Oligonucleotide design for PCR primers and microarray probes

Related Terms

Acid alpha-glucosidase, acid maltase deficiency, chromosome, clotting factor VIII, diagnosis, DNA, drug, gene, genotype, hemophilia, hybridize, infection, microarray, PCR, primer, probe, polymerase chain reaction, sequencing.

Background

An individual's genes are present in molecules of DNA (deoxyribonucleic acid). A DNA molecule resembles a twisted ladder and is called a double helix. The sides of the double helix are made of alternating sugar and phosphate molecules, while the "rungs" of the "ladder" are made of bases. Researchers often refer to DNA as being double-stranded, with each strand containing a sugar-phosphate backbone with attached bases.
DNA contains four different chemical compounds called bases, which include cytosine, thymine, guanine, and adenine. In any given person, these bases are found in a particular order along the chromosomes, the structures in which DNA is packaged. It is the order of these bases that stores information for making genes. In the laboratory, researchers have the ability to create short segments of DNA with any sequence of bases.
An oligonucleotide is a short section of DNA that can be synthetically produced in a laboratory. Primers and probes are types of oligonucleotides used by researchers to facilitate experiments. Primers may be used in a type of experiment called polymerase chain reaction (PCR), which is a method that uses proteins called enzymes to amplify and detect genes. Probes may be used when performing microarray experiments. DNA microarrays are small chips that have many probes attached, and they are able to detect a wide variety of genes in a sample.
When researchers make oligonucleotides to use for PCR or microarrays, they typically design the oligonucleotides to detect specific genes. They can design oligonucleotides that are specific for a particular gene based on the properties of DNA structure and the DNA sequence. It is known that the base cytosine binds to the base guanine and that the base thymine binds to the base adenine. Additionally, guanine can bind to cytosine, and adenine can bind to thymine. Therefore, if a piece of DNA has the sequence guanine-guanine-guanine-adenine-guanine, then an oligonucleotide with the sequence cytosine-cytosine-cytosine-thymine-cytosine would be able to bind to that piece of DNA.
PCR and microarray technology have many valuable applications. For example, researchers may use PCR to help diagnose a genetic disease by detecting mutations, or errors, in the DNA of a gene known to be involved in that disease. Microarray technology may be used to study diseases, for example, by detecting genes that become abnormally regulated in the cells of a patient with cancer. The successful application of these methods relies on using primers or probes that are able to specifically detect the gene or genes that a researcher is interested in studying.

Methods

Polymerase chain reaction (PCR): PCR is a method of multiplying a piece of DNA millions of times. To perform PCR, DNA is first extracted from a biological sample, such as a cell or tissue. Primers, which are small sections of DNA that are usually made in a laboratory and are 20-30 bases in length, are then added to the sample to specifically recognize the DNA of interest. Researchers commonly select two different primers, both of which are specific for a particular region of DNA, when performing PCR. It is common for a researcher to select two distinct primers, which are designed to bind to each end of the DNA sequence when performing PCR. The DNA sequence could be hundreds or thousands of bases long. Additionally, because a DNA molecule has two strands, it is vital that each primer is designed to recognize a different strand.
Another protein, called DNA polymerase, is then added to the sample. DNA polymerases are enzymes that are able to synthesize DNA, and in a PCR reaction, the DNA polymerase makes thousands or millions of copies of the DNA sequence located between the two primers. DNA polymerase is typically purified from cells, but is obtained from a cell different from the target DNA. DNA polymerase can copy a strand of DNA, beginning at the location where a primer is bound. Because each primer is designed to bind to a different strand of DNA, the DNA polymerase is able to duplicate both strands.
Once the DNA sequence is amplified, researchers can place the DNA products in a gel and stain them with a blue dye for observation. By attaching the gel to an electric power box, a charge is applied, which causes the DNA to move at different speeds based on its charge and size. DNA is negatively charged, so it will move through the gel when a charge is applied. Larger DNA molecules will move more slowly, and smaller ones will move more rapidly. This allows researchers to separate DNA based on size.
Sometimes PCR is used to determine the genotype based on the sequence at one location in the DNA. The genotype is the specific base that an individual has at a particular location in the DNA. For example, some individuals may have the base adenine at a specific location in the DNA, while other individuals may have the base cytosine. PCR may be used to determine the genotypes of these individuals by choosing primers that can specifically recognize this genetic difference. By designing the primer so it has thymine at a specific location, for example, a primer could be designed to recognize only individuals with adenine at that location. In this case, the primer may not be able to recognize the DNA of an individual with the thymine base, and that individual's DNA would not be amplified.
DNA microarray: DNA microarrays are small pieces of glass or silicon that have many short pieces of DNA (about 30-80 bases long) attached. Each short piece of DNA attached to a microarray is called a probe. Every probe is a different sequence, and microarrays are often designed to contain enough probes to detect hundreds or thousands of different genes. If a microarray contains a probe for a specific gene, then it can be used to check for the presence of that gene in a sample.
When using microarrays to detect a genetic mutation, multiple probes for the same gene may be added to the microarray. Each probe could be designed to detect a separate region or variant of that gene, so a microarray experiment could be used to distinguish a substantial number of mutations simultaneously.
To detect the genes in a biological sample using a microarray, ribonucleic acid (RNA) from a sample is attached to a fluorescent dye. The fluorescently labeled RNA is then put onto the DNA microarray. If a probe designed specifically to identify a particular RNA detects the RNA on the microarray, the RNA will stick to the microarray. This process is called hybridization.
After the RNA is given a chance to stick to the microarray, all of the RNA that did not stick is washed off. RNA sticks when the bases in the RNA bind to complementary bases on the probe. Researchers may then measure how much RNA from a specific gene was present in the biological sample by measuring the amount of fluorescent signal produced by the microarray with RNAs sticking to it. Each probe on a microarray is contained in a known location. If the probe for a certain gene has a strong fluorescent signal, it means that a large amount of RNA from that gene was present in the cell. If a certain probe is producing a low or no fluorescent signal, it reveals that the RNA from that gene was not present or was present in low amounts in the biological sample.

Research

Diagnosing diseases: For many diseases, researchers have identified genetic mutations that are responsible for causing the disease. If a patient displays symptoms of a particular genetic disease, polymerase chain reaction (PCR) or DNA microarrays may be used to help diagnose that patient.
For example, mutations in clotting factor VIII are known to cause hemophilia, a disease in which blood does not clot properly. Clotting factor VIII functions in coagulation, a step in the clotting process when a protein net is formed around torn blood vessels to stop the bleeding. Microarray technology could be used to check for these mutations and to diagnose hemophilia. To perform these tests, DNA from patients could be hybridized to a customized DNA microarray that contains many short probes for the factor VIII gene. By checking for a probe where the DNA from a patient does not hybridize strongly, researchers may be able to identify which part of the factor VIII gene contains a mutation. This is because a mutation in the factor VIII gene will prevent the gene from binding as strongly to the probe that was designed to detect the sequence of the normal gene.
PCR may be used to diagnose viral infections. Because viruses have their own unique DNA and RNA, doctors can determine whether an individual has been infected with a virus by checking for the presence of viral nucleic acids (DNA or RNA). For example, PCR using primers specific for RNA from the hepatitis C virus (HCV) has been used to detect the virus in the blood of infected individuals. Additionally, genotyping by selecting specific primers may be used to determine which specific strain of a virus has infected an individual.
Understanding diseases: Microarray technology is a powerful way to identify possible disease-causing genes because it allows researchers to check the levels of hundreds or thousands of genes in one experiment. This practice is vital for researchers because they may not know which exact gene causes a specific disease and therefore may not know which genes to focus on in advance. In many human diseases, cells no longer carry out their normal function, and they may make more or less of specific genes. By measuring the levels of different RNAs in diseased cells and comparing them to the levels of the same RNAs in healthy cells, researchers can learn which specific genes may be involved in a particular disease. RNA levels can be measured by looking at the strength of a fluorescent signal for a particular RNA in a microarray experiment.
Microarray technology has been used to study prostate cancer cells in order to identify specific genes that may be involved in prostate cancer progression. The levels of different RNAs from prostate cancer cells were compared to the levels of RNAs from normal cells using a DNA microarray. Using this approach, researchers were able to identify many new genes that may be involved in prostate cancer metastasis. Metastasis is a process in which a tumor leaves its original site and invades other sites in the body. DNA microarrays have been used in a similar way to identify genes involved in other types of cancer, such as brain cancer, and other diseases, such as Huntington's disease (a condition in which brain cells degenerate).

Implications

Genetic counseling: If prospective parents have a family history of a genetically inherited disease, they may choose to undergo genetic testing to determine their chances of passing a disease on to their children. Each parent may have a specific gene sequenced to determine whether they are carrying a disease-causing mutation in the gene. Polymerase chain reaction (PCR) using primers specific for that gene can be used to amplify the gene from each parent so that enough DNA is available to sequence the gene and check for mutations.
For example, it is known that the acid alpha-glucosidase gene is mutated in patients with acid maltase deficiency (AMD), a recessive genetic condition that causes a buildup of glycogen. The acid alpha-glucosidase gene normally functions in muscle cells to break down glycogen, a form of starch that is used to store short-term energy. Individuals have two copies of the acid alpha-glucosidase gene, one from the mother and one from the father, and both inherited copies must be defective in order to develop AMD. PCR can be used to help check for mutations in this gene. People who have only one mutated gene are called "carriers;" carriers generally do not show symptoms of AMD but can pass on the mutated gene to their children. If only one parent is a carrier, none of the children will have AMD, but each child has a 50% chance of being a carrier. If both parents are carriers, then there is a 50% chance that each child will be a carrier, and a 25% chance that each child will have AMD.
Fighting diseases: DNA microarray technology can be used to better understand some diseases. By understanding the specific changes in gene levels between an individual with a disease and a healthy individual, researchers can better understand the disease. This is because changes in the levels of genes can alter the function or behavior of cells, and this may lead to a disease. As a result of identifying the genes that become improperly regulated, scientists are better able to understand how the disease is caused. This information may further aid in treatment if used to develop medication to fight the disease.

Limitations

DNA microarrays may be used to detect the expression of hundreds or thousands of genes in just one experiment. In order to detect a specific gene, however, researchers need to have prior knowledge of the gene and the gene sequence to design a probe for that gene. Therefore, researchers cannot use microarrays to discover new genes. Instead, microarrays are used to detect genes that have already been characterized. Similarly, to perform polymerase chain reaction (PCR) to amplify a specific gene, some knowledge of the sequence of that gene, or the DNA near that gene, is generally needed.
A probe or primer is a sequence of DNA that can recognize and bind to the gene or region of DNA a researcher is interested in studying. Some regions of one gene may be very similar to regions on a different gene. Therefore, researchers may need to generate a probe or primer that is specific to just the gene they are interested in, but that is not similar to other genes. Otherwise, researchers may think they have identified the gene they are interested in when they have really identified a different, but related, gene. Nevertheless, researchers must be careful when designing primers or probes to use for PCR or microarray analysis.
If prospective parents have a family history of a genetically inherited disease, they may choose to undergo genetic testing to determine what the chance is that they may pass a disease on to their children. While genetic testing can be performed using PCR, the cost may be a limitation.

Future research

Research in this field is ongoing. As new discoveries and advancement are made, researchers may use polymerase chain reaction (PCR) to help diagnose genetic disorders by detecting mutations, or errors, in the DNA of a gene known to be involved in that disease. Microarray technology may be used to study diseases, for example, by detecting genes that become abnormally regulated in the cells of a patient with cancer.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

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