Allele-specific Oligonucleotide
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An anti-sense oligonucleotide (ASO) is a short piece of synthetic
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a
Southern blot Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample (such as blood or tissue) is digested with res ...
assay or, more commonly, in the simpler
dot blot A dot blot (or slot blot) is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by electrophoresis. Ins ...
assay. It is a common tool used in
genetic testing Genetic testing, also known as DNA testing, is used to identify changes in DNA sequence or chromosome structure. Genetic testing can also include measuring the results of genetic changes, such as RNA analysis as an output of gene expression, or ...
,
forensics Forensic science combines principles of law and science to investigate criminal activity. Through crime scene investigations and laboratory analysis, forensic scientists are able to link suspects to evidence. An example is determining the time and ...
, and
molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
research. An ASO is typically an
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, Recombinant DNA, research, and Forensic DNA, forensics. Commonly made in the laboratory by Oligonucleotide synthesis, solid-phase ...
of 15–21 nucleotide bases in length. It is designed (and used) in a way that makes it specific for only one version, or
allele An allele is a variant of the sequence of nucleotides at a particular location, or Locus (genetics), locus, on a DNA molecule. Alleles can differ at a single position through Single-nucleotide polymorphism, single nucleotide polymorphisms (SNP), ...
, of the DNA being tested. The length of the ASO, which strand it is chosen from, and the conditions by which it is bound to (and washed from) the target DNA all play a role in its specificity. These probes can usually be designed to detect a difference of as little as 1 base in the target's genetic sequence, a basic ability in the assay of
single-nucleotide polymorphism In genetics and bioinformatics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a ...
s (SNPs), important in genotype analysis and the
Human Genome Project The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a ...
. To be detected after it has bound to its target, the ASO must be labeled with a radioactive, enzymatic, or fluorescent tag. The Illumina Methylation Assay technology takes advantage of ASO to detect one base pair difference (cytosine versus thymine) to measure methylation at a specific CpG site.


Example

The human disease
sickle cell anemia Sickle cell disease (SCD), also simply called sickle cell, is a group of inherited haemoglobin-related blood disorders. The most common type is known as sickle cell anemia. Sickle cell anemia results in an abnormality in the oxygen-carrying ...
is caused by a genetic
mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, ...
in the
codon Genetic code is a set of rules used by living cells to translate information encoded within genetic material (DNA or RNA sequences of nucleotide triplets or codons) into proteins. Translation is accomplished by the ribosome, which links prote ...
for the sixth
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
of the blood protein beta-hemoglobin. The normal DNA sequence G-A-G codes for the amino acid
glutamate Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a Essential amino acid, non-essential nutrient for humans, meaning that ...
, while the mutation changes the middle
adenine Adenine (, ) (nucleoside#List of nucleosides and corresponding nucleobases, symbol A or Ade) is a purine nucleotide base that is found in DNA, RNA, and Adenosine triphosphate, ATP. Usually a white crystalline subtance. The shape of adenine is ...
to a
thymine Thymine () (symbol T or Thy) is one of the four nucleotide bases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine ...
, leading to the sequence G-T-G (G-U-G in the
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
). This altered sequence substitutes a
valine Valine (symbol Val or V) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- carboxylic acid group (which is in the deproton ...
into the final protein, distorting its structure. To test for the presence of the mutation in a DNA sample, an ASO probe would be synthesized to be complementary to the altered sequence,Studencki AB, Conner BJ, Impraim CC, Teplitz RL, and Wallace RB "Discrimination among the human beta A, beta S, and beta C-globin genes using allele-specific oligonucleotide hybridization probes." Am J Hum Genet vol. 37(1), pp. 42–51 (1985). here labeled as "S". As a control, another ASO would be synthesized for the normal sequence "A". 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 weaker interaction). The first diagram shows how the "S" probe is fully complementary to the "S" target (top), but is partially mismatched against the "A" target (bottom). 350px, A segment of the beta-hemoglobin genes in the sample DNA(s) would be amplified by PCR, and the resulting products applied to duplicate support membranes as
Dot blot A dot blot (or slot blot) is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by electrophoresis. Ins ...
s. The sample's DNA strands are separated with alkali, and each ASO probe is applied to a different blot. After hybridization, a washing protocol is used which can discriminate between the fully complementary and the mismatched hybrids. The mismatched ASOs are washed off of the blots, while the matched ASOs (and their labels) remain. In the second diagram, six samples of amplified DNA have been applied to each of the two blots.
Detection {{Unreferenced, date=March 2018 In general, detection is the action of accessing information without specific cooperation from with the sender. In the history of radio communications, the term "detector" was first used for a device that detected ...
of the ASO label that remains after washing allows a direct reading of the
genotype The genotype of an organism is its complete set of genetic material. Genotype can also be used to refer to the alleles or variants an individual carries in a particular gene or genetic location. The number of alleles an individual can have in a ...
of the samples, each with two copies of the beta-hemoglobin gene. Samples 1 and 4 only have the normal "A" allele, while samples 3 and 5 have both the "A" and "S" alleles (and are therefore
heterozygous Zygosity (the noun, zygote, is from the Greek "yoked," from "yoke") () is the degree to which both copies of a chromosome or gene have the same genetic sequence. In other words, it is the degree of similarity of the alleles in an organism. Mos ...
carriers of this recessive mutation). Samples 2 and 6 have only the "S" allele, and would be affected by the disease. The small amount of 'cross hybridization' shown is typical, and is considered in the process of interpreting the final results.


Alternatives

ASO analysis is only one of the methods used to detect genetic polymorphisms. Direct
DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, thymine, cytosine, and guanine. The ...
is used to initially characterize the mutation, but is too laborious for routine screening. An earlier method,
Restriction Fragment Length Polymorphism In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence. T ...
(RFLP) didn't need to know the sequence change beforehand, but required that the mutation affect the cleavage site of a
Restriction Enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
. The RFLP assay was briefly adapted to the use of oligonucleotide probes, but this technique was quickly supplanted by ASO analysis of
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...
(PCR) amplified DNA. The PCR technique itself has been adapted to detect polymorphisms, as allele-specific PCR. However, the simplicity and versatility of the combined PCR/ASO method has led to its continued use, including with non-radioactive labels, and in a "reverse dot blot" format where the ASO probes are bound to the membrane and the amplified sample DNA is used for hybridization.


History

The use of synthetic oligonucleotides as specific probes for genetic sequence variations was pioneered by R. Bruce Wallace, working at the
City of Hope National Medical Center City of Hope is a private, non-profit clinical research center, hospital and graduate school located in Duarte, California, United States. The center's main campus resides on of land adjacent to the boundaries of Duarte and Irwindale, California ...
in
Duarte, California Duarte () is a city in Los Angeles County, California, United States. As of the 2020 United States census, 2020 census, the city population was 21,727. It is bounded to the north by the San Gabriel Mountains, to the north and west by the cities ...
. In 1979 Wallace and his coworkers reported the use of ASO probes to detect variations in a single-stranded bacterial virus, and later applied the technique to cloned human genes. In 1983 and 1985 Wallace's lab reported the detection of the mutation for
sickle cell anemia Sickle cell disease (SCD), also simply called sickle cell, is a group of inherited haemoglobin-related blood disorders. The most common type is known as sickle cell anemia. Sickle cell anemia results in an abnormality in the oxygen-carrying ...
in samples of whole genomic DNA, although this application was hampered by the small amount of label that could be carried by the ASO. Fortunately PCR, a method to greatly amplify a specific segment of DNA, was also reported in 1985. In less than a year PCR had been paired with ASO analysis.Saiki RK, Bugawan TL, Horn GT, Mullis KB, and Erlich HE "Analysis of enzymatically amplified beta-globin and HLA-DQ DNA with allele-specific oligonucleotide probes" Nature vol. 324(6093) pp. 163–166 (1986). This combination solved the problem of ASO labeling, since the amount of target DNA could be amplified over a million-fold. Also, the specificity of the PCR process itself could be added to that of the ASO probes, greatly reducing the problem of spurious binding of the ASO to non-target sequences. The combination was specific enough that it could be used in a simple
Dot blot A dot blot (or slot blot) is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by electrophoresis. Ins ...
, avoiding the laborious and inefficient
Southern blot Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample (such as blood or tissue) is digested with res ...
method.


Other uses

ASO-PCR may also be used to detect
minimal residual disease Minimal residual disease (MRD), also known as molecular residual disease, is the medical condition in which small number of cancer cells persist in a patient either during or after treatment when the patient is in Remission (medicine), remission ...
in blood cancers such as
multiple myeloma Multiple myeloma (MM), also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibody, antibodies. Often, no symptoms are noticed initially. As it progresses, bone ...
.


References


External links


Image of ASO autoradiogram
{{DEFAULTSORT:Anti-Sense Oligonucleotide DNA profiling techniques Molecular biology Laboratory techniques Molecular biology techniques