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In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a specific protein in a cell that does not normally express that protein, or to sequence or quantify mRNA molecules using DNA based methods. cDNA that codes for a specific protein can be transferred to a recipient cell for expression, often bacterial or yeast expression systems. cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays, qPCR, and RNA-seq.
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 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 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.
Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017.
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). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes. These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. The original nucleic acid arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based analysis was published in 1981. It was invented by Patrick O. Brown. An example of its application is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. It is also used for the identification of structural variations and the measurement of gene expression.
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."
Rapid amplification of cDNA ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies. The amplified cDNA copies are then sequenced and, if long enough, should map to a unique genomic region. RACE is commonly followed up by cloning before sequencing of what was originally individual RNA molecules. A more high-throughput alternative which is useful for identification of novel transcript structures, is to sequence the RACE-products by next generation sequencing technologies.
Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.
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.
Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism.
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.
Webtag is an on-line bioinformatics tool providing oligonucleotide sequences that are absent from a specified genome. These tags can be appended to gene specific primers for reverse transcriptase polymerase chain reaction (RT-PCR) experiments, circumventing genomic DNA contamination.
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.
Cap analysis of gene expression (CAGE) is a gene expression technique used in molecular biology to produce a snapshot of the 5′ end of the messenger RNA population in a biological sample. The small fragments from the very beginnings of mRNAs are extracted, reverse-transcribed to cDNA, PCR amplified and sequenced. CAGE was first published by Hayashizaki, Carninci and co-workers in 2003. CAGE has been extensively used within the FANTOM research projects.
The reverse northern blot is a method by which gene expression patterns may be analyzed by comparing isolated RNA molecules from a tester sample to samples in a control cDNA library. It is a variant of the northern blot in which the nucleic acid immobilized on a membrane is a collection of isolated DNA fragments rather than RNA, and the probe is RNA extracted from a tissue and radioactively labelled. A reverse northern blot can be used to profile expression levels of particular sets of RNA sequences in a tissue or to determine presence of a particular RNA sequence in a sample. Although DNA Microarrays and newer next-generation techniques have generally supplanted reverse northern blotting, it is still utilized today and provides a relatively cheap and easy means of defining expression of large sets of genes.
Single-cell sequencing examines the sequence information from individual cells with optimized next-generation sequencing technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. For example, in cancer, sequencing the DNA of individual cells can give information about mutations carried by small populations of cells. In development, sequencing the RNAs expressed by individual cells can give insight into the existence and behavior of different cell types. In microbial systems, a population of the same species can appear genetically clonal. Still, single-cell sequencing of RNA or epigenetic modifications can reveal cell-to-cell variability that may help populations rapidly adapt to survive in changing environments.
G&T-seq is a novel form of single cell sequencing technique allowing one to simultaneously obtain both transcriptomic and genomic data from single cells, allowing for direct comparison of gene expression data to its corresponding genomic data in the same cell...
This glossary of genetics is a list of definitions of terms and concepts commonly used in the study of genetics and related disciplines in biology, including molecular biology, cell biology, and evolutionary biology. It is intended as introductory material for novices; for more specific and technical detail, see the article corresponding to each term. For related terms, see Glossary of evolutionary biology.
References
- 1 2 3 4 5 Peale FV, Jr; Gerritsen, ME (September 2001). "Gene profiling techniques and their application in angiogenesis and vascular development". The Journal of Pathology. 195 (1): 7–19. doi: 10.1002/path.888 . PMID 11568887.
- ↑ Pandey, A; Mann, M (15 June 2000). "Proteomics to study genes and genomes". Nature. 405 (6788): 837–46. doi:10.1038/35015709. PMID 10866210.
- ↑ Reece, Richard J. (2004). Analysis of genes and genomes. Chichester: Wiley. ISBN 978-0-470-84380-2.
- ↑ Liang, P; Pardee, AB (14 August 1992). "Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction". Science. 257 (5072): 967–71. PMID 1354393.