Padlock probes
Related Terms
Acquired immune deficiency syndrome, AIDS, Alzheimer's disease, arthritis, circularizable oligonucleotide, C-probe, cystic fibrosis, disease detection, DNA library, DNA ligation, genotyping, hemoglobin, human immunodeficiency virus type 1, HIV-1, hybridization, in-situ detection, Marfan syndrome, microarray, mutation, padlock probe, pathogens, PCR, polymerase chain reaction, RCA, rolling circle amplification, sickle cell anemia, single-nucleotide polymorphism, SNP, supramolecular DNA assembly.
Background
A padlock probe (PLP), a long oligonucleotide whose ends are complementary to an adjacent target sequence, is used to detect and identify a target molecule in a sample. PLP is used in genetic analysis techniques. Genetic analysis is performed to detect variations, or mutations, in the sequence of a gene, which may be associated with the development of certain diseases in humans, plants, and/or animals. Thus, detection of mutations may help in the early identification and prevention of these associated diseases. Padlock probes have a very good distinguishing quality (specificity), which makes them significantly advantageous over other conventional oligonucleotide probes.
Genes are the functional units of deoxyribonucleic acid (DNA) and are considered the building blocks of life because they provide instructions for all the cells in the body. Genes, which are located inside cells, control an organism's development and functions by instructing cells to make new molecules (usually proteins).
DNA is a long thread-like molecule made up of large numbers of nucleotides. Nucleotides are molecules composed of a nitrogen base, a 5-carbon sugar, and one or more phosphate groups. Long strands of nucleotides form nucleic acids, such as (DNA) and ribonucleic acid (RNA).
RNA is a single chain of nucleotides that contains ribose as its sugar (ribonucleotides). RNA determines the protein synthesis and transmission of genetic information.
Nitrogen bases are of two types; purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T) in DNA, and cytosine and uracil (U) in RNA. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information.
A probe is a synthetic nucleic acid sequence that selectively binds with complementary nucleic acid sequences on the target DNA from the sample of interest; this allows the target sequence or DNA to be isolated and identified. The probe is usually attached (labeled) with a fluorescent marker. If the target sequence of interest is present in the sample, the probe emits a colored light, thus helping in identification. Oligonucleotide probes are short sequences of nucleotides, usually of 20-50 bases that are complementary to a specific DNA sequence or a region where a mutation is known to occur. Some of the genetic analysis techniques that use oligonucleotide probes include polymerase chain reaction and hybridization reactions.
Polymerase chain reaction (PCR): PCR is an efficient and sensitive laboratory technique to amplify (by replication) a specific sequence of DNA into billions of its copies. It is conducted in the presence of sequence specific oligonucleotide primers and DNA polymerase enzymes. An oligonucleotide primer is a sequence of nucleotides, usually of 20-50 bases, that is complementary to a specific DNA sequence and assists in DNA replication and/or multiplication.
Hybridization: Hybridization is a process of binding or bonding between probe and sample DNA or RNA. This laboratory technique is used to detect specific DNA and RNA sequences in a biological sample utilizing the probe. The probe, when exposed to cells or sample of interest, will bind with the target sequence of the DNA or RNA. This forms a hybrid (a double strand of nucleotides containing one strand of DNA and one strand of RNA), and the reaction process is called a hybridization reaction.
Structure of padlock probes: Padlock probes are made of linear oligonucleotides (typically of 70-100 nucleotides in length) that have complementary sequences to the target at both its ends, and a nucleotide sequence in between that can be used for amplification or identification via DNA tag (attached) sequences. During the genetic analysis techniques using the padlock probes, the end- segments of these probes get hybridized with the complementary target sequence, forming a circle around the target. Hence, the padlock probes are also called circularizable oligonucleotide probes or C-probes.
Methods
Probes are sequences of nucleotides that selectively bind with complementary nucleotide sequences on the target deoxyribonucleic acid (DNA) from the sample of interest. This enables the target sequence or DNA to be isolated and identified by the nucleic acid analysis techniques (e.g., polymerase chain reaction/PCR). Nucleotides are the building blocks of DNA in a sequence and are made of nitrogenous bases, sugars, and phosphate. An oligonucleotide probe or primer is a sequence of nucleotides, usually of 20-50 bases.
General: A padlock probe is a linear oligonucleotide sequence that has complementary sequences to the target at both of its ends, and a nucleotide sequence in between that can be used for amplification or identification via DNA tag (attached) sequences. The nucleic acid analysis techniques utilizing padlock probes involve isolation (extraction) of DNA or RNA from the sample of interest, followed by hybridization, as well as ligation reaction processes and rolling-circle amplification (RCA)-polymerase chain reactions (PCRs).
Hybridization and ligation reaction: Hybridization is a laboratory technique in which a probe is used to detect specific DNA and RNA sequences in a biological sample.. The probe, when exposed to cells or samples of interest (DNA or RNA sequences), will bind with the target sequence of the DNA or RNA, thereby forming a hybrid. This reaction process is called a hybridization reaction. Both ends of the padlock probes get hybridized with the target DNA sequence, and in the presence of a DNA ligase (enzyme), form a firm bond between the complementary sequences. Thus, a circular closed structure is formed around the target structure. The probe locks onto its target due to helical turns (spiral form) formed between complementary sequences of the target and the padlock probe.
The nature of padlocks and stringent requirements for ligation (binding) make the padlock probe especially useful for in-situ hybridization and detection of single-nucleotide polymorphisms (SNPs). In-situ hybridization is a technique where the probe is hybridized with the target DNA or RNA sequence that is to be detected. This sequence is present at its original place (in-situ), i.e., within the cell, tissue sections, or isolated chromosomes, thereby aiding in localizing the target sequence at its original place. SNP refers to DNA sequence variations that occur when a single nucleotide in the genome sequence is altered. SNPs have been associated with the development of several diseases.
Rolling circle replication (RCA)-PCR: PCR is an efficient and sensitive laboratory technique to amplify a specific sequence of DNA into billions of its copies in the presence of sequence-specific oligonucleotide primers and DNA polymerase enzymes. An oligonucleotide primer is complementary to a target DNA sequence and serves as a starting point for DNA replication. DNA polymerase is an enzyme that creates new DNA strands using preexisting DNA strands as a template, thereby assisting in DNA replication. A fluorescent marker is also incorporated into the PCR product during the amplification process. This assists in visualizing the PCR products at a later stage.
The RCA is a nucleic acid amplification process that uses the circular molecules of single-stranded DNA as a template. In the presence of a DNA polymerase enzyme and oligo- or polynucleotide primer, a billion-fold copies of linear replicas of the DNA circle are synthesized at a constant temperature within 1-2 hours. Thus, RCA methodology is an ultra-sensitive and robust diagnostic analytical technique to detect specific DNA markers.
Advantages: The padlock probe offers a number of advantages in comparison to standard hybridization probes and PCR. The ligation reaction process forms a firm bond between probes and long target sequences that can withstand washing procedures to remove the un-bound sequences and un-reacted probes; this preserves the reacted or bound probes. The RCA process may provide the laboratory researchers and clinical diagnosticians with a highly sensitive and efficient contamination-resistant diagnostic technique that is capable of high multiplexity (amplification). The RCA process may be used in a variety of testing formats, as well. Also, this methodology has proved to be a rapid, accurate, and efficient technique to detect and identify SNPs that may be associated with the development of certain diseases/disorders.
Research
Disease detection: Because nucleic acid detection methods that use padlock probes are fast,, are accurate, and easy to automate while remaining economical, they are being studied to diagnose several viral diseases in animals, which come under the World Organization for Animal Health's (OIE) list of notifiable diseases. The selected diseases include foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, classical swine fever, African swine fever, bluetongue, African horse sickness, Newcastle disease, and highly pathogenic avian influenza. These are highly contagious.. Rapid and accurate detection and diagnosis of these diseases allows for the rapid prohibition of livestock exportation, and may help prevent high economical losses caused by the spread of disease. Livestock exportation refers to exporting farm animals, such as cattle, horses, sheep, etc. to different countries and regions.
Strand-specific techniques: the human immunodeficiency virus type 1 (HIV-1) causes acquired immune deficiency syndrome (AIDS), a debilitating and fatal disease of the human immune system. HIV-1 has two strands of ribonucleic acid (RNA). The polymerase chain reactions (PCR) used to amplify the DNA strands cannot distinguish between the two DNA strands. A system for quantitative (measurement of quantity of viruses), strand-specific analysis of reverse transcription during the course of viral infection is now available.
Reverse transcription is a process of the synthesis of DNA under the direction of RNA. Generally, transcription is the process of RNA synthesis from a DNA template. A novel strand-specific amplification method using padlock probes has been developed for site-specific amplification and the quantificationof each strand during HIV-1 reverse transcription. This is usedto measure the relative abundance of HIV-1 reverse transcriptionproducts generated at distinct steps over the course ofinfection. This provides insights into the viral replication process and the course of viral infection, which may help in developing newer drugs suitable to the specific targets. This, in turn, may help in limiting the progression of HIV-1 infection.
Microarray: DNA microarrays or chips are miniaturized chemical reaction areas used to test DNA/RNA fragments by immobilizing the target sequence, along with a fluorescent marker on the surface of the chip, and hybridizing them with a probed sample. Fluorescent color is emitted when the specific target is hybridized and scanned. The data are then analyzed by a computer, which aids in identifying the target sequence. Hybridization refers to binding of the probe with the target complementary sequence forming a hybrid. The padlock probes have been used with the microarray output to detect several multiplex reactions. Thus, multiple reactions to detect the target sequences, which may be associated with several diseases or markers in a single disease at the same time, provide timely treatment decisions and management of disease with high efficiency and specificity.
Implications
General: Padlock probes are made of linear oligonucleotides that have complementary sequences to the target at both ends and a nucleotide sequence in between that can be used for amplification or identification via DNA tag (attached) sequences. Nucleotides are considered the building blocks of deoxyribonucleic acid (DNA) in a sequence and are made of nitrogenous bases, sugars, and phosphate. An oligonucleotide probe is a sequence of nucleotides, usually of 20-50 bases. Mutations are variations in the sequence of a gene, which may be associated with the development of certain diseases.
There is vast availability of information about nucleic acid sequences in humans, as well as other organisms; identification of these sequences on a large scale is generally considered important. However, methods currently used cannot offer the required combination of high-throughput (fast), sensitivity, and specificity of detection. Padlock probes may provide a means to detect, distinguish, quantitate, and locate very large numbers of DNA or RNA sequences.
Genetic disease diagnosis: A genetic disease or disorder is a condition that is caused by an abnormal expression of one or more genes. This occurs when the chemicals that make up an individual's genes are incorrectly deleted, added, or substituted. If the mutation causes the cells in the body to stop functioning properly, the person may develop a disease or disorder. Some examples include cystic fibrosis, sickle cell anemia, and Marfan syndrome. Cystic fibrosis is one of the most common genetic diseases, affecting the lungs, digestive system, sweat glands, and male infertility. Sickle cell anemia is a blood disorder affecting the hemoglobin, which may be fatal. Hemoglobin is an oxygen carrying protein in the blood. Marfan syndrome is a connective tissue disorder chiefly involving the eyes, heart, blood vessels, bones, and muscles. Early detection and diagnosis of these genetic disorders using the padlock probe may help physicians decide how best to treat or manage particular conditions.
Carrier detection: Genetic disease carriers are individuals who do not express or present with disease symptoms, but who do carry the mutated genes associated with the disease. These individuals may pass the mutated genes on to their children. A child can develop a recessive disorder if they inherit one mutated from each of his/her parents. Genetic analysis techniques using padlock probes may help determine whether an individual is a carrier for a disease-causing gene. As a result, this may help prevent the transmission of disease to the next generation.
Prenatal detection: Some of the genetic disorders and infectious diseases (e.g. viral infections) may be passed on from the mother to the baby during pregnancy. Prenatal tests may be performed to determine if a fetus has a particular disease or disorder. Prenatal detection techniques that use padlock probes are generally rapid, highly specific, and robust, and thus, more efficient than many other types of prenatal tests.
Evaluation of multifactorial disorders: Multifactorial disorders are caused by a combination of environmental factors and mutations in multiple genes. The presence of particular genetic mutations may make patients more susceptible to developing disorders. Some of these examples include: heart disease, obesity, Alzheimer's disease, high blood pressure, arthritis, diabetes, and cancer. Alzheimer's disease is a progressive disorder of the nervous system associated with aging and memory loss. Diabetes mellitus is a chronic (long-term) condition associated with abnormally high levels of glucose (sugar) in the blood. Detection of the gene mutations, most often the single nucleotide polymorphisms (SNPs) associated with such disorders, understanding the genetic basis of these diseases, and the drug response to them may aids in the prevention and monitoring of multifactorial diseases. An SNP is a DNA sequence variation that occurs when a single nucleotide in the genome sequence is altered.
Detection of pathogens: An agent or an organism capable of causing disease in humans, animals, or plants is known as a pathogen (for example, certain bacteria, viruses, and fungi). Detection and identification of these pathogens, which require genetic analysis techniques, may involve padlock probes for fast and efficient detection. Padlock probes also help in identification or screening for multiple pathogens in a single reaction. Padlock probes may also aid in suitable therapeutic management and disease monitoring (e.g., HIV).
Genotyping: Genotype refers to the genetic makeup of an organism, and genotyping refers to the process of determining the genotype of an organism. Employing the use of padlock probes in genotyping techniques aids in detecting and identifying the different species and/or strains (subspecies) of microbial organisms. This, in turn, helps researchers trace the origin of disease-causing organisms and may help preventing the spread of diseases.
Forensic detection: DNA analysis techniques that use padlock probes help confirm the identification of individual polynucleotides isolated from the biological matter (e.g. blood) collected from crime scenes or victims. This technique may assist in criminal cases because it may be used to provide evidence about the identity of both crime victims and perpetrators of crimes who leave any biological material at the crime scene.
Limitations
Using long padlock probes, those containing about 80 nucleotides or more, is expensive and may result in synthesis failures during the amplification process. This may be overcome by using shorter padlock probes. The size of the padlock probes also depends on the target sequence to be detected.
The ligation reaction process of circularization of the padlock probes at constant temperature may sometimes result in weak bonds. This may be overcome by using a multiple cycle ligation process that results in more effective circularization of padlock probes, and one that shows higher specificity in the identification of mismatches. The conventional methods are time-consuming, but have been overcome with recent advances, such as real time-polymerase chain reaction (RT-PCR) and DNA microarray technology. RT-PCR is a PCR-based laboratory technique that enables both detection and quantification of a specific sequence in a DNA sample simultaneously. This follows the general principle of PCR with a key feature, i.e., the amplification or increase in the amount of DNA can be measured and also viewed as it gets collected in the reaction in real time (at the same time during which the process occurs) after each round of the amplification cycle. DNA microarrays or chips are miniaturized chemical reaction areas used to test DNA/RNA fragments by immobilizing the target sequence, along with a fluorescent marker on the surface of the chip, and hybridizing them with a probed sample. Fluorescent color is emitted when the specific target is hybridized. The target is then scanned and the data are analyzed by a computer, thus aiding in the identification of the target sequence. Hybridization refers to binding of the probe with the target complementary sequence, thereby forming a hybrid.
Future research
The applications of padlock probes have potential in several nucleic acid analysis techniques, such as supramolecular DNA assemblies and construction of DNA libraries. Supramolecular DNA assembly is a process of putting the fragments of DNA that have been sequenced into their correct chromosomal locations. The pieces of DNA are assembled to reconstitute the sequence of the chromosome from which they came. This helps to study the form and structure of an organism in relation to its function. A DNA library is a collection of one organism's DNA fragments that are stored within a host organism. For example, the long strands of chromosomal DNA are cut into thousands of fragments and are stored in a separate host organism. In this library form, the fragments can be screened to identify individual genes.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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