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Allele-specific oligonucleotide hybridization

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Also listed as: ASO hybridization
Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Allele, ASO hybridization, DNA, genetic epidemiology, genotyping, hybridization, mutation analysis, oligonucleotide, PCR, polymorphism.

Background
  • An allele-specific oligonucleotide (ASO) is a short, single-strand deoxyribonucleic acid (DNA) molecule, usually 15-21 nucleotides, or bases, in length.
  • DNA makes up the genetic "blueprint" that contains all the information necessary to make a living being. The unique twisted ladder shape of DNA is called a double helix. The sides of the double helix are made of alternating sugar and phosphate molecules. The "rungs" of the "ladder" are made of small molecules called bases. There are four different types of these bases in DNA: adenine, thymine, cytosine, and guanine. These bases are arranged in a unique order, or sequence, in the DNA. Because they contain nitrogen, these molecules are sometimes called nitrogen bases. DNA provides instructions for the production of a molecule called mRNA (messenger ribonucleic acid), which then controls the production of protein encoded by the gene.
  • Alleles are variations of a single gene; each gene contains two alleles because one is inherited from each parent. A mutation occurs when the chemicals that make up an individual's genes are incorrectly deleted, added, or substituted.
  • An ASO is complementary, meaning that it pairs with the sequence of a variable target DNA. The base pairs in a DNA molecule bind to each other in a particular way. The adenine base is complementary to thymine and the cytosine to guanine. The sequence of the bases defines the information contained in the DNA. A large proportion of DNA is the same in all human beings, but there are some regions of DNA that show considerable variation among individuals in the population.
  • An ASO also corresponds to either the normal sequence of the target or a specific known mutation. An ASO can function as a probe, which is a single-stranded DNA molecule that can detect the presence of a complementary sequence among a mixture of other single-stranded DNA sequences. A DNA probe can be used for a specific DNA target sequence in a Southern blot assay or in a simple dot blot assay. A Southern blot assay is used to detect a sequence of DNA in a sample. In this assay, the DNA is separated by size using agarose gel electrophoresis, which separates DNA by moving negatively charged nucleic acid molecules through an agarose matrix, a gel-like substance, through an electric field. Shorter molecules move faster and migrate farther than longer ones. A dot blot assay is similar to Southern blotting in that DNA is detected but instead of being separated by size, total DNA is pooled on a single spot on a membrane.
  • ASO hybridization is a technique commonly used in basic molecular biology research, genetic testing, and in forensics, which is the application of science to answer questions of interest to the criminal justice system. Forensics may use ASO to determine the DNA present in a blood sample left at a crime scene, for example. In genetic testing, ASO is used to detect the presence of a mutation in one's DNA and can be used to establish whether an individual has a certain genetic disease.
  • The DNA sequence of an ASO is typically designed to be specific for only one allele, which is one member of a pair of different forms of a gene of the DNA target being tested. Although many alleles of a gene exist, one individual inherits two alleles, one from the mother and one from the father. ASO probes are able to detect as little as a single nucleotide mismatch, which is when only one base in a DNA molecule is altered in some way. ASO technology is an important tool in studying gene variations as part of the Human Genome Project, an international scientific research project with a primary goal to determine and identify the about 20,000-25,000 genes of the human genome.

Methods
  • General: Typically, a panel of allele-specific oligonucleotide (ASO) probes is required for mutational screening, which is used to detect a mutation in a gene based on the known mutations found in the ethnic group of an individual. For prenatal diagnosis and adult patients, two oligonucleotide probes (one complimentary to the mutated DNA sequence and the other to the normal, or wild-type, gene at the same position) are needed to screen for each mutation. The patient's genotype is determined by the detection of signal from both the mutated and normal ASO probes. ASO is most straightforward for studying populations with only one common mutation.
  • Polymerase chain reaction (PCR): DNA is present in small amounts. PCR uses special enzymes to make millions of copies of a specific piece of DNA for research purposes. As PCR progresses, the DNA that is generated is used as a template for replication. This sets in motion a chain reaction in which the DNA template is amplified. With PCR it is possible to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the piece of DNA. PCR is used to amplify specific regions of a target DNA strand. This can be a single gene, or part of a gene. Reverse transcription PCR (RT-PCR) is a method used to amplify, isolate, or identify a known sequence from RNA rather than DNA. The PCR is preceded by a reaction using an enzyme called reverse transcriptase, which converts RNA to cDNA (complementary DNA). The PCR part of the process makes many copies of the resulting piece of DNA.
  • Southern blot assay: A Southern blot assay is used to detect a sequence of DNA in a sample. An ASO can function as a probe, which is a single-stranded DNA molecule used in molecular biology to detect the presence of a complementary sequence among a mixture of other single-stranded DNA sequences. In this assay, the DNA is separated by size using agarose gel electrophoresis. This method separates DNA by moving negatively charged nucleic acid molecules through a gel-like substance in an electrical field. Shorter molecules move faster and migrate farther than longer ones.
  • Dot blot assay: A dot blot assay is used to detect a sequence of DNA in a sample. A DNA probe can be used for a specific DNA target sequence in a simple dot blot assay. A dot blot assay is similar to Southern blotting in that DNA is detected, but instead of being separated by size, total DNA is pooled on a single spot on a membrane. To detect an ASO after it has bound to its target, the ASO must be labeled. ASO labeling is typically done with a radioactive, enzymatic, or fluorescent molecule that is linked to the probe. A researcher can then observe the size of the dots on the membrane in order to detect variation among samples.
  • Reverse dot blot assay: This technique allows several mutations to be tested in a single hybridization reaction. In this method, unlabelled ASO probes, specific to various mutations and to the normal DNA sequence, are bound to nylon membrane strips in the form of dots or slots. PCR-amplified genomic DNA is then labeled and hybridized to the nylon membrane. This procedure may require the use of several filters, initially screening for the more common mutations observed in the patient's ethnic background, and later blots screening for the rarer gene allele abnormalities.
  • DNA microarray: DNA microarray is a technique that consists of a series of thousands of spots of DNA oligonucleotides, called features, each containing tiny amounts of a specific DNA sequence. An ASO can be used as the DNA probe used during a process called hybridization, in which the complementary single-stranded nucleic acids are combined to make a double-stranded molecule. Once the probe and target are joined, they are detected by attaching a fluorescent label so the amount of the target can be determined.

Research
  • Increasingly, allele-specific oligonucleotide (ASO) techniques have been automated for rapid screening of a growing list of human genetic diseases. In genetic testing, ASO is used to detect the presence of a mutation in an individual's DNA and can be used to establish whether a person has a certain genetic disease. The Human Genome Project, an international scientific research project to determine the sequence of chemical base pairs of DNA and to identify the about 20,000-25,000 genes of the human genome, is expanding the potential list of target DNA sequences.
  • One genetic disease that can be diagnosed using ASO hybridization is sickle cell anemia, which is caused by a genetic mutation in the blood protein beta-hemoglobin. The normal DNA sequence G-A-G codes for the amino acid glutamate; the mutation changes the adenine to a thymine, leading to the sequence G-T-G. This altered sequence substitutes a valine into the final protein, distorting its structure. To test for the presence of the mutation in a DNA sample, an ASO probe complementary to the altered sequence would be made. As a control, another ASO would be synthesized for the normal sequence. Each ASO is fully complementary to its target sequence and will bind strongly, but has a single mismatch against its non-target allele, leading to a weaker interaction.
  • Commercial assays for many of the more common genetic diseases are available for rapid screening. For example, beta-thalassemia strips screen for the most frequent mutations observed in various regions of the world are commercially available.

Implications
  • Through allele-specific oligonucleotide (ASO) technology, the sequencing of an individual's entire genome, that is, all of the genes that make up that individual, may become more practical. As researchers identify potential DNA sequences that become mutated as a result of a genetic disease, the potential exists for medicines and treatments to be tailored to individuals based on their particular needs. For example, a group of enzymes called cytochrome P450 enzymes control drug metabolism, breakdown, and clearance. Patients with known mutations in these enzymes metabolize drugs more slowly. Knowledge of P450 enzyme mutations allows physicians to adjust drug dosages accordingly to help prevent adverse reactions.

Limitations
  • This method is not readily adapted to screening populations carrying a large number of different gene alleles because each mutation requires a separate hybridization and washing step. However, increased automation of the allele-specific oligonucleotide (ASO) assay process is providing more rapid results.
  • ASO screening detects only known gene mutations. Other "silent" DNA mutations, which are DNA changes that do not affect the structure of the coded protein, may complicate ASO screening.
  • Tests for many genetic diseases are restricted to proprietary DNA assays. This means that the assay is not available for public use and therefore may have associated high costs.

Safety




Future research
  • In the future, personalized genetic testing for a wide array of inherited diseases and predispositions will likely become more routine because . However, such testing is not widespread today. Allele-specific oligonucleotide (ASO) assays are only one technique for determining a patient's genetic makeup. Because it is a well-established and automated technique, ASO will likely be a common genetic testing strategy for some time to come.
  • With the use of ASO hybridization techniques, researchers and clinicians may be able to provide patients with personalized medicine, meaning that a patient's genotype could be used to tailor medical care to his or her specific needs. Such information could be used to help stratify disease status, select different medications, tailor dosages of medications, provide a specific therapy for an individual's disease, or initiate a preventive measure that is particularly suited to a specific patient at the time of administration.

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

Bibliography
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  5. Natural Standard: The Authority on Integrative Medicine. . Copyright © 2008. Accessed July 22, 2008.
  6. Oak Ridge National Laboratory. . Accessed July 22, 2008.
  7. Saiki RK, Bugawan TL, Horn GT, et al. Analysis of enzymatically amplified beta-globin and HLA-DQ DNA with allele-specific oligonucleotide probes. Nature 1986 324(6093):163-6.
  8. Saiki RK; Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985 230(4732):1350-4.
  9. Studencki AB, Conner BJ, Impraim CC, et al. Discrimination among the human beta A, beta S, and beta C-globin genes using allele-specific oligonucleotide hybridization probes. Am J Hum Genet 1985 37(1):42-51.
  10. Tomaszewski P, Kubiak-Tomaszewska G, Lukaszkiewicz J, et al. Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. III. Influence of CYP genetic polymorphism on population differentiation of drug metabolism phenotype. Acta Pol Pharm. 2008 May-Jun;65(3):319-29.
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