MICROARRAY TECHNOLOGY By Sehreen Saddique Ibtesam Riaz Bakhtawer Nasrullah University of Gujrat.
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Transcript of MICROARRAY TECHNOLOGY By Sehreen Saddique Ibtesam Riaz Bakhtawer Nasrullah University of Gujrat.
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MICROARRAY TECHNOLOGY
BySehreen Saddique
Ibtesam Riaz Bakhtawer NasrullahUniversity of Gujrat
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OUTLINE
1. Introduction of microarray1. Introduction of microarray
3. Applications of microarray3. Applications of microarray
2. Types of microarray 2. Types of microarray
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DNA MICROARRAY
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Gene expression
is used to measure the levels of gene expression DNA MICROARRAY
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If the hybridised DNA or RNA is labelled fluorescently it can be quantified by scanning of the chip.
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DNA microarrays can be manufactured by:
• Photolithography
• Robot spotting
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Photolithography
• Each probe 25 bp long• 22-40 probes per gene• Perfect Match (PM) as
well as MisMatch (MM) probes
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Robot Spotting
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DNA MICROARRAY TECHNIQUE
• Presynthesized DNA strands or synthesis of oligonuleotide sequence
• Spotting of DNA strands to plastic plate or glass plate
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DNA MICROARRAY TECHNIQUE
• The two samples to be compared are grown/acquired. In this example treated sample (case) and untreated sample (control).
• Extraction of mRNA material from cells
• Formation of complementary DNA through reverse transcription
• Labeling with fluorescent dyes with Cy3 or Cy5
• Mixing of labeled samples & presenting to microarray spots again by spotters
• Hybridization to a DNA Microarray
• Scanning the Hybridized Array
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ANALYSIS OF GENE EXPRESSION DATA
• Analyzing issues
• Different environmental conditions
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TYPES OF MICROARRAY
SEHREEN SADDIQUE
ROLL NO:08030614-045
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Types of microarray
• There are two types of DNA microarray
• c DNA
• Oligoneuclotide array
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c DNA MICROARRAY
1. Prepare sample.
Test Reference
2. Label with fluorescent dyes.
3. Combine cDNAs.
4. Print microarray.
5. Hybridize to microarray.
6. Scan.
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Nelzo ErefulCrop Bioinformatics
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cDNA Array Sample Preparation
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APPLICATIONS OF MICROOARRAY
BAKHTAWER
ROLL:08030614-047
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Applications of microarray
Drug Discovery and development
Evolutionary and ecological
genomics
Tiling array Tumor classification
Gene expression
(stress)
Microarray
SNP detection
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Applications of microarray
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Application in ecology
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Microarray and asthma
Disease discovery
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Medical application of microarray analysis
treatment of cancer may be tailored to the needs of that individual’s gene expression profile.
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Tumor classification
Quackenbush 2006; Retrieved from http://content.nejm.org/cgi/content/full/354/23/2463
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tolerant ATGTGTGGAGGAGAAGTGATCCCCGCCGACATGCCGGCGGCGCCGTTCACGCCACGCCAC 60 susceptible ------------------------------ATGCCGGCGGCGCCGTTCACGCCACGCCAC 30 ******************************
tolerant GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAGGAAGCGCGGC 120 susceptible GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAGGAAGCGCGGC 90 ************************************************************
tolerant GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGACGACGACGAC 180 susceptible GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGACGACGACGAC 150 ************************************************************
tolerant GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 240 susceptible GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 210 ************************************************************
tolerant GCAGGCCGGGGCGCCGCCGCCACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 300 susceptible GCAGGCCGGGGCGCCGCCGCCACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 270 ************************************************************
tolerant GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 360 susceptible GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 330 ************************************************************
tolerant GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 420 susceptible GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 390 ************************************************************
tolerant GTCCGCCTCTGGCTCGGCACCTTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 480 susceptible GTCCGCCTCTGGCTCGGCACCTTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 450 ************************************************************
tolerant GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 540 susceptible GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 510 ************************************************************
tolerant TCGCCGCCGCCGCCGTCGCAGCCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 600 susceptible TCGCCGCCGCCGCCGCCGCAGCCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 570 *************** ********************************************
tolerant ATCGGAGAAGCGCCGACCGACGACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 660 susceptible ATCGGAGAAGCGCCGACCGACGACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 630 ************************************************************
tolerant GGCGACGCGCGCATACAACTGGAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 720 susceptible GGCGACGCGCGCATACAGCTGGAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 690 ***************** ******************************************
tolerant GGCTACGACGTGGCCAGCGGCGACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 780 susceptible GGCTACGACGTGGCCAGCGGCGACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 750 ************************************************************
tolerant TCTGTTAACCAAGAGATCAAGACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 840 susceptible TCTGTTAACCAAGAGATCAAGACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 810 ************************************************************
tolerant CGCCCCATGACTTGTCACTTTAAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 900 susceptible CGCCCCATGACTTGTCACTTTAAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 870 ************************************************************
tolerant ACCATGCTAAACTTACTCAAAGGCCACAAACAATAA 936 susceptible ACCATGCTAAACTTACTCAAAGGCCACAAACAATAA 906 ************************************
CCGTCGC
CCGCCGC
tolerant ATGTGTGGAGGAGAAGTGATCCCCGCCGACATGCCGGCGGCGCCGTTCACGCCACGCCAC 60 susceptible ------------------------------ATGCCGGCGGCGCCGTTCACGCCACGCCAC 30 ******************************
tolerant GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAGGAAGCGCGGC 120 susceptible GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAGGAAGCGCGGC 90 ************************************************************
tolerant GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGACGACGACGAC 180 susceptible GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGACGACGACGAC 150 ************************************************************
tolerant GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 240 susceptible GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 210 ************************************************************
tolerant GCAGGCCGGGGCGCCGCCGCCACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 300 susceptible GCAGGCCGGGGCGCCGCCGCCACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 270 ************************************************************
tolerant GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 360 susceptible GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 330 ************************************************************
tolerant GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 420 susceptible GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 390 ************************************************************
tolerant GTCCGCCTCTGGCTCGGCACCTTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 480 susceptible GTCCGCCTCTGGCTCGGCACCTTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 450 ************************************************************
tolerant GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 540 susceptible GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 510 ************************************************************
tolerant TCGCCGCCGCCGCCGTCGCAGCCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 600 susceptible TCGCCGCCGCCGCCGCCGCAGCCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 570 *************** ********************************************
tolerant ATCGGAGAAGCGCCGACCGACGACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 660 susceptible ATCGGAGAAGCGCCGACCGACGACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 630 ************************************************************
tolerant GGCGACGCGCGCATACAACTGGAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 720 susceptible GGCGACGCGCGCATACAGCTGGAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 690 ***************** ******************************************
tolerant GGCTACGACGTGGCCAGCGGCGACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 780 susceptible GGCTACGACGTGGCCAGCGGCGACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 750 ************************************************************
tolerant TCTGTTAACCAAGAGATCAAGACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 840 susceptible TCTGTTAACCAAGAGATCAAGACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 810 ************************************************************
tolerant CGCCCCATGACTTGTCACTTTAAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 900 susceptible CGCCCCATGACTTGTCACTTTAAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 870 ************************************************************
tolerant ACCATGCTAAACTTACTCAAAGGCCACAAACAATAA 936 susceptible ACCATGCTAAACTTACTCAAAGGCCACAAACAATAA 906
************************************
CCGTCGC = tolerant (S186)
CCGCCGC = intolerant (P186)
ACAACTG = tolerant
ACAGCTG = intolerant
SNP
SNP detection using Microarray
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Generating DNA Sequence
base callingquality clippingvector clippingcontig assembly
chromatogram files
automatedsequencer
software pipeline
>GENE01
ACCTGTCAGTGTCAACTGCTTCAATAGCTAATGCTAGGCTCGATAATCGCTGGCCTCAGCTCAGTCTAGCATTACGATTACGGAGACCTATGCTTTAGCTAGTAGGAACCTCAGCTCAGTACCTGTCAGTGTCAACTGCTTCAATAGCTAATGCTACTC
>GENE01
ACCTGTCAGTGTCAACTGCTTCAATAGCTAATGCTAGGCTCGATAATCGCTGGCCTCAGCTCAGTCTAGCATTACGATTACGGAGACCTATGCTTTAGCTAGTAGGAACCTCAGCTCAGTACCTGTCAGTGTCAACTGCTTCAATAGCTAATGCTACTC
output
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