DNA chips

 

It is widely believed that thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain.

In the past few years, as a result of the Human Genome Project, there has been an explosion in the amount of information available about the DNA sequence of the human genome. Consequently, researchers have identified a large number of novel genes within these previously unknown sequences. The challenge currently facing scientists is to find a way to organize and catalog this vast amount of information into a usable form. Only after the functions of the new genes are discovered will the full impact of the Human Genome Project be realized, so that researchers can have a better picture of the interactions among thousands of genes simultaneously.

The microarrays theory relies on the following theory - every cell of the body contains a full set of chromosomes and identical genes (with only few exceptional cells). Only a fraction of these genes are turned on, however, and it is the subset that is "expressed" that confers unique properties to each cell type.

"Gene expression" is the term used to describe the transcription of the information contained within the DNA, the repository of genetic information, into messenger RNA (mRNA) molecules that are then translated into the proteins that perform most of the critical functions of cells. Scientists study the kinds and amounts of mRNA produced by a cell to learn which genes are expressed, which in turn provides insights into how the cell responds to its changing needs. Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. This mechanism acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes as necessary.

Why Are Microarrays Important?

What are Microarrays will be described in the next paragraph. Before the actual definition, it is important to understand that 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 tissue. 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. General studies of the expression levels in various kinds of cells represent an important and necessary first step in our understanding and cataloging of the human genome.

As more information accumulates, scientists will be able to use microarrays to ask increasingly complex questions and perform more intricate experiments. With new advances, researchers will be able to infer probable functions of new genes based on similarities in expression patterns with those of known genes. Ultimately, these studies promise to expand the size of existing gene families, reveal new patterns of coordinated gene expression across gene families, and uncover entirely new categories of genes. Furthermore, because the product of any one gene usually interacts with those of many others, our understanding of how these genes coordinate will become clearer through such analyses, and precise knowledge of these inter-relationships will emerge. The use of microarrays may also speed the identification of genes involved in the development of various diseases by enabling scientists to examine a much larger number of genes. This technology will also aid the examination of the integration of gene expression and function at the cellular level, revealing how multiple gene products work together to produce physical and chemical responses to both static and changing cellular needs. The microarray (DNA chip) technology is having a significant impact on genomics study. Drug discovery and toxicological research, will certainly benefit from the use of DNA microarray technology.

 

 

What Exactly Is a DNA Microarray?

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, the size of two side-by-side pinky fingers, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support. With the aid of a computer, the amount of mRNA bound to the spots on the microarray is precisely measured, generating a profile of gene expression in the cell.

The American Heritage Dictionary defines "array" as "to place in an orderly arrangement". It is important that the gene sequences in a microarray are attached to their support in an orderly or fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides.

There are two major application forms for the DNA microarray technology:

1) Identification of sequence (gene / gene mutation)

2) Determination of expression level (abundance) of genes of one sample or comparing gene transcription in two or more different kinds of cells.

In our work we will refer to the second application - determining gene expression level.

 

Building the array - in more details:

probe cDNA (500~5,000 bases long) is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, "traditionally" called DNA microarray, is widely considered as developed at Stanford University. An article by R. Ekins and F.W. Chu (Microarrays: their origins and applications. Trends in Biotechnology, 1999, 17, 217-218) seems to provide some generally forgotten facts.

After preparing the chip surface and incubating it with the wanted samples we can see different color dots.

The red dots imply expression of a specific gene in sample no 1. Green dots imply expression of the gene in sample no 2, and yellow dots imply that the gene is expressed in both samples.

 

To summaries the major steps of a comparative cDNA hybridization experiment are

  1. Choosing Cell Populations
  2. mRNA Extraction and Reverse Transcription
  3. Fluorescent Labeling of cDNA's
  4. Hybridization to a DNA Microarray
  5. Scanning the Hybridized Array
  6. Interpreting the Scanned Image

 

 

A flow chart of the microarray process can be seen: