Fundamental Skills in DNA sequence analysis · Fundamental Skills in DNA sequence analysis Based on publicly available tools Ning Jiang, Dept. of Horticulture

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.

html

>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

http://www.cbs.dtu.dk/index.shtml


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

http://www.genomatix.de/cgi-bin/tools/tools.pl




>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

work)

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

work)


How to design primers for PCR

T

T

TT

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/


Documents

Fundamental Skills in DNA sequence analysis · Fundamental Skills in DNA sequence analysis Based on publicly available tools Ning Jiang, Dept. of Horticulture