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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.
The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.
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.
Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the assay. When an assay for measuring the infective virus particle is done, viral titre often refers to the concentration of infectious viral particles, which is different from the total viral particles. Viral load is measured using body fluids sputum and blood plasma. As an example of environmental specimens, the viral load of norovirus can be determined from run-off water on garden produce. Norovirus has not only prolonged viral shedding and has the ability to survive in the environment but a minuscule infectious dose is required to produce infection in humans: less than 100 viral particles.
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.
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication. In this context, amplification refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As it refers to the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as "PCR product."
The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.
A nuclear run-on assay is conducted to identify the genes that are being transcribed at a certain time point. Approximately one million cell nuclei are isolated and incubated with labeled nucleotides, and genes in the process of being transcribed are detected by hybridization of extracted RNA to gene specific probes on a blot. Garcia-Martinez et al. (2004) developed a protocol for the yeast S. cerevisiae that allows for the calculation of transcription rates (TRs) for all yeast genes to estimate mRNA stabilities for all yeast mRNAs.
In biology, a branched DNA assay is a signal amplification assay that is used to detect nucleic acid molecules.
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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.
Nucleic acid sequence-based amplification, commonly referred to as NASBA, is a method in molecular biology which is used to produce multiple copies of single stranded RNA. NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.
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 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.
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.
Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.
Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation sequencing. Some of these technologies emerged between 1993 and 1998 and have been commercially available since 2005. These technologies use miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads per instrument run.
In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. Though a double-stranded DNA sequence is generally stable under physiological conditions, changing these conditions in the laboratory will cause the molecules to separate into single strands. These strands are complementary to each other but may also be complementary to other sequences present in their surroundings. Lowering the surrounding temperature allows the single-stranded molecules to anneal or “hybridize” to each other.
References
- ↑ Tang, Wei; Hu, Shichao; Wang, Huaming; Zhao, Yan; Li, Na; Liu, Feng (23 September 2014). "A universal molecular translator for non-nucleic acid targets that enables dynamic DNA assemblies and logic operations". Chem. Commun. 50 (92): 14352–14355. doi:10.1039/C4CC07041K. PMID 25295484.
- ↑ Ihalainen, Petri; Pettersson, Fredrik; Pesonen, Markus; Viitala, Tapani; Määttänen, Anni; Österbacka, Ronald; Peltonen, Jouko (7 March 2014). "An impedimetric study of DNA hybridization on paper-supported inkjet-printed gold electrodes". Nanotechnology. 25 (9): 094009. Bibcode:2014Nanot..25i4009I. doi:10.1088/0957-4484/25/9/094009. PMID 24522116. S2CID 206070778.
- ↑ Berney, H.; Oliver, K. (2005). "Dual polarization interferometry size and density characterisation of DNA immobilisation and hybridisation". Biosensors and Bioelectronics. 21 (4): 618–626. doi:10.1016/j.bios.2004.12.024. PMID 16202875.
- ↑ Dixon, Matthew C. (July 2008). "Quartz Crystal Microbalance with Dissipation Monitoring: Enabling Real-Time Characterization of Biological Materials and Their Interactions". Journal of Biomolecular Techniques. 19 (3): 151–158. PMC 2563918 . PMID 19137101.
- ↑ Hannon, Gregory J. (July 2002). "RNA interference". Nature. 418 (6894): 244–251. Bibcode:2002Natur.418..244H. doi:10.1038/418244a. ISSN 1476-4687. PMID 12110901. S2CID 4426281.
