Bioinformatics: Microarray Technology. Assc.Prof. Chuchart Areejitranusorn AMS. KKU.

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1 Introduction to Bioinformatics: Microarray Technology Assc.Prof. Chuchart Areejitranusorn AMS. KKU.

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3 ความจร งเก ยวก บ ความจรงเกยวกบ Cell and DNA Cell Nucleus Chromosome Protein Gene (mrna), single strand Gene (DNA)

4 DNA RNA polymerase Transcription RNA Reverse Transcription RNA dependent DNA polymerase Transla ation PROTEIN

5 ความจร งเก ยวก บ Cell and DNA All living organisms consist of cells. Humans have trillions of cells; Yeast - one cell. Cells are of many different types (blood, skin, nerve), but all arose from a single cell (the fertilized egg)

6 ความจร งเก ยวก บ Cell and DNA Each cell contains a complete copy of the genome (the program for making the organism), encoded in DNA. A gene is a segment of DNA that specifies how to make a protein. Human DNA has about 30-35,000 genes

7 Gene Expression Cells are different because of differential gene expression. About 40% of human genes are expressed at one time.

8 Gene Expression Gene is expressed by transcribing DNA into single-stranded mrna mrna is later translated t into a protein Microarrays measure the level l of mrna expression

9 Microarray Device A Microarray is a device detects the presence and abundance of labelled nucleic acids in a biological l sample. In the majority of experiments, the labelled nucleic acids are derived from the mrna of a sample or tissue.

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11 Designing the Probes high specificity to avoid hybridization with wrong target molecules. an output that is easy to read high sensitivity to detect the mrna and the intensity of the spot light must be differentiable from background noise.

12 Designing the Probes The intensity of a spot light also needs to correlate with the abundance of the target molecule in the sample. Results must be reproducible across multiple l experiments.

13 cdna Probe polymerase l chain reaction (PCR) products (cdnas) amplified DNA is purified, the clones are typically long sequences

14 Oligo probe(in-situ Synthesis Affymetrix)

15 The Array An oligonucleotide, l or oligo as it is commonly called, is a short fragment of a single-stranded DNA that is typically y 5 to 50 nucleotides long.

16 The Operation of the Spotting Robot The pins are dipped into the wells to collect the first batch of DNA. This DNA is spotted onto a number of different arrays, depending on the number of arrays being made and the amount of liquid the pins can hold. The pins are washed to remove any residual solution and ensure no contamination of the next sample. The pins are dipped into the next set of wells. Return to step 2 and repeat until the array is complete.

17 Comparison of Probe Types Oligos probe Advantages No need to isolate and purify cdnas Short oligonucleotides are less likely to have cross-reactivity with other sequences in the target DNA. Density of chips is higher than with cdnas. cdna Probes Advantages Flexibility to study cdnas from any source. cdnas do not require any a priori information about the corresponding genes. Longer sequences increase hybridization specificity, c which reduces false positives.

18 Comparison of Probe Types cdna Probes Oligos probe Limitations The sequence has to be known. Synthesis can be expensive and time- consuming. The short sequences are not as specific for target DNA, so appropriate controls must be added. Limitations Isolation of individual cdnas to immobilize on each spot can be cumbersome. Density is lower cdnas are longer sequences and are more likely to randomly contain sequences found in target DNA, which results in cross-

19 Steps of a Microarray Experiment choosing probes. Generate a hybridization solution containing a mixture of fluorescently labelled targets. Incubate hybridization mixture. Detect probe hybridization using laser technology Analyze data using computational y g p methods.

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21 In Single label l experiments, only one sample is hybridised to the arrays labelled with one dye. (in which case control needs to be measured using a separate chip).

22 Dual Label Experiments Most laboratories use fluorescent labelling, with the two dyes Cy3 (excited by a green laser) and Cy5 (excited by a red laser). In Dual label experiments, two samples are hybridised to the arrays, one labelled with each dye; this allows the simultaneous measurement of two samples (e.g. for differential analysis)

23 Dual Label Experiments + Green label + Red label RNA sample 1 RNA sample 2 Typically used to study one sample (e.g. diseased tissue) vs. a control sample (e.g. normal tissue)

24 The Process elled targets in solution Probes on array Heteroduplexes Hybridisation

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26 Qualitative Interpretation of Double Label Experiments GREEN = High Control hybridization RED = High Sample hybridization YELLOW = combination of Control and Sample where both hybridized d equally. BLACK = neither the Control nor Sample hybridized.

27 Spot Quality Problems

28 From Microarray images to Gene Expression Matrices Raw data Array scans Intermediate data Images Final data Gene Expression Matrix Samples Spot ts Gen nes Spot/Image quantiations Gene expression levels

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30 PM to maximize hybridization MM to ascertain the degree of cross-hybridization Affymetrix Gene Chips Perfect Matches and Mismatches

31 Microarray reader

32 Influenza strains

33 Commercial kits SARS Hepatitis TB Mycoplasma pneumonea Entero virus infection Papilloma virus Food Milk Etc.

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37 References สว สด สวสด com/ chip.comcom p// om