Allele-specific oligonucleotide

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An anti-sense 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.

Contents

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. [1] 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.

Example

Binding of the "S" ASO probe to "S" DNA (top) or "A" DNA (bottom). Allele-specific oligonucleotide (sample).jpg
Binding of the "S" ASO probe to "S" DNA (top) or "A" DNA (bottom).

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, [2] 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).

Schematic of dot-blots using the "A" or "S" ASO probes. DotBlotDemo.jpg
Schematic of dot-blots using the "A" or "S" ASO probes.

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 blots. 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.

Alternatives

ASO analysis is only one of the methods used to detect genetic polymorphisms. Direct DNA sequencing 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, [3] 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.

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 in Duarte, California. In 1979 Wallace and his coworkers reported the use of ASO probes to detect variations in a single-stranded bacterial virus, [4] and later applied the technique to cloned human genes. In 1983 [5] and 1985 [2] 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. [2]

Fortunately PCR, a method to greatly amplify a specific segment of DNA, was also reported in 1985. [3] In less than a year PCR had been paired with ASO analysis. [6] 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.

Other uses

ASO-PCR may also be used to detect minimal residual disease in blood cancers such as multiple myeloma. [7]

Related Research Articles

Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.

<span class="mw-page-title-main">Polymerase chain reaction</span> Laboratory technique to multiply a DNA sample for study

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 sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

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. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

<span class="mw-page-title-main">Reverse transcription polymerase chain reaction</span> Laboratory technique to multiply an RNA sample for study

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

<span class="mw-page-title-main">Molecular beacon</span>

Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel non-radioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes.

In biology, a branched DNA assay is a signal amplification assay that is used to detect nucleic acid molecules.

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

<span class="mw-page-title-main">Nucleic acid test</span> Group of techniques to detect a particular nucleic acid sequence

A nucleic acid test (NAT) is a technique used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissue, urine, etc. NATs differ from other tests in that they detect genetic materials rather than antigens or antibodies. Detection of genetic materials allows an early diagnosis of a disease because the detection of antigens and/or antibodies requires time for them to start appearing in the bloodstream. Since the amount of a certain genetic material is usually very small, many NATs include a step that amplifies the genetic material—that is, makes many copies of it. Such NATs are called nucleic acid amplification tests (NAATs). There are several ways of amplification, including polymerase chain reaction (PCR), strand displacement assay (SDA), transcription mediated assay (TMA), and loop-mediated isothermal amplification (LAMP).

<span class="mw-page-title-main">Oligomer restriction</span>

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. If, however, the target DNA does not exactly match the probe, the restriction enzyme will have no effect on the length of the probe. The OR technique, now rarely performed, was closely associated with the development of the popular polymerase chain reaction (PCR) method.

<span class="mw-page-title-main">History of polymerase chain reaction</span>

The history of the polymerase chain reaction (PCR) has variously been described as a classic "Eureka!" moment, or as an example of cooperative teamwork between disparate researchers. Following is a list of events before, during, and after its development:

The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.

The ligase chain reaction (LCR) is a method of DNA amplification. The ligase chain reaction (LCR) is an amplification process that differs from PCR in that it involves a thermostable ligase to join two probes or other molecules together which can then be amplified by standard polymerase chain reaction (PCR) cycling. Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR. Thus, LCR requires two completely different enzymes to operate properly: ligase, to join probe molecules together, and a thermostable polymerase to amplify those molecules involved in successful ligation. The probes involved in the ligation are designed such that the 5′ end of one probe is directly adjacent to the 3′ end of the other probe, thereby providing the requisite 3′-OH and 5′-PO4 group substrates for the ligase.

The cleaved amplified polymorphic sequence (CAPS) method is a technique in molecular biology for the analysis of genetic markers. It is an extension to the restriction fragment length polymorphism (RFLP) method, using polymerase chain reaction (PCR) to more quickly analyse the results.

<span class="mw-page-title-main">MAGIChip</span>

MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads of 100x100x20 μm or smaller size. This technology is used for analysis of nucleic acid hybridization, specific binding of DNA, and low-molecular weight compounds with proteins, and protein-protein interactions.

Diversity Arrays Technology (DArT) is a high-throughput genetic marker technique that can detect allelic variations to provide comprehensive genome coverage without any DNA sequence information for genotyping and other genetic analysis. The general steps involve reducing the complexity of the genomic DNA with specific restriction enzymes, choosing diverse fragments to serve as representations for the parent genomes, amplify via polymerase chain reaction (PCR), inserting fragments into a vector to be placed as probes within a microarray, and then fluorescent targets from a reference sequence will be allowed to hybridize with probes and put through an imaging system. The objective is to identify and quantify various forms of DNA polymorphism within genomic DNA of sampled species.

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.

COLD-PCR is a modified polymerase chain reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA. The ability to preferentially amplify and identify minority alleles and low-level somatic DNA mutations in the presence of excess wildtype alleles is useful for the detection of mutations. Detection of mutations is important in the case of early cancer detection from tissue biopsies and body fluids such as blood plasma or serum, assessment of residual disease after surgery or chemotherapy, disease staging and molecular profiling for prognosis or tailoring therapy to individual patients, and monitoring of therapy outcome and cancer remission or relapse. Common PCR will amplify both the major (wildtype) and minor (mutant) alleles with the same efficiency, occluding the ability to easily detect the presence of low-level mutations. The capacity to detect a mutation in a mixture of variant/wildtype DNA is valuable because this mixture of variant DNAs can occur when provided with a heterogeneous sample – as is often the case with cancer biopsies. Currently, traditional PCR is used in tandem with a number of different downstream assays for genotyping or the detection of somatic mutations. These can include the use of amplified DNA for RFLP analysis, MALDI-TOF genotyping, or direct sequencing for detection of mutations by Sanger sequencing or pyrosequencing. Replacing traditional PCR with COLD-PCR for these downstream assays will increase the reliability in detecting mutations from mixed samples, including tumors and body fluids.

Suspension array technology is a high throughput, large-scale, and multiplexed screening platform used in molecular biology. SAT has been widely applied to genomic and proteomic research, such as single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, screening drug discovery and clinical diagnosis. SAT uses microsphere beads to prepare arrays. SAT allows for the simultaneous testing of multiple gene variants through the use of these microsphere beads as each type of microsphere bead has a unique identification based on variations in optical properties, most common is fluorescent colour. As each colour and intensity of colour has a unique wavelength, beads can easily be differentiated based on their wavelength intensity. Microspheres are readily suspendable in solution and exhibit favorable kinetics during an assay. Similar to flat microarrays, an appropriate receptor molecule, such as DNA oligonucleotide probes, antibodies, or other proteins, attach themselves to the differently labeled microspheres. This produces thousands of microsphere array elements. Probe-target hybridization is usually detected by optically labeled targets, which determines the relative abundance of each target in the sample.

<span class="mw-page-title-main">Molecular diagnostics</span> Collection of techniques used to analyze biological markers in the genome and proteome

Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. In medicine the technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients, and in agricultural biosecurity similarly to monitor crop- and livestock disease, estimate risk, and decide what quarantine measures must be taken.

<span class="mw-page-title-main">Surveyor nuclease assay</span>

Surveyor nuclease assay is an enzyme mismatch cleavage assay used to detect single base mismatches or small insertions or deletions (indels).

References

  1. ↑ Monga, Isha; Qureshi, Abid; Thakur, Nishant; Gupta, Amit Kumar; Kumar, Manoj (September 2017). "ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy". G3. 7 (9): 2931–2943. doi: 10.1534/g3.117.044024 . PMC   5592921 . PMID   28696921.
  2. 1 2 3 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).
  3. 1 2 Saiki, RK; Scharf S; Faloona F; Mullis KB; Horn GT; Erlich HA; Arnheim N (20 Dec 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science . 230 (4732): 1350–4. Bibcode:1985Sci...230.1350S. doi:10.1126/science.2999980. PMID   2999980. Archived from the original on 19 December 2008.
  4. ↑ Wallace, RB; Shaffer, J; Murphy, RF; Bonner, J; Hirose, T; Itakura, K (1979). "Hybridization of synthetic oligodeoxyribonucleotides to Phi-X 174 DNA: the effect of single base pair mismatch". Nucleic Acids Research. 6 (11): 3543–3558. doi:10.1093/nar/6.11.3543. PMC   327955 . PMID   158748.
  5. ↑ Conner BJ, Reyes AA, Morin C, Itakura K, Teplitz RL, and Wallace RB "Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides." Proc Natl Acad Sci USA. vol. 80(1), pp. 278–282 (1983).
  6. ↑ 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).
  7. ↑ Caers, Jo; Garderet, Laurent; Kortüm, K. Martin; O’Dwyer, Michael E.; van de Donk, Niels W.C.J.; Binder, Mascha; Dold, Sandra Maria; Gay, Francesca; Corre, Jill; Beguin, Yves; Ludwig, Heinz (November 2018). "European Myeloma Network recommendations on tools for the diagnosis and monitoring of multiple myeloma: what to use and when". Haematologica. 103 (11): 1772–1784. doi:10.3324/haematol.2018.189159. ISSN   0390-6078. PMC   6278986 . PMID   30171031.
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