Fundamental Skills in DNA sequence analysis · Fundamental Skills in DNA sequence analysis Based on...

transcript

Fundamental Skills in DNA sequence analysis

Based on publicly available tools

Ning Jiang, Dept. of Horticulture

Outline for today

• Identifying gene structure – promoter, introns, Open reading frame

• Editing DNA sequence and mapping restriction sites

• Primer design

Donor site

Acceptor site

Donor site

Acceptor site

Transcription Start Site

Transcription Stop Site

An example of gene

AT1G09590.1 60S ribosomal protein L21

Promoter finding

• Find promoter (BDGP)• http://www.fruitfly.org/seq_tools/promoter.

>chr01 CHROMOSOME dumped from ADB: Jun/20/09 14:53; last updated: 2009-02-02CAGTATAATGTCACTAGGTGTTTGCATTGCTGTCTCTGGTTTCCAGGTACTCTACTTGAAGCAGTACTTTGAGAAGAAGAAGCTTATATAGAGATTCCCTGATATTCAGGTCCTTTAATGAGAGTTTCTTCTTAAAGAATAATATTCAATTCATCTCCTTTCTTTCCTCTTTACACGCCTCCTTTTTACTGACGCTGTAAAATTTTTGTTCATGGATTGTTTATGTAGCTTCTCTCTGATAAGAAGTCATTATTTCTGATTCTGAAATAATAATATTAACTTCTTGTCATCTCCAAAGTCCAAACGAAAGAGATTCTAGAACCATTGATATTGGGTTGTAACTGATACTTTATGTTAGTTTGGGGCTATAACACGTAAGTTTGACAGTACAAGGGTTAAGATTACAAGAAAAAACTAATCAAATGGGTATTACATACAAATCAGCCCAAATTTCTGACCCAACAGGCCCGTTAAGAGCAAACCCTAATTTCAAAGAGAATATATATAAACCCTAATCACATTTCGCAACCACCAAAGCGGAGGAAAAATGCCGGCTGGACATGGAGTTCGAGCGAGAACGAGGGATCTGTTCGCGAGGCCTTTCAGGAAGAAGGGTTACATTCCTCTATCGACTTACCTGAGGACCTTCAAGGTCGGCGATTACGTCGATGTGAAGGTGAATGGTGCGATCCACAAGGGTATGCCTCACAAGTTCTACCATGGTCGTACTGGTCGTATCTGGAACGTCACCAAGCGCGCCGTCGGTGTTGAAGTCAACAAACAGGTCTGATGATCTCTCACTTCCCTTAAACTTTTGTGTCCTTGAATTAGGATTTGAGCTTGTTGTGCTATTTGGATTGTTGATCTGTTGATTTGATGTTGAAATATGTGATTGAGTATTAGGTTTGTAGAATGTAAACCTTCTACTGTGCTCCAATTTAGCTCAGATTGTTAGTTTTGGCTCAAAGATCTTATCTTCTATTATGCAGTAGTGTGGTTCTTGATCCTGTTTCAGATTTGAAGTTTGATTGCTTAGGTCTCGTTTAGTCTTTAGCCTTAGCATGCATTGCGTTGTATCAATTTGGCCCTTTTTGCATCACAGTTCTAGCTAAGCATCTAGTTTTACTTCTCCATTGAACTGCTAGTATGTTTGGTTTCATGACTATCGCATTGCTCTAGTTTTGCTTTAAATGACCACCAACATTCGTATATTAATCTTGTTTCTATAAGTGTCTTAGAGATGTTATTGTGTAATGCTGATTAGCCTATGACCAGAACACAAACATTTTTGCTAGTACTACTTATATTGGGTATTATGACTGACATATGATGGTGGGGTTGTTTCAGATTGGGAACAGGATCATAAGGAAGAGGATTCATGTTCGTGTGGAGCATGTGCAGCAGTCAAGATGTGCCGAGGAGTTCAAGCTGAGGAAGAAGAAAAACGATGAGCTCAAGGCTGCAGCCAAAGCCAATGGTGAGACCATCAGCACCAAGAGACAGCCTAAAGGACCCAAACCAGGATTCATGGTCGAAGGAATGACCTTGGAGACTGTCACTCCAATCCCCTACGATGTCGTCAACGATCTCAAAGGAGGCTATTAGTTCTATCTTCTTGTGCTTTAGAACTCTTTTTCATTTGTTTTGTAGCAGACTTAAAACTAAGAATTATGATTTACTATATAACAGGAGTTTGCAATCCAGATTTTGTAAGGATGGTTTATTTTTATTATTGGTATTTTTGATCTTCCAACTATTGTTCACTTTTGTC

Promoter finding

• Find promoter (BDGP)• http://www.fruitfly.org/seq_tools/promoter.

html• Transcription Start Site at 532 bp

Finding introns

• Finding intron (CBS)• http://www.cbs.dtu.dk/index.shtml• CBS prediction server• NetPlantGene

Finding introns

• Intron position 785 -1347

Donor site

Acceptor site

Donor site

Acceptor site

Editing DNA sequence

• http://www.genomatix.de/cgi-bin/tools/tools.pl (Genomatix)

• Extracting sequence• Genomatix TOOLS form• Remove intron and untranscribed region.

Region retained: 532-784, 1348 - 1771

>chr01 CHROMOSOME dumped from ADB: Jun/20/09 14:53; last updated: 2009-02-02

>chr01 CHROMOSOME dumped from ADB: Jun/20/09 14:53; last updated: 2009-02-02CCAAAGCGGAGGAAAAATGCCGGCTGGACATGGAGTTCGAGCGAGAACGAGGGATCTGTTCGCGAGGCCT TTCAGGAAGAAGGGTTACATTCCTCTATCGACTTACCTGAGGACCTTCAAGGTCGGCGATTACGTCGATG TGAAGGTGAATGGTGCGATCCACAAGGGTATGCCTCACAAGTTCTACCATGGTCGTACTGGTCGTATCTG GAACGTCACCAAGCGCGCCGTCGGTGTTGAAGTCAACAAACAG ATTGGGAACAGGATCATAAGGAAGAGGATTCATGTTCGTGTGGAGCATGTGCAGCAGTCAAGATGTGCCG AGGAGTTCAAGCTGAGGAAGAAGAAAAACGATGAGCTCAAGGCTGCAGCCAAAGCCAATGGTGAGACCAT CAGCACCAAGAGACAGCCTAAAGGACCCAAACCAGGATTCATGGTCGAAGGAATGACCTTGGAGACTGTC ACTCCAATCCCCTACGATGTCGTCAACGATCTCAAAGGAGGCTATTAGTTCTATCTTCTTGTGCTTTAGA ACTCTTTTTCATTTGTTTTGTAGCAGACTTAAAACTAAGAATTATGATTTACTATATAACAGGAGTTTGC AATCCAGATTTTGTAAGGATGGTTTATTTTTATTATTGGTATTTTTGATCTTCCAACTATTGTTCACTTT TGTC

Editing DNA sequence

• Reverse–complement sequence: DNA has two strands

• Reformat sequence• Sequence statistics

Work on a single DNA sequence

• http://www.restrictionmapper.org/• Restriction Map (For DNA recombination

Donor site

Acceptor site

Donor site

Acceptor site

Finding Open Reading Frame

• http://www.ncbi.nlm.nih.gov/ • ORF finder

>chr01 CHROMOSOME dumped from ADB: Jun/20/09 14:53; last updated: 2009-02-02CCAAAGCGGAGGAAAAATGCCGGCTGGACATGGAGTTCGAGCGAGAACGAGGGATCTGTTCGCGAGGCCT TTCAGGAAGAAGGGTTACATTCCTCTATCGACTTACCTGAGGACCTTCAAGGTCGGCGATTACGTCGATG TGAAGGTGAATGGTGCGATCCACAAGGGTATGCCTCACAAGTTCTACCATGGTCGTACTGGTCGTATCTG GAACGTCACCAAGCGCGCCGTCGGTGTTGAAGTCAACAAACAG ATTGGGAACAGGATCATAAGGAAGAGGATTCATGTTCGTGTGGAGCATGTGCAGCAGTCAAGATGTGCCG AGGAGTTCAAGCTGAGGAAGAAGAAAAACGATGAGCTCAAGGCTGCAGCCAAAGCCAATGGTGAGACCAT CAGCACCAAGAGACAGCCTAAAGGACCCAAACCAGGATTCATGGTCGAAGGAATGACCTTGGAGACTGTC ACTCCAATCCCCTACGATGTCGTCAACGATCTCAAAGGAGGCTATTAGTTCTATCTTCTTGTGCTTTAGA ACTCTTTTTCATTTGTTTTGTAGCAGACTTAAAACTAAGAATTATGATTTACTATATAACAGGAGTTTGC AATCCAGATTTTGTAAGGATGGTTTATTTTTATTATTGGTATTTTTGATCTTCCAACTATTGTTCACTTT TGTC

Work on a single DNA sequence

• http://www.restrictionmapper.org/• Restriction Map (For DNA recombination

How to design primers for PCR

Annealing issues with primers

Stem loop Self dimer heterodimer

Primer issues

• Whether it is unique in the genome –specificity check

• Stem loop • Self dimer• Heterodimer

Primer design

• http://www.ncbi.nlm.nih.gov/tools/primer-blast/

• Primer design • Custom primer examined with IDT DNA

site• http://www.idtdna.com/analyzer/Applicatio

ns/OligoAnalyzer/

Fundamental Skills in DNA sequence analysis · Fundamental Skills in DNA sequence analysis Based on...

Documents