Research Technology - Page 2

Fluorescence In Situ Hybridization (FISH)

FISH is a technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes.

FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosome.

In medicine, FISH can be used to form or confirm a diagnosis, to evaluate a prognosis, or to evaluate remission of a disease, such as cancer. Treatment can then be specifically tailored to the specific situation. In reseach, FISH can be used to measure chromosome defects like gene deletions and duplications or fusions that can be associated with cancer.

Microarrays

Because this technique is so often discussed in research, this section contains a little more detailed information than the others.

Background:

With only a few exceptions, every cell of the body contains a full set of chromosomes and identical genes. Only a fraction of these genes are turned on or "expressed", 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; these molecules are then transformed into the proteins that perform most of the critical functions of cells.

Scientists study the kinds and amounts of mRNA produced by a cell in order to learn which genes are expressed; this in turn, provides insights into how the cell responds to its changing needs.

	Using traditional methods to assay gene expression, researchers were able to survey a relatively small number of genes at a time. The emergence of new microarray tools enables researchers to address previously intractable problems and to uncover novel potential targets for therapies. Now microarrays allow scientists to analyze expression of many genes in a single experiment quickly and efficiently. This represents a major methodological advance and illustrates how the advent of new technologies provides powerful tools for researchers.
Image courtesy of Dr. Larssono for use in the public domain

A microarray works by exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated.

By using an array containing many DNA samples, scientists can determine in a single experiment the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array.

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 each of the samples.

Explanation of the microarray technique

• An array is a collection of DNA spots attached to a solid support such as a microscope slide. The solid surface can be glass or a silicon chip, in which case it is commonly called a gene chip or colloquially Affy chip when an Affymetrix chip is used.
• Each spot contains one or more single-stranded DNA oligonucleotide (a short nucleic acid polymer, typically with twenty or fewer bases) fragments.
• The affixed DNA segments are known as probes. Thousands of them can be placed in known locations on a single DNA microarray.
• Since an array can contain tens of thousands of probes, a microarray experiment can ask many questions at once. Thus arrays have dramatically accelerated the progress of many types of studies.
• Typically, many samples are simultaneously studied in parallel arrays. Thus, 'heat maps' show patterns of gene expression with one dimension being the different genes whose expression is being measured, and the other being individual patient samples. This type of test often finds subgroups of patients with distinct gene expression patterns.

See explanation of image below showing map of patients with Lymphoma

	Image courtesy of the National Cancer Institute

• Each column represents tissue from a patient
• Each row represents a different gene
• Green pixels represent genes that are over-expressed compared to normal
• Red pixels represent genes that are under-expressed
• As seen in the image patients divide into two major classes - those with Burkitt lymphoma versus those with Diffuse Large B_Cell Lymphoma.

DNA microarrays can be used:

• To measure changes in expression levels
• To detect single nucleotide polymorphisms (SNPs)
• To categorize different subgroups of patients - ie 'heat maps'
• In genotyping
• In resequencing mutant genomes.

Types of microarrays:

There are three basic types of samples that can be used to construct DNA microarrays, two are genomic (DNA) and the other measures mRNA levels.

The Promise of Microarray Technology in Treating Disease

"Now that you understand the concept behind array technology, picture this: a hand-held instrument that a physician could use to quickly diagnose cancer or other diseases during a routine office visit. What if that same instrument could also facilitate a personalized treatment regimen, exactly right for you? Personalized drugs. Molecular diagnostics. Integration of diagnosis and therapeutics. These are the long-term promises of microarray technology.

Maybe not today or even tomorrow, but someday. For the first time, arrays offer hope for obtaining global views of biological processes-simultaneous readouts of all the body's components-by providing a systematic way to survey DNA and RNA variation." - Quote from the National Center for Biotechnology Information

CISN Summary

We have described some of the techniques used in research. It is not necessary to memorize them; they are mentioned so you have some context and if need be, you can come back to this section for details.

This chart lists many of the currently used technologies arranged by tissue type utilized. Several have been described in more detail.