Allele specific oligonucleotide
Encyclopedia
An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA
complementary to the sequence of a variable target DNA. It acts as a probe
for the presence of the target in a Southern blot
assay or, more commonly, in the simpler Dot blot
assay. It is a common tool used in genetic testing
, forensics
, and Molecular Biology
research.
An ASO is typically an oligonucleotide
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
, 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 polymorphisms (SNPs), important in genotype analysis
and the Human Genome Project
. 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.
The human disease sickle cell anemia
is caused by a genetic mutation
in the codon for the sixth amino acid
of the blood protein beta-hemoglobin
. The normal DNA sequence G-A-G codes for the amino acid glutamate, while the mutation changes the middle adenine
to a thymine
, leading to the sequence G-T-G (G-U-G in the mRNA
). 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 would be synthesized to be complementary to the altered sequence, 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).
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
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 of the ASO label that remains after washing allows a direct reading of the genotype
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 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.
is used to initially characterize the mutation, but is too laborious for routine screening. An earlier method, Restriction Fragment Length Polymorphism
(RFLP) didn't need to know the sequence change beforehand, but required that the mutation affect the cleavage site of a Restriction Enzyme
. 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
(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.
in Duarte, California
. 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
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. 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
, avoiding the laborious and inefficient Southern blot
method.
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
complementary to the sequence of a variable target DNA. It acts as a probe
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
for the presence of the target in a Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
assay or, more commonly, in the simpler Dot blot
Dot blot
right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate Dot-blot filters.A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods...
assay. It is a common tool used in genetic testing
Genetic testing
Genetic testing is among the newest and most sophisticated of techniques used to test for genetic disorders which involves direct examination of the DNA molecule itself. Other genetic tests include biochemical tests for such gene products as enzymes and other proteins and for microscopic...
, forensics
Forensics
Forensic science is the application of a broad spectrum of sciences to answer questions of interest to a legal system. This may be in relation to a crime or a civil action...
, and Molecular Biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
research.
An ASO is typically an oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
of 15–21 nucleotide bases
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
in length. It is designed (and used) in a way that makes it specific for only one version, or allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
, 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 polymorphisms (SNPs), important in genotype analysis
Genotype
The genotype is the genetic makeup of a cell, an organism, or an individual usually with reference to a specific character under consideration...
and the Human Genome Project
Human Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...
. 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
Illumina Methylation Assay
The Illumina Methylation Assay using the Infinium II platform uses 'BeadChip' technology to generate a comprehensive genome wide profiling of human DNA methylation. Similar to bisulfite sequencing and pyrosequencing, this method quantifies methylation levels at specific loci within the genome...
technology takes advantage of ASO to detect one base pair difference (cytosine versus thymine) to measure methylation at a specific CpG site.
Example
Sickle-cell disease
Sickle-cell disease , or sickle-cell anaemia or drepanocytosis, is an autosomal recessive genetic blood disorder with overdominance, characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the cells' flexibility and results in a risk of various...
is caused by a genetic mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
in the codon for the sixth amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
of the blood protein beta-hemoglobin
HBB
Beta globin Beta globin Beta globin (HBB, β-globinprotin that, along with alpha globin (HBA), makes up the most common form of hemoglobin in adult humans. The normal adult hemoglobin tetramer consists of two alpha chains and two beta chains.-Gene locus:...
. The normal DNA sequence G-A-G codes for the amino acid glutamate, while the mutation changes the middle adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
to a thymine
Thymine
Thymine is one of the four nucleobases 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 nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
, leading to the sequence G-T-G (G-U-G in the mRNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
). This altered sequence substitutes a valine
Valine
Valine is an α-amino acid with the chemical formula HO2CCHCH2. L-Valine is one of 20 proteinogenic amino acids. Its codons are GUU, GUC, GUA, and GUG. This essential amino acid is classified as nonpolar...
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, 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).
Dot blot
right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate Dot-blot filters.A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods...
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 of the ASO label that remains after washing allows a direct reading of the genotype
Genotype
The genotype is the genetic makeup of a cell, an organism, or an individual usually with reference to a specific character under consideration...
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 carriers
Genetic carrier
A genetic carrier , is a person or other organism that has inherited a genetic trait or mutation, but who does not display that trait or show symptoms of the disease. They are, however, able to pass the gene onto their offspring, who may then express the gene...
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 sequencingDNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
is used to initially characterize the mutation, but is too laborious for routine screening. An earlier method, Restriction Fragment Length Polymorphism
Restriction fragment length polymorphism
In molecular biology, restriction fragment length polymorphism, or RFLP , is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related...
(RFLP) didn't need to know the sequence change beforehand, but required that the mutation affect the cleavage site of a Restriction Enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
. The RFLP assay was briefly adapted to the use of oligonucleotide probes
Oligomer restriction
Oligomer Restriction is a procedure to detect an altered DNA sequence in a genome. A labeled oligonucleotide probe is hybridized to a target DNA, and then treated with a restriction enzyme. If the probe exactly matches the target, the restriction enzyme will cleave the probe, changing its size...
, but this technique was quickly supplanted by ASO analysis of polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) amplified DNA. The PCR technique itself has been adapted to detect polymorphisms, as allele-specific PCR
Variants of PCR
The versatility of PCR has led to a large number of variants:-Basic modifications:Often only a small modification needs to be made to the standard PCR protocol to achieve a desired goal:...
. 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 CenterCity of Hope National Medical Center
City of Hope National Medical Center, is a private, not-for-profit clinical research center, hospital and graduate medical school located in Duarte, California, United States...
in Duarte, California
Duarte, California
Duarte is a city in Los Angeles County, California, United States. As of the 2010 census, the city population was 21,321, down from 21,486 at the 2000 census....
. 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
Sickle-cell disease , or sickle-cell anaemia or drepanocytosis, is an autosomal recessive genetic blood disorder with overdominance, characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the cells' flexibility and results in a risk of various...
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. 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
Dot blot
right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate Dot-blot filters.A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods...
, avoiding the laborious and inefficient Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
method.