for T7 Primed Sequences

Cut at the ends of PCR Product for T7 Primed Sequences

Example: Tube 8 Reverse Complement with right and left ends cut

TATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCA

Finding where the sequences overlap

Finding where the sequences overlapTube 7ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCCGTAAGAACCTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTCTCTGGTGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCANGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTNAGCTGATCGTGCT

Tube 8 TATCCAGATCCACAGATACAAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCA

Putting the Two Partial Sequences Together

ACACCATGGTGCGTAGACCTCGAGAAGCACATCTTTAGAATCGTCCAGAACAATTTCATCAAAGTTATCTCCAACCACTATTTTCACATCTTCATCGTTCTTTTCAGGAATGGGGTCTGACTTATAGAAAGGCTTTAACTTGTCGTTCAGGAAATCCTCCCCAAATATCTTAATTTTATCTGACTGGATCTCGCCATCGAAAAAGTATTTTTTAGGGTCTTCATTCCCTGTGTAGCCAATAAGTTTAGGACCATTTCCAGACACACCAAAGTATTCAGCGACTGGCTTCCCATAATCCTCATTATCCAGATCCACAGATACAAAGATGAGCTTTCCTTTGAATGATTTCGCTGCTTCTTGAAATTCNGTAAGAACNTTTTCAGATTCATTTTTGGTTACAAACAACAACAACTGTTTCTTGATTGCACTCTCAAAAATTTCCGGAGCAGTCTTTCTGGGAAAACAGAGACCAAAGCAAGCTTGTTGGCAGACACAAAACTAACTAGAGCAGACTTAACAAATTCTCCATCAAAATGGCTAATTTTCTCCTCTTCCTTCTTAACTAGGACAAGAGCAGGCCTTTTAGACTCCGGATCAAGGTGAAACATCTTTGCAACATCAGGATTCACTGTTTGATAAAAGTTCACATCGTCTTCAGCTTTGGAAGCAGCATTAAGCTGATCGTGCTCAACACCCACCAAGGAGTTCAAGTATCCCAAAACAACTTTGTTCCCAGAAGTCAACACTTTCTCAGCATCATCTAATGTAGTTAGATTATACACACCAGGACCAATCTTNTTCTTCACCCATGTCACAATTGTTTCTTTAGTCCTTCCTCCAGTGTAAGGCTTGTGCTCACCATCAACGAAGAAAAGAAGCGTCGGAAAGCCCTGAACACGATACTCCTGAGCTAACTCATTCTCCTCCGTCGCATCAATCTTAGCCAAAACAACACCATCTTCCTTAAGCTCCGTGGCA

BLAST Search for the combined sequence in tubes 7 and 8

http://ncbi.nih.gov/BLAST/

Sequences with Significant Alignments

Sequence Alignments

Arabidopsis thaliana At5g60640 Gene Information

Translation

Full Gene Sequence

Blast search results for combined sequences from tubes 3 and 4

GFP

GFP Sequence Alignment Results

Summary

• Arabidopsis thaliana

• Total RNA

• cDNA

• tDNA

• DNA Sequencing

• Identification of Genes– PDI2– GFP