Single-strand conformation polymorphism (SSCP), or single-strand chain polymorphism, is defined as a conformational difference of single-stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions. This property allows sequences to be distinguished by means of gel electrophoresis, which separates fragments according to their different conformations. [1]
A single nucleotide change in a particular sequence, as seen in a double-stranded DNA, cannot be distinguished by gel electrophoresis techniques, which can be attributed to the fact that; the physical properties of the double strands are almost identical for both alleles. After denaturation, single-stranded DNA undergoes a characteristic 3-dimensional folding and may assume a unique conformational state based on its DNA sequence. The difference in shape between two single-stranded DNA strands with different sequences can cause them to migrate differently through an electrophoresis gel, even though the number of nucleotides is the same, which is, in fact, an application of SSCP.
SSCP used to be a way to discover new DNA polymorphisms apart from DNA sequencing but is now being supplanted by sequencing techniques on account of efficiency and accuracy. [2] These days, SSCP is most applicable as a diagnostic tool in molecular biology. It can be used in genotyping to detect homozygous individuals of different allelic states, as well as heterozygous individuals that should each demonstrate distinct patterns in an electrophoresis experiment. [3] SSCP is also widely used in virology to detect variations in different strains of a virus, the idea being that a particular virus particle present in both strains will have undergone changes due to mutation, and that these changes will cause the two particles to assume different conformations and, thus, be differentiable on an SSCP gel. [4]
Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is deoxyribose, a variant of ribose, the polymer is DNA.
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.
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.
In genetics and bioinformatics, a single-nucleotide polymorphism 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 sufficiently large fraction of the population, many publications do not apply such a frequency threshold.
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, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.
Multilocus sequence typing (MLST) is a technique in molecular biology for the typing of multiple loci, using DNA sequences of internal fragments of multiple housekeeping genes to characterize isolates of microbial species.
Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method for approximately 40 years. An automated instrument using slab gel electrophoresis and fluorescent labels was first commercialized by Applied Biosystems in March 1987. Later, automated slab gels were replaced with automated capillary array electrophoresis. More recently, higher volume Sanger sequencing has been replaced by next generation sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use for smaller-scale projects and for validation of deep sequencing results. It still has the advantage over short-read sequencing technologies in that it can produce DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%. Sanger sequencing is still actively being used in efforts for public health initiatives such as sequencing the spike protein from SARS-CoV-2 as well as for the surveillance of norovirus outbreaks through the Center for Disease Control and Prevention's (CDC) CaliciNet surveillance network.
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation, though this term is used for other procedures as well. In a restriction digest, DNA molecules are cleaved at specific restriction sites of 4-12 nucleotides in length by use of restriction enzymes which recognize these sequences.
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change, or a long one, like minisatellites.
TILLING is a method in molecular biology that allows directed identification of mutations in a specific gene. TILLING was introduced in 2000, using the model plant Arabidopsis thaliana, and expanded on into other uses and methodologies by a small group of scientists including Luca Comai. TILLING has since been used as a reverse genetics method in other organisms such as zebrafish, maize, wheat, rice, soybean, tomato and lettuce.
A restriction fragment is a DNA fragment resulting from the cutting of a DNA strand by a restriction enzyme, a process called restriction. Each restriction enzyme is highly specific, recognising a particular short DNA sequence, or restriction site, and cutting both DNA strands at specific points within this site. Most restriction sites are palindromic,, and are four to eight nucleotides long. Many cuts are made by one restriction enzyme because of the chance repetition of these sequences in a long DNA molecule, yielding a set of restriction fragments. A particular DNA molecule will always yield the same set of restriction fragments when exposed to the same restriction enzyme. Restriction fragments can be analyzed using techniques such as gel electrophoresis or used in recombinant DNA technology.
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.
Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.
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.
Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. The temperature at which 50% of DNA is denatured is known as the melting temperature. Measurement of melting temperature can help us predict species by just studying the melting temperature. This is because every organism has a specific melting curve.
Experimental approaches of determining the structure of nucleic acids, such as RNA and DNA, can be largely classified into biophysical and biochemical methods. Biophysical methods use the fundamental physical properties of molecules for structure determination, including X-ray crystallography, NMR and cryo-EM. Biochemical methods exploit the chemical properties of nucleic acids using specific reagents and conditions to assay the structure of nucleic acids. Such methods may involve chemical probing with specific reagents, or rely on native or analogue chemistry. Different experimental approaches have unique merits and are suitable for different experimental purposes.
A gene is said to be polymorphic if more than one allele occupies that gene's locus within a population. In addition to having more than one allele at a specific locus, each allele must also occur in the population at a rate of at least 1% to generally be considered polymorphic.
Surveyor nuclease assay is an enzyme mismatch cleavage assay used to detect single base mismatches or small insertions or deletions (indels).
Ground squirrel hepatitis virus, abbreviated GSHV, is a partially double-stranded DNA virus that is closely related to human Hepatitis B virus (HBV) and Woodchuck hepatitis virus (WHV). It is a member of the family of viruses Hepadnaviridae and the genus Orthohepadnavirus. Like the other members of its family, GSHV has high degree of species and tissue specificity. It was discovered in Beechey ground squirrels, Spermophilus beecheyi, but also infects Arctic ground squirrels, Spermophilus parryi. Commonalities between GSHV and HBV include morphology, DNA polymerase activity in genome repair, cross-reacting viral antigens, and the resulting persistent infection with viral antigen in the blood (antigenemia). As a result, GSHV is used as an experimental model for HBV.
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