DNA MICROARRAY TECHNOLOGY: APPLICATION IN CANCER GENE DETECTION AND DISCOVERY

A SEMINAR PRESENTED BY

FATOKI JOHN OLABODE 153475

CANCER RESEARCH AND MOLECULAR BIOLOGY UNIT DEPARTMENT OF BIOCHEMISTRY FACULTY OF BASIC MEDICAL SCIENCES COLLEGE OF MEDICINE UNIVERSITY OF IBADAN
FEBRUARY, 2011

OUTLINE
Introduction What is DNA Microarray Technology Principle of DNA Microarray Technology Microarray Production Process Target Preparation Hybridization Slide Scanning and Data Analysis Application in Cancer Gene Discovery and Detection.

INTRODUCTION The American Heritage Dictionary defines "array" as "to place in an orderly arrangement".

A microarray therefore, is an analytical device that comprises an array of molecules immobilized at discrete ordered locations on the surface of an insoluble solid support.

INTRODUCTION CONTD These devices have been highly effective for the simultaneous detection of large numbers of analytes in a sample, Microarrays have quickly emerged as important analytical tools in many branches of the biological sciences. A microarray-based analytical strategy is quicker and more convenient than serial testing for each analyte.

INTRODUCTION CONTD The current scope of microarray applications includes;

sequencing by hybridization, resequencing, mutation detection, assessment of gene copy number, comparative genome hybridization, drug discovery, expression analysis, etc.

INTRODUCTION CONTD Microarrays are a significant advance both

because they may contain a very large number of genes and because of their small size. Microarrays are therefore useful when one wants to survey a large number of genes quickly or when the sample to be studied is small. Microarrays may be used to assay gene expression within a single sample or to compare gene expression in two different cell types or tissue samples, such as in healthy and diseased (for instance cancer) tissue.

INTRODUCTION CONTD Because a microarray can be used to examine the expression of hundreds or thousands of genes at once, it promises to revolutionize the way scientists examine gene expression.

What is DNA Microarray Technology

DNA Microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations.

The supports themselves are usually glass microscope slides, but can also be silicon chips or nylon membranes.
The DNA is printed, spotted, or actually synthesized directly onto the support.

With DNA Microarray Technology, researchers can measure changes in gene expression and are therefore able to learn how cells respond to a disease or to some other challenges

DEFINITION OF TERMS TARGET The target is the DNA molecule that is attached to the microarray substrate that reacts with a complementary probe molecule in solution. PROBE The probe is the sample (mRNA molecule) in solution that is labeled with fluorescent tags, and reacts with a complementary target molecule on the substrate.

Principle of DNA Microarray.

DNA Microarray Technology works by exploiting the ability of an mRNA to bind specifically to, or hybridized to, the DNA template from which it was originated (Kane et al, 2000). In other words, the whole process of DNA Microarray is based on hybridization probing.

Each single-stranded DNA fragment is made up of four different nucleotides, adenine (A), thymine (T), guanine (G), and cytosine (C). Adenine is the complement of, or will always pair with, thymine, and guanine is the complement of cytosine.

Therefore, the complementary sequence to G-T-C-C-T-A will be C-A-G-G-A-T. When two complementary sequences find each other, such as the immobilized target and the mobile probe, they will hybridize.

Figure 1 DNA denaturation and renaturation. Source: Nelson and Cox 2005.

Microarray Production Process (Target):

DNA microarrays are created by spotting every gene in a genome onto a glass microscope slide (probe). The targets use are usually cDNA, DNA fragments that are amplified by PCR technique, or presynthesized oligonucleotides. Either of these are spotted on the microscopic glass slide coated with polylysine prior to spotting process.

Figure 2 Array Fabrication Process Source: http://www.gene-chips.com/

Figure 3: DNA Microarray by Affymetrix

Figure 4 DNA Microarray

Probe Preparation:
Messenger RNA are extracted from the tissues of interest, from which the gene expression level is to be compared. Messenger RNA are then transcribed to cDNA by reverse transcription.

DNA from the first tissue of interest (normal cell line) is labeled with a green dye.

DNA from the second tissue of interest (for instance, cancer cell line) is labeled with a red dye.

 Figure 5: Probe Preparation Process. Source: http://www.gene-chips.com/

Hybridization: cDNAs are then hybridized with the microarray. The chip is then incubated for one night at 42 oC. At this temperature, a DNA strand that encounter the complementary strand bind together to create a double strand DNA. The fluorescent DNA (probe) will then hybridizes on the spotted ones. The microarray is then washed and scanned for red and green cDNA (spot on the microarray).

Figure 6: Hybridization Process

Source:
http://learn.genetics.utah.edu/content/labs/microarra y/

Figure 7 Hybridization

 Slide Scanning: A laser (Light Amplification by Stimulated Emission of Radiation) is use to excites each spot and the fluorescent emission gather through a photo-multiplicator (PMT) coupled to a confocal microscope.
Two images will then be obtained. By superimposing the two images, one image composed of spots going from green spots (where only DNA from the first condition is fixed) to red (where only DNA from the second condition is fixed) passing through the yellow color (where DNA from the two conditions are fixed on equal amount) is obtained.

Figure 8: A scanned slide

Source: http://learn.genetics.utah.edu/content/labs/microarray/

 Data Analysis: This can be done using statistical analysis. However, since statistical analysis is complicated, a number of software have been develop. The differences in gene expression can be visualize and/or analyze using any of these software; which is composed of program that run data analysis automatically on a computer system. An example is the one developed in the laboratory called ArrayPlot. This software is use to analyze and measure the intensities of each spot and display the red intensities of each spot as a function of the green intensities.

 If a particular gene is highly expressed, it synthesized many molecules of messenger RNA, which hybridize to the DNA on the microarray and generate a very bright fluorescent area. Genes that are not highly expressed synthesized fewer mRNAs, which results in dimmer fluorescent spots. If there is no fluorescence, none of the mRNA molecules have probably hybridized to the DNA, indicating that the gene is relatively inactive.

Figure 8: Overview of DNA Microarray Technology Process.

Source: http://www.gene-chips.com/

 DNA Microarray Technology: Application in Cancer Gene Discovery and Detection:

To better understand, diagnose and treat liver cancer, Li and co-workers at Fudan University, China used microarrays to identify genes that show elevated or reduced expression in cancerous liver tissue isolated from patients afflicted with the disease.

Six patients were screened using microarrays containing cDNA derived from 12,800 human genes, and analysis of the data indicated changes in gene expression of many human genes when normal and cancerous tissues were compared.
Of the genes examined, 199 showed elevated expression in liver cancer tissue and 1,633 exhibited reduced expression

In a seminal publication, Golub et al. [1999] analyzed the expression of ~6,800 genes in bone marrow from 38 patients with acute leukemia (27 with the acute lymphoblastic form, ALL, and 11 with the acute myeloid form, AML). Fifty genes whose levels of expression differed most between AML and ALL cells were selected. Using this subset of genes, the investigators were able to correctly identify which patients had AML and which had ALL

 In an attempt to identify the primary origin of metastatic cancer, Su et al.[2001] used the information gained from the gene expression profile analysis of 100 primary cancers as a training set.
A further 75 blinded primary and metastatic tumors were used as a test set. Prediction accuracies of 97% and 95% were obtained in the training and test sets, respectively. With as few as 11 genes, the authors could predict the anatomic origin of 91% and 83% of the training and blinded samples, respectively.

 DNA Microarray Technology therefore, offers the potential to identify, in an unbiased manner, genes expressed specifically in cancer or at least genes showing the greatest difference in expression between malignant and corresponding benign or normal tissue.
The major advantage of DNA microarray over more traditional approaches is that, it identified combinations of tumor markers. And this will enhance the positive predictive value of diagnostic testing and minimize the proportion of false-positive and false-negative results [Srinivas et al., 2001].

Although DNA microarray technology is unlikely to replace conventional histopathology in the foreseeable future for the primary diagnosis of cancer.
It could in certain situations provide more detailed diagnostic information, classify or differentiate between morphologically similar types of malignancy and help identify metastatic cancers of unknown primary origin.

CONCLUSION DNA microarrays can be used to aid the differentiation of tumors with similar morphological appearance, independently of conventional prognostic factors.

If confirmed and transferred to the clinic, DNA microarray technology will undoubtedly result in improved diagnosis and management of certain groups of patients with specific cancers.
It is unlikely, however, that DNA microarray technology will fully replace existing procedures, at least in the near future. Rather, the old and new approaches are likely to be combined resulting in a more individualized approach to cancer patient management.

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