A common Gram-negative bacterium useful for cloning experiments. Present in human intestinal tract.
It describes the expression of a gene in a tissue in which it is not usually expressed.
Efficiency of the Reaction:
The efficiency of the reaction can be calculated by the following equation: E = 10(-1/slope) –1. The efficiency of the PCR should be 90-100% meaning doubling of the amplicon at each cycle.
A method for introducing foreign nucleic acid into bacterial or eukaryotic cells that uses a brief, high voltage DC charge which renders the cells permeable to the nucleic acid. Also useful for introducing synthetic peptides into eucaryotic cells.
A method of separating large molecules from a mixture of similar molecules. An electric current is passed through a medium containing the mixture, and each kind of molecule travels through the medium at a different rate, depending on its electrical charge and size. Separation is based on these differences. Agarose and acrylamide gels are the media commonly used for electrophoresis of proteins and nucleic acids.
A protein(s) that associates with the ribosome cyclically to assist in loading tRNA into the the A site of the ribosome.
The technique of adding a radioactively labeled group to one end (5' or 3' end) of a DNA strand.
This is an RNA or DNA that is naturally present in each experimental sample. By using an invariant endogenous control as an active 'reference', quantitation of a messenger RNA (mRNA) target can be normalized for differences in the amount of total RNA added to each reaction and correct for sample-to-sample variations in reverse transcriptase PCR efficiency.
As opposed to quantitative analysis using the data collected during exponential phase of PCR, real-time applications can also be used to collect end-point data for qualitative assays. These are either allelic discrimination assays (genotyping) or absence/presence assays (pathogen detection).
An enzyme which digests nucleic acids starting in the middle of the strand. Examples include the restriction enzymes, DNase I and RNase A.
A specialized membranous organelle within eukaryotic cells responsible for synthesis of membrane-inserted proteins, and for proteins to be exported of proteins to the cell surface or beyond.
An enhancer is a nucleotide sequence to which transcription factor(s) bind, and which increases the transcription of a gene. It is NOT part of a promoter; the basic difference being that an enhancer can be moved around anywhere in the general vicinity of the gene and it will still function. It can even be clipped out and spliced back in backwards, and will still operate. A promoter, on the other hand, is position- and orientation-dependent. Some enhancers are "conditional" - in other words, they enhance transcription only under certain conditions, for example in the presence of a hormone.
A protein that acts as a catalyst, speeding the rate at which a biochemical reaction proceeds but not altering the direction or nature of the reaction.
A change in phenotype brought about by changes in gene regulation rather than by a change in genotype.
Extrachromosomal genetic element. Generally used synonymously for plasmid.
Estrogen Response Element: A binding site in a promoter to which the activated estrogen receptor can bind. The estrogen receptor is essentially a transcription factor which is activated only in the presence of estrogens.
Intercalates within the structure of nucleic acids in such a way that they fluoresce under UV light. Ethidium bromide staining is commonly used to visualize RNA or DNA in agarose gels placed on UV light boxes.
The gene-rich regions of a genome.
Cell or organism with membrane- bound, structurally discrete nucleus and other well- developed subcellular compartments. Eukaryotes include all organisms except viruses, bacteria, and blue- green algae.
Defined by the rate at which mutations accumulate within a given gene.
One can infer which portions of a gene are important by comparing the sequence of that gene with its cognates from other species. A plot showing the regions of high conservation will presumably reflect the regions that are functional in all the test species.
It removes a single stranded sequence of DNA containing damaged or mispaired bases and replace it in the duplex by synthesizing a sequence complementary to the remaining strand.
It is the process of secreting proteins from a cell into the medium.
Those portions of a genomic DNA sequence which will be represented in the final, mature mRNA. The term "exon" can also be used for the equivalent segments in the final RNA. Exons may include coding sequences, the 5' untranslated region or the 3' untranslated region.
This is a characterized RNA or DNA spiked into each sample at a known concentration. An exogenous active reference is usually an in vitro construct that can be used as an internal positive control (IPC) to distinguish true target negatives from PCR inhibition. An exogenous reference can also be used to normalize for differences in efficiency of sample extraction or complementary DNA (cDNA) synthesis by reverse transcriptase. Whether or not an active reference is used, it is important to use a passive reference dye (usually ROX) in order to normalize for non-PCR-related fluctuations in fluorescence signal.
DNA originating outside an organism.
Expressed Sequence Tag (EST):
A short piece of DNA sequence corresponding to a fragment of a complementary DNA (made from a cell's messenger RNA). ESTs have been used to hunt for genes, so hundreds of thousands are present in sequence databases. (See Sequence tagged sites)
An enzyme which digests nucleic acids starting at one end. An example is Exonuclease III, which digests only double-stranded DNA starting from the 3' end.
To "express" a gene is to cause it to function. A gene which encodes a protein will, when expressed, be transcribed and translated to produce that protein. A gene which encodes an RNA rather than a protein will produce that RNA when expressed.
This is a clone which is designed to produce a protein from the DNA insert.
A plasmid or phage designed for production of a polypeptide from inserted foreign DNA under specific controls. Often an inducer is used. The vector always provides a promoter and often the transcriptional start site, ribosomal binding sequence, and initiation codon. In some cases the product is a fusion protein.