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Oligonucleotides are short DNA or RNA molecules used in molecular biology and medicine for disease screening, gene identification, DNA sequencing, and PCR. They are synthesized chemically and identified by their chain length. Oligonucleotides are used as primers in DNA sequencing and PCR, and as probes in microarray chips for genetic disorder screening and viral infection identification. Antisense oligonucleotides are used to block gene expression and as new types of drugs to block toxic RNAs.
An oligonucleotide is a short chain of DNA or RNA molecules that has many uses in molecular biology and medicine. It is used as a probe for screening for disease, viral infections, and for identifying genes in molecular biology experiments. It is also used as a primer in one type of DNA sequencing.
To understand an oligonucleotide, it helps to understand the structure of DNA. DNA molecules are very long coils of two strands, made up of four different nucleotide base units, arranged in varying orders. Each unit has a complementary base that it will bind to, so each strand has an opposite set of bases that bind to it. These bases can form a great variety of different combinations, and it is the combination of bases that gives the genetic code. DNA is transcribed to make messenger RNA (mRNA), which is then translated to make proteins.
Oligonucleotides are identified by their chain length. For example, an oligonucleotide ten nucleotide bases long would be called ten mer. They are generally synthesized chemically and the type of synthesis limits the chain length to less than 60 bases.
In a type of DNA sequencing known as dideoxy sequencing, oligonucleotides are used as primers, so that the enzyme that makes the DNA has a template to work from. Single-stranded DNA is used, and an oligonucleotide complementary to the DNA strand is synthesized using an automated machine. The DNA synthesizing DNA polymerase continues to add to the primer and synthesizes the opposite strand of DNA from it. This reaction produces double-stranded DNA.
A more recent use of oligonucleotides as primers is in polymerase chain reaction (PCR) which is used to amplify small fragments of DNA. This technique has highly practical uses, such as in forensics and paternity testing. It has also revolutionized research in medicine and the life sciences, as it is often used in genetic engineering experiments.
An array of oligonucleotide probes is often used to isolate genes from a library of complementary genes or DNA (cDNA). cDNA libraries consist of double-stranded DNA, where one strand is derived from an mRNA strand and the other is complementary to it. Such libraries have the advantage of not having the gaps frequently found in genes from higher organisms.
The structure of the genes of many organisms is known, thanks to sequencing projects, and is publicly available. If you want to clone a gene from another organism, you can see what is known about the gene in other organisms and design probes based on common areas in those sequences. The researchers then synthesized a set of oligonucleotide probes that account for possible variations in the joint area. They scan the library with those probes and look for oligonucleotides that bind. Many genes have been identified this way.
An antisense oligonucleotide contains a single strand of RNA or DNA which is the complement of a sequence of interest. Once a particular gene encoding a protein has been cloned, antisense RNA is often used to block its expression by binding to the mRNA that synthesizes it. This allows researchers to determine the effects on the body when it is not producing that protein. Antisense oligonucleotides are also being developed as new types of drugs to block toxic RNAs.
Microarray chips have been another area where oligonucleotides have been of great use. These are slides, or some other matrix, that have spots containing thousands of different DNA probes, in this case made of oligonucleotides. They are a very efficient way to test for changes in a number of different genes at once. The DNA is bound to a compound that changes color, or fluoresces, if complementary DNA binds to it, so the spots change color if there is a reaction with the test DNA.
Some of the things oligonucleotide microarrays are used for include screening for genetic disorders. For example, there are small probes that represent the activity of genes involved in breast cancer, BRCA1 and BRCA2. It is possible to find out if a woman has a mutation in one of these genes and test it further to see if she has a predisposition to breast cancer.
There is a microarray called the ViroChip that has probes for about 20,000 genes from various pathogenic viruses that have been sequenced. Body secretions, such as phlegm, can be analyzed with the chip, which can often identify what type of virus a person is infected with. Identifying viral infections can be quite difficult, as symptoms are often similar with different types of viruses.
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