DNA Microarray and its application in DNA Microarray and its application in Cancer diagnosis Cancer diagnosis

ByBy

Brijesh Singh YadavBrijesh Singh YadavDivision of Biochemistry Division of Biochemistry

Indian Veterinary Research instituteIndian Veterinary Research institute

Izatnagar,India.243122Izatnagar,India.243122

What we will be discussingWhat we will be discussing……

• What is DNA microarray?• The purpose of using DNA microarray• Oligonucleotide Probes• The plate.• Steps to perform a microarray.• Application in Cancer Biology.• Future perspectives.

What is DNA Microarray?What is DNA Microarray?

• Scientists used to be able to perform genetic analysis of a few genes at once. DNA microarray allows us to analyze thousands of genes in one experiment!

• It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, each containing a specific DNA sequence, known as probes.

PurposesPurposes• So why do we use DNA microarray?

– To measure changes in gene expression levels – two samples’ gene expression can be compared from different samples, such as from cells of different stages of mitosis.

– To observe genomic gains and losses. Microarray Comparative Genomic Hybridization (CGH)

– To observe mutations in DNA.

Oligonucleotide ProbesOligonucleotide Probes

A piece of DNA that binds specifically to a particular gene or part of a gene.

• The sequence of the gene probe must be complementary to the nucleotide bases of the specific mRNA or DNA sequence of interest.

• Gene probes can be as small as 20-40 base pairs or be up to 1000bp long.

The PlateThe Plate• Usually made commercially,made of glass, silicon, or nylon.• Each plate contains thousands of spots, and each spot

contains a probe for a different gene.• Probes can either be attached by robotic means, where a

needle applies the cDNA to the plate, or by a method similar to making silicon chips for computers. The latter is called a Gene Chip.

Let’s perform a microarray!Let’s perform a microarray!

1) Probes Designing2) Collect Samples.3) Isolate mRNA.4) Create Labelled DNA.5) Hybridization.6) Microarray Scanner.7) Analyze Data.

STEP 1: STEP 1: Probes DesigningProbes Designing

• DNA probes should be designed considering the various aspect such as melting temperature (Tm),guanine /cytonine (G/C)content, secondary structure, length and binding position within the target DNAs

• These factor can effect the signal intensity, specificity and sensitivity following hybridization.

• Longer probes are more specific to individual target genes, and shorter probes may be spotted across the array in higher densities and are cheaper to manufacture.

STEP 2:STEP 2: Isolate mRNAIsolate mRNA..• Extract the RNA from the samples. Using either a column,

or a solvent such as phenol-chloroform.

• After isolating the RNA, we need to isolate the mRNA from the rRNA and tRNA. mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind the mRNA.

• Rinse with buffer to release the mRNA from the beads. The buffer disrupts the pH, disrupting the hybrid bonds.

STEP 3a: Collect SamplesSTEP 3a: Collect Samples

This can be from a variety of organisms. We’ll use two samples – cancerous human skin tissue & healthy human skin tissue

STEP 3bSTEP 3b: Create Labelled : Create Labelled DNA.DNA.

Add a labelling mix to the RNA. The labelling mix contains poly-T (oligo dT) primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides.

We will add cyanine 3 (fluoresces green) to the healthy cells and cyanine 5 (fluoresces red) to the cancerous cells.

The primer and RT bind to the mRNA first, then add the fluorescently dyed nucleotides, creating a complementary strand of DNA

STEP 4: HybridizationSTEP 4: Hybridization..

• Apply the cDNA we have just created to a microarray plate.

• When comparing two samples, apply both samples to the same plate.

• The ssDNA will bind to the cDNA already present on the plate.

STEP 5a: LASERS!STEP 5a: LASERS!

STEP 5b: Microarray STEP 5b: Microarray Scanner.Scanner.

The scanner has a laser, a computer, and a camera.

The laser causes the hybrid bonds to fluoresce.

The camera records the images produced when the laser scans the plate.

The computer allows us to immediately view our results and it also stores our data.

STEP 6: Analyze the STEP 6: Analyze the DataData..

GREEN – the healthy sample hybridized more than the diseased sample.

RED – the diseased/cancerous sample hybridized more than the non diseased sample.

YELLOW - both samples hybridized equally to the target DNA.

BLACK - areas where neither sample hybridized to the target DNA.

By comparing the differences in gene expression between the two samples, we can understand more about the genomics of a disease.

Application in Cancer BiologyApplication in Cancer Biology

CancerCancer

• CANCER is a genetic disorder, mostly resulting from acquired mutations and epigenetic changes that influence gene expression.

• The use of DNA microarrays for the comprehensive analysis of RNA expression (expression profiling) in human tumor sample should much promise.

Electron Microscope Photographs of Cancer CellsElectron Microscope Photographs of Cancer Cells

Breast Cancer Cell Brain Cancer Cell 

Cancer cell being attacked by the immune system Prostate cancer cell

Microarray in CancerMicroarray in Cancer

• The advent of the technology of DNA microarrays constitutes an epochal change in the classification and discovery of different types of cancer because the information provided by DNA microarrays allows an approach to the problem of cancer analysis from a quantitative rather than qualitative point of view.

• Cancer classification requires well founded mathematical methods which are able to predict the status of new specimens with high significance levels starting from a limited number of data.

Working with cancerWorking with cancer

• The mRNA is a copy of the DNA and, therefore, is complimentary to the DNA it was copied from.

• mRNA is placed onto the chip, the mRNA that was expressed from the DNA will bind onto the DNA that it is complementary.

• As a result, the mRNA that binds with the DNA on the chip will give off a fluorescent dye that can be analyzed by a computer. The brighter the color that is present on the computer screen, the more active the gene is and the less color, or even no color, means that a gene is not being expressed.

DNA MicroarrayDNA Microarraychromosome

PCR

Gene A Gene C

Arrayer

Gene A Gene BGlassSlide

Purification of mRNA

Labeling during RT

Gene A Gene B

16 hr, 42C Wash

Scan

Image Analysis:

(Probe)(Target)

samplle-1 samplle-2

Gene C

Gene C

Gene B

samplle-2 samplle-1,2 samplle-1

Microarray

Cancer Data AnalysisCancer Data Analysis

Expression data

Microarray

Feature Selection

Cancer Predictor

Tumor Normal

Pearson's correlation coefficientSpearman's correlation coefficientEuclidean distanceCosine coefficientInformation gainMutual informationSignal to noise ratio

3-layered MLP with back propagationk-nearest neighborSupport vector machineStructure adaptive self-organizing mapEnsemble classifier

Cancer classification system

The Future of DNA MicroarrayThe Future of DNA Microarray

• Gene discovery. DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels .

• Disease diagnosis. classify the types of cancer on the basis of the patterns of gene activity in the tumor cells.

• Pharmacogenomics. is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients.

• Toxicogenomics. microarray technology allows us to research the impact of toxins on cells. Some toxins can change the genetic profiles of cells, which can be passed on to cell progeny.

Personalized MedicinePersonalized Medicine

• Expression patterns and levels, referred to as the brightness from the fluorescent dye, allows scientists to be able to determine which genes are being active in a cancerous cell.

• Personalized treatment plans can be developed for each individual patient.

Sources…..Sources….. DNA Microarray Technology. National Human Genome Research Institute, 17

Dec. 2009. 19 Feb. 2010 <http://www.genome.gov/10000533>

Microarrays: Chipping Away at the Mysteries of Science and Medicine. National Center for Biotechnology Information, 27 July 2007. 19 Feb. 2010. <http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html>

Brown, P.O. & Botstein, D. Exploring the New World of the Genome with DNA Microarrays. Nature Genetics Supplement. 21. (1999): 33-37. <http://www.ctu.edu.vn/~dvxe/Bioinformatic/PDF%20Files/Volume21/ng0199supp_33.pdf>

Simon, R., Radmacher, M.D., Dobbin, K., & McShane, L.M. Pitfalls in the Use of DNA Microarray Data for Diagnostic and Prognostic Classification. Journal of the National Cancer Institute. 95. (2003): 14-18. http://jnci.oxfordjournals.org/cgi/content/full/95/1/14

Holloway, A.J., Van Laar, R.K., Tothill, R.W., & Bowtell, D.D.L. Options Available – From Start to Finish – For Obtaining Data From DNA Microarrays II. Nature Genetics Supplement. 32. (2002): 482-489. <http://web.cs.mun.ca/~harold/Courses/Old/CS6754.W04/Diary/ng1030.pdf>

Special Thanks to…………Special Thanks to…………

• Dr.Bhaskar Sharma(ICAR, National Professor, IVRI)

• Dr.K.P.Mishra (VC, Nehru Gram Bharti University )

• Dr.Ajay Kumar(Scientist, Division of Biotechnology, IVRI)

• Dr.Meeta Saxena(Scientist, Division of Biochemistry,IVRI)

• Mr.Mayank Pokhriyal (RA, Division of Biochemistry,IVRI)

• Dr.Barkha Ratta (RA, Division of Biochemistry,IVRI)

• Mr.Gaurav Rai (SRF, Division of Biochemistry,IVRI)

Send your suggetion to me at my Send your suggetion to me at my email:-brijeshbioinfo@gmail.comemail:-brijeshbioinfo@gmail.com