Table of Contents > Genomics > Genotyping Print



Related terms
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Related Terms
  • Chromosome, diagnosis, disease, DNA, drug, gene, genotype, hybridize, infection, microarray, PCR, primer, probe, polymerase chain reaction, polymorphism, sequencing.

  • The genotype of an individual is that individual's deoxyribonucleic acid (DNA) sequence, while genotyping is the method by which one's DNA sequence is determined. Often, genotyping is not used to determine the entire DNA sequence of an individual, but rather is used to determine the DNA sequence of one or a few genes or regions of DNA.
  • DNA is located in a compartment of the cell called the nucleus and is packaged in structures called chromosomes. Human cells contain 46 chromosomes, and each chromosome has hundreds of genes. Genes contain the instructions for making the proteins that perform all the functions in the human body. Chromosomes also contain many other regulatory sequences that control how much of a gene will be made, when it will be made, and where in the body it will be made.
  • DNA contains four different chemical compounds called nucleotide bases: cytosine, thymine, guanine, and adenine. In any given person, these nucleotide bases are found in a particular order along the chromosomes, and it is the order of these nucleotide bases that stores information for making genes. Even though the DNA sequences of different people are similar (on average, DNA is about 99.9% identical between two people), differences in DNA between people are important. Differences in DNA may be caused by genetic changes, called mutations. When genotyping individuals, these differences in DNA may be identified.
  • Researchers may use several different methods to determine an individual's genotype, including polymerase chain reaction (PCR), DNA sequencing, and DNA microarray technology. PCR is a method that uses proteins called enzymes to amplify and detect genes. DNA microarrays are small chips called probes that have many short pieces of DNA attached, and which are able to detect a wide variety of genes in a sample. DNA sequencing is a technique researchers may use in order to determine the sequence of bases along a chromosome or in a gene.
  • Differences in DNA between people may be responsible for differences in traits, such as height or hair texture. Changes in DNA may also cause some people to develop genetic diseases. For example, Duchenne muscular dystrophy is a disease caused by a genetic change that leads to loss of muscle function. DNA may change in a variety of ways. For instance, bases may become deleted, or one base may become substituted with another base. Genotyping individuals may help researchers detect differences in DNA that are related to a particular genetic disease or that are responsible for affecting other human traits, such as height. By detecting these changes, researchers may be able to better understand and treat a disease; for example, by using gene therapy, in which a defective gene is replaced with a functioning gene.

  • Researchers may use several different methods to determine an individual's genotype, including polymerase chain reaction (PCR), DNA sequencing, and DNA microarray technology.
  • Polymerase chain reaction (PCR): PCR is a method of multiplying a piece of DNA millions of times. PCR can be used to determine the genotype of an individual at a specific gene (or at any other specific location in the DNA). Researchers may use PCR alone to determine an individual's genotype. Alternatively, they may use PCR to amplify the DNA, and then sequence the DNA to determine an individual's genotype.
  • To perform PCR, DNA is first extracted from a biological sample, such as cells or tissue. Different methods, such as a mouth swab or blood sample, may be used to obtain the DNA. Then, pieces of DNA called primers that can specifically recognize the DNA of interest are added to the sample. Primers can recognize the DNA because they are designed such that the bases in the primer bind to the bases in the DNA sample. Researchers commonly select two different primers when performing PCR, and these two primers typically are located at each end of the DNA sequence the researcher is interested in studying.
  • Next, another protein, called DNA polymerase, is 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 that is located between the two primers. Once the DNA sequence is amplified, researchers can place the DNA products in an agarose gel and stain it to visualize them. A charge is applied to the gel, which causes the DNA to move at different speeds based on its charge and size. DNA is negatively charged, so a positive charge will cause the DNA to move through the gel. Smaller DNA pieces will move more rapidly, while larger ones will move more slowly because they encounter more resistance moving through the agarose. Typically, a charge needs to be applied for about one hour.
  • PCR may be used to determine the genotype based on the length of DNA that was between the two primers. This is because some genetic changes between individuals result in a region of DNA having extra bases and thus being longer in one individual than in another. In these cases, the genotype can be determined by observing how quickly the piece of DNA is able to move through the gel. For example, individuals with a deletion mutation in a gene will have a shorter gene. If PCR is used to amplify that gene, the PCR product will be smaller and will move through a gel more quickly than DNA from an individual who did not have a deletion. Samples of DNA without the deletion are run with the test sample to use as a comparison.
  • PCR may be used to determine the genotype based on the sequence at one specific location in the DNA. For example, some individuals may have the base adenine at a specific location in the DNA, while others may have the base thymine. PCR may be used to determine the genotypes of these individuals by choosing primers that can specifically recognize this genetic difference. For example, a primer could be designed to recognize only individuals with the base adenine at this specific 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 sequencing: Researchers may sequence a region of DNA to determine the genotype. A sequencing reaction is typically performed on DNA after PCR has been used to amplify the DNA. In a sequencing reaction, an enzyme called a polymerase is used that is able to replicate, or synthesize, DNA. The new DNA is synthesized by the polymerase in a test tube using free bases (cytosine, thymine, guanine, and adenine).The sequencing reaction is designed so that the target DNA molecule will be replicated many times, but each time the replication occurs, synthesis of the new DNA strand will be stopped at a different position in the DNA sequence. At the end of the sequencing reaction, many different DNA products of different lengths that are all shorter than the original product will have been generated.
  • Researchers can use the DNA products from the sequencing reaction to determine the sequence of the original piece of DNA. During the sequencing reaction, each DNA product is labeled with one of four fluorescent dyes, and each of the four bases has its own unique dye. Based on which of the four dyes a DNA product is labeled with, researchers can determine which base (cytosine, thymine, guanine, or adenine) was the last to be added to the DNA strand during the reaction. This is because the DNA sequencing reaction is designed such that only the last base to be added to a newly synthesized strand will have a fluorescent tag and these tagged bases are designed to stop the synthesis of the new strand after they are added.
  • By arranging all the differently sized products from the DNA sequencing reaction in order from smallest to largest and then determining what the last base to be added to each product was using the fluorescent tag, researchers can determine the sequence of the original DNA strand. This is because the starting base in the synthesis is always the same, and only the final base is different. In order to separate the DNA products by size, researchers may put the DNA products in agarose gel and use an electric field to separate the products. This procedure is identical to that used for separating PCR products in a gel. A machine called an automated DNA sequencing machine is commonly used to separate the DNA pieces and determine the order of bases in the DNA based on the fluorescent tags.
  • After a DNA sequence is obtained, it will appear as long line of letters with A corresponding to adenine, T to thymine, G to guanine, and C to cytosine. For example, a short DNA sequence may appear as AGCCTGATCCGGGATCAGCTTAAAGCTTAGCCGTAAAAAGT. By reading the letters in this sequence, researchers are able to determine the genotype of an individual for that region of DNA.
  • DNA microarray: DNA microarrays are small pieces of glass or silicon that have many short pieces of DNA attached. DNA microarrays are commonly found on chips. Each short piece of DNA that is attached to a microarray is called a probe, which contains bases that are complementary to the genes they are designed to detect. Every probe is different, and microarrays are often designed so that they 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 determine an individual's genotype, many different probes for the same gene may be added to the microarray, with each probe designed to detect a different region or genetic variant of that gene. Microarrays may therefore be used to detect a large number of different genotypes all at once. Even though microarray experiments are costlier than other methods, they may be preferred in situations in which a large number of genes need to be studied at once.
  • To detect the genes in a biological sample using a microarray, DNA from the sample is attached to a fluorescent dye. The labeled DNA is then placed onto the DNA microarray. If a probe designed to detect a specific DNA is present on the microarray, then that DNA will stick to the microarray. DNA can find a specific probe if the probe was designed with bases that will recognize that piece of DNA.
  • After the DNA is given a chance to stick to the microarray, a process called hybridization, all of the DNA that did not stick is washed off. Researchers may then determine how much DNA from a specific gene was present in the biological sample by using a machine called a fluorescent plate reader to measure the amount of fluorescent signal the microarray produced. 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 DNA from that gene was present in the cell. If a certain probe is producing a low fluorescent signal or none at all, it means DNA from that gene was not present or was present in low amounts in the biological sample.

  • Differences in DNA between people may be responsible for differences in traits, or changes in DNA may cause some people to develop genetic diseases. Genotyping individuals may help researchers detect changes in DNA that are related to a particular genetic disease or that are responsible for affecting other human traits. Often, genotyping is used to diagnose genetic diseases that are known to be caused by mutations in specific genes.
  • 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, genotyping may be used to help diagnose that patient.
  • For example, genetic mutations that affect production of clotting factor VIII are known to cause hemophilia, a disease in which blood does not clot properly, causing patients to experience excessive 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 at which 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 cause the sequence of the gene to change, preventing it from binding as strongly to the probe, which was designed to detect the sequence of the normal gene.
  • Genotyping may be used to detect the presence of a viral infection in an individual. This is because viruses have their own unique DNA and RNA (ribonucleic acid, a nucleotide intermediate made from DNA that is used by cells to generate proteins). By checking for the presence of viral nucleic acids in a patient, doctors can determine whether that individual has been infected with a virus. For example, PCR using primers specific for the hepatitis C virus (HCV) has been used to detect HCV 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 because each strain of virus has a slightly different DNA sequence.
  • Identifying polymorphisms: Although the DNA sequence between people is almost identical, about 0.1% of DNA is different between two individuals. A polymorphism is a difference in the sequence of DNA between different individuals. It is thought that some polymorphisms do not cause any differences in the physical or mental traits between people. However, some polymorphisms may change the function of a gene or the amount of protein that a gene produces, and these polymorphisms may have an impact on the physical and mental traits of people. By sequencing DNA from many people, it is possible to find genetic differences between people that may affect human traits. For example, it has been found that polymorphisms in specific genes involved in odor detection cause some individuals to perceive odors differently from other people.
  • Researchers have identified a genetic variant in the gene PON1, which may be involved in disease susceptibility. PON1 makes an enzyme that helps the body break down toxic compounds called organophosphates, which are present in many products, such as insecticides and solvents. Organophosphate poisoning may result in a variety of symptoms, including fatigue, headache, seizures, or coma. By performing research in animals, researchers found that PON1 genes with a specific polymorphism were more effective at breaking down a type of toxic organophosphate. This research suggests that individuals who have this PON1 polymorphism are less at risk for being poisoned by some types of organophosphates.
  • Response to drugs: Patients respond in a variety of different ways to the same medication. For instance, some patients may see improvement, others may not see any effect, and others may experience serious side effects. It is known that genetics may play a role in these different responses. In some cases, specific genes have been found that influence how a patient will react to a drug. For example, some patients have variants of a gene that makes the protein CYP2D6, also known as the cytochrome P450 CYP2D6 enzyme, which breaks down drugs in the body. Some of these genetic variants cause the protein to be less functional, so it is not able to break down the drug as well, which may lead to toxic side effects. Genotyping may be used to determine which specific variant of this gene a patient has and whether he or she would benefit or be at risk from taking a certain drug. For example, the genotype of CYP2D6 may affect how patients react to tamoxifen, a drug used to treat breast cancer.

  • Diagnosing disease: For many different genetic diseases, researchers have identified genetic mutations that are responsible for causing the disease. If a patient displays symptoms of a particular genetic disease, genotyping may be used to help diagnose that patient. For example, mutations in the frataxin gene are known to cause Friedreich's ataxia, a disease in which nerve tissue progressively degenerates. Genetic tests can be used to check for these mutations and diagnose Friedreich's ataxia. To perform these tests, blood may be withdrawn from a patient, and the frataxin gene could be sequenced to check for mutations.
  • Infection: Genotyping may be used to better understand how some microorganisms, such as bacteria, cause infection. For example, there are several different but closely related strains of E. coli bacteria, some of which can cause infection and some of which cannot. By sequencing and comparing the DNA of these different strains, researchers have been able to identify differences in genes that may be involved in infection. By studying these different genotypes, researchers may be able to develop new strategies to combat infection by developing drugs that target these genes.
  • Genetic counseling: If prospective parents have a family history of a genetically inherited disease, they may choose to undergo genetic counseling to determine what the chance is that they may pass a disease on to their offspring. Each parent may get a specific gene sequenced to determine whether they are carrying a disease-causing mutation in the gene.
  • For example, it is known that the acid alpha-glucosidase gene has a mutation in patients with acid maltase deficiency (AMD), a recessive genetic condition that causes a buildup of glycogen. AMD is one of a group of diseases called glycogen storage diseases. Individuals have two copies of the acid alpha-glucosidase gene, and both copies must be defective for a person to develop AMD. People who have only one mutated gene are called "carriers." If only one parent is a carrier, none of the children will have AMD, but each child has a 50% chance to be 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.

  • One limitation of genotyping is that a genotype can tell a researcher what the DNA sequence of a gene is, but cannot tell a researcher what the functional consequence is of having that particular genotype. Not all genetic changes will have functional consequences. Whether a genetic change has a functional consequence is usually dependent on how the genetic change affects the production or function of a protein. Once researchers have determined that an individual has a particular genotype, they may need to perform additional experiments to better understand how that genotype affects an individual and how it may differ from other genotypes.
  • Researchers must also be careful when designing primers or probes to use for genotyping. A probe or primer is a sequence of DNA that can recognize and bind to the gene or to a region of DNA that a researcher is interested in studying. Probes may be made by researchers in a lab using a special machine that controls the synthesis of DNA, one base at a time. Some regions of one gene may be very similar to regions on another gene. Therefore, researchers may need to generate a probe that is specific to only 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. By using longer primers or probes, researchers may be able to obtain a higher degree of specificity.


Future research
  • New DNA sequencing methods have recently been developed that offer some advantages over traditional sequencing methods. Traditional sequencing methods are generally slow and expensive. Additionally, they usually require an amplification step, such as PCR or cloning, before the DNA can be sequenced. Newer methods have been developed that allow for faster and cheaper sequencing of DNA. In the newer sequencing methods, for example, pyrosequencing, many copies of DNA are synthesized from one strand onto a small bead. Many individual beads, each with a different DNA sequence, can then be simultaneously analyzed. These newer sequencing methods should increase the speed and decrease the cost for genotyping individuals.
  • Researchers have already identified through genotyping a number of genetic differences that may be involved in human disease or human traits. For example, many genetic changes that may lead to cancer have been identified. However, for many genetic diseases and human traits it is still unclear what genetic factors are involved. In some cases, multiple genes may be involved in determining the trait. In the future, researchers will continue to test individuals through genotyping to identify additional genetic variants that have an effect on human health.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (

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