Question

How do researchers know if a particular gene examined via a Microarray scan is producing protein?

Is it possible for a person who is colorblind to successfully interpret a Microarray scan? Please explain.

Answer 1

DNA microarrays exploit the ability of complementary strands of nucleic acids to base-pair with each other and bind. For example, ATATGCGC will bind to its complement (TATACGCG) with a certain affinity. This method was first used by Sol Spiegelman to measure the homology (similarity) of two different nucleic acids; Spiegelman called the method "hybridization" of nucleic acids. Later, the developers of the DNA microarray dotted an array of DNA copies (cDNAs) corresponding to a large number of different mRNAs of known sequence onto a glass slide. Because this array was so tiny, it was termed a microarray. Although the cDNAs were double-stranded, they could be melted, or denatured, to single strands, which could then be used to bind, or hybridize, to fluorescently labelled nucleic acid samples from cancerous or normal cells. After washing away the unbound molecules, bound fluorescent nucleic acid samples were identified by laser microscopy. Fluorescent dots indicated expressed genes, and differences in microarray patterns between normal and cancerous cells could be quickly identified.

In these early microarray experiments, mRNA from one cell type was made into cDNA labelled with a red fluorescent dye, and mRNA from another cell type was made into cDNA labelled with a green fluorescent dye. The two cDNAs were then mixed and hybridized to the same DNA microarray, resulting in red, green, and yellow dots (caused by a combination of red and green), as well as black dots. Comparative gene expression in the two samples could easily be determined by quantitating the ratio of red and green fluorescence in the spot corresponding to each gene.

Microarray Technique

An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots.

Thousands of spotted samples known as probes (with known identity) are immobilized on the solid support (microscope glass slides or silicon chips or nylon membrane). The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene.

Types of Microarrays

Depending upon the kind of immobilized sample used construct arrays and the information fetched, the Microarray experiments can be categorized in three ways:

1. Microarray Expression Analysis: In this experimental setup, the cDNA derived from the mRNA of known genes is immobilized. The sample has genes from both the normal as well as the diseased tissues. Spots with more intensity are obtained for diseased tissue gene if the gene is overexpressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease.

2. Microarray for Mutation Analysis: For this analysis, the researchers use gDNA. The genes might differ from each other by as less as a single nucleotide base.

A single base difference between two sequences is known as Single Nucleotide Polymorphism (SNP) and detecting them is known as SNP detection.

3. Comparative Genomic Hybridization: It is used for the identification in the increase or decrease of the important chromosomal fragments harbouring genes involved in a disease.

Applications of Microarrays

Gene Discovery: DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels under different conditions.

Disease Diagnosis: DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumours develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumour cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.

Drug Discovery: Microarray technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.

Toxicological Research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes the correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants.

In each type of cell, like a muscle cell or a skin cell, different genes are expressed (turned on) or silenced (turned off). If the cells that are turned on mutate, they could—depending on what role they play in the cell—trigger the cell to become abnormal and divide uncontrollably, causing cancer. By identifying which genes in the cancer cells are working abnormally, doctors can better diagnose and treat cancer. One way they do this is to use a DNA microarray to determine the expression levels of genes. When a gene is expressed in a cell, it generates messenger RNA (mRNA). Overexpressed genes generate more mRNA than underexpressed genes. This can be detected on the microarray. The first step in using a microarray is to collect healthy and cancerous tissue samples from the patient. This way, doctors can look at what genes are turned on and off in the healthy cells compared to the cancerous cells. Once the tissues samples are obtained, the messenger RNA (mRNA) is isolated from the samples. The mRNA is colour-coded with fluorescent tags and used to make a DNA copy (the mRNA from the healthy cells is dyed green; the mRNA from the abnormal cells is dyed red.)

The DNA copy that is made, called complementary DNA (cDNA), is then applied to the microarray. The cDNA binds to complementary base pairs in each of the spots on the array, a process known as hybridization. Based on how the DNA binds together, each spot will appear red, green, or yellow (a combination of red and green) when scanned with a laser. • A red spot indicates that that gene was strongly expressed in cancer cells. (In your experiment these spots will be dark pink.) • A green spot indicates that that gene was strongly repressed in cancer cells. (In your experiment these spots will be light pink.) • If a spot turns yellow, it means that that gene was neither strongly expressed nor strongly repressed in cancer cells. (In your experiment these spots will be clear.) • A black spot indicates that none of the patient’s cDNA has bonded to the DNA in the gene located in that spot. This indicates that the gene is inactive. (All of the genes in your experiment are active.)