Molecular Genetic Techniques

Determining Gene Function

Molecular genetic technology can determine the activity of a particular gene, thus enabling researchers to discern the gene's function.

DNA technology can be used to discover the function of a particular gene. Molecular genetic technology can do this by "turning off" genes that surround the gene of interest, thus forcing the desired gene to work in isolation. The most common way to isolate a gene is by studying the mRNA, or messenger RNA, molecules that are transcribed from that gene. Remember that transcription is the formation of mRNA from a template DNA strand; after transcription, cells use the information in the newly made mRNA to synthesize proteins.

When a gene is expressed, the mRNA molecules that are complementary to the gene can be identified, serving as evidence that the gene is active. A DNA microarray assay is a technique that uses a large number of single-stranded DNA fragments attached to a microscope slide in a grid pattern (a microarray). The microarray represents all the genes an organism has. Each DNA fragment in the microarray acts as a nucleic acid probe to find the complementary mRNA produced by a specific gene. Each nucleic acid probe in a microarray assay is dyed with a different color so that multiple samples can be analyzed at the same time.

For example, an mRNA sequence is recorded as follows:

C G G C C A C U A C U C

A nucleic acid probe that is tagged with dye is added to the array:

G C C G G T G A T G A G

The probe is added into the developing gene expression of a DNA sample and allowed to hybridize, or incorporate, itself into the organism's genome. It matches up with any complementary mRNA sequences as the gene is being transcribed. Nucleic acid probes can use different colors of fluorescence at the same time, so the isolated genes often appear as a rainbow, allowing for easy identification.

Process of DNA Microarray Assay

A DNA microarray assay uses mRNA and fluorescent strands of complementary DNA to isolate particular genes in an organism's genome. The genes are highlighted for better identification.
Genes can be inactivated through the use of a biological process called RNA interference (RNAi). RNA interference (RNAi) is the blocking of a gene's expression by a small segment of interfering RNA. There are currently two general forms of RNAi inside cells, exogenous double-stranded RNA (siRNA) and endogenous single-stranded RNA (miRNA). Either siRNA or the miRNA molecules with a sequence that matches the targeted gene are introduced into the cell or organism. The siRNA or miRNA is cleaved into shorter segments called small interfering RNAs (siRNAs), which hybridize with the target cell's mRNAs and cause them to break down. In this way, the expression of the target gene is halted, causing the gene to be inactivated. siRNA molecules bind perfectly to their mRNA targets in cells. In contrast, miRNA binds imperfectly to its target, and therefore inhibits translation of numerous mRNA sequences. It is thought the RNAi pathway evolved as a defense mechanism against infection by viruses, some of which have double-stranded RNA. An invading virus can destroy a cell's RNA, preventing the cell from producing certain proteins. But RNAi can enable the cell to destroy the viral RNA, helping the cell resist viral attack. The use of RNAi can occur in simple organisms, such as bacteria and invertebrates, and in nonhuman mammals.

Once the gene of interest has been isolated and silenced (turned-off), scientists can determine the gene's function by comparing the sequence to that of other species. Logically, similar sequences most likely indicate similar functionality. A reporter gene is a gene that encodes a protein that can be easily tracked and, when attached to another gene or sequence of genes, the reporter gene will alert researchers to their expression. Reporter genes enable the detection of other genes via expression of visually identifiable characteristics, such as through a color change.Examples include green fluorescent protein, luciferase, or GUS protein. Additionally, scientists can disable the gene to observe the consequences in the organism's cells.

Another area of gene manipulation involves a reporter gene called the green fluorescent protein (GFP), which is a protein common in jellyfish that glows green in the presence of ultraviolet light and is often inserted into other organisms. This protein absorbs ultraviolet radiation from the sun and then rebroadcasts it as green light. The jellyfish use the light to attract prey in dark ocean water. GFP is of use in molecular biology because it makes anything to which it is attached glow. This is especially helpful when trying to isolate particular genes or other proteins in a cell. Just shining ultraviolet (UV) light on the GFP at any time will indicate where it is located because of the fluorescent green color. Some research has transplanted this gene into small mammals, such as mice and rabbits. The fur color of these animals is white under white light, but under UV light, they glow in the dark. While there is not a high demand for glow-in-the-dark animals, the ability to transfer the GFP gene shows how genetic technology is able to manipulate genes for other uses.

New technologies have allowed scientists to measure gene expression thousands of genes at a time. This allows for more comprehensive treatments for diseases and other medical conditions. For example, DNA microarray assay has allowed for the identification of several patterns in the genes of breast cancer cells that have resulted in the development of more targeted and effective treatments.