Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.
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.
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.
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, and ultimately affect a phenotype. These products are often proteins, but in non-protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the product is a functional non-coding RNA. The process of gene expression is used by all known life—eukaryotes, prokaryotes, and utilized by viruses—to generate the macromolecular machinery for life.
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins produce messenger RNA (mRNA). Other segments of DNA are transcribed into RNA molecules called non-coding RNAs (ncRNAs).
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.
This is a list of topics in molecular biology. See also index of biochemistry articles.
An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA gang replication, DNA repair or RNA processing and maturation, as well as pre-mRNA splicing. Although precursors can be found in earlier literature, most current assays are based on methods described by Garner and Revzin and Fried and Crothers.
DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells.
A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. In molecular biology, restriction maps are used as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA. There are other ways of mapping features on DNA for longer length DNA molecules, such as mapping by transduction.
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.
The L-arabinose operon, also called the ara or araBAD operon, is an operon required for the breakdown of the five-carbon sugar L-arabinose in Escherichia coli. The L-arabinose operon contains three structural genes: araB, araA, araD, which encode for three metabolic enzymes that are required for the metabolism of L-arabinose. AraB (ribulokinase), AraA, and AraD produced by these genes catalyse conversion of L-arabinose to an intermediate of the pentose phosphate pathway, D-xylulose-5-phosphate.
DNA adenine methyltransferase identification, often abbreviated DamID, is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip or ChIP-seq.
Promoter bashing is a technique used in molecular biology to identify how certain regions of a DNA strand, commonly promoters, affect the transcription of downstream genes. Under normal circumstances, proteins bind to the promoter and activate or repress transcription. In a promoter bashing assay, specific point mutations or deletions are made in specific regions of the promoter and the transcription of the gene is then measured. The contribution of a region of the promoter can be observed by the level of transcription. If a mutation or deletion changes the level of transcription, then it is known that that region of the promoter may be a binding site or other regulatory element.
Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.
Recombinant DNA (rDNA), or molecular cloning, is the process by which a single gene, or segment of DNA, is isolated and amplified. Recombinant DNA is also known as in vitro recombination. A cloning vector is a DNA molecule that carries foreign DNA into a host cell, where it replicates, producing many copies of itself along with the foreign DNA. There are many types of cloning vectors such as plasmids and phages. In order to carry out recombination between vector and the foreign DNA, it is necessary the vector and DNA to be cloned by digestion, ligase the foreign DNA into the vector with the enzyme DNA ligase. And DNA is inserted by introducing the DNA into bacteria cells by transformation.
The lacUV5 promoter is a mutated promoter from the Escherichia coli lac operon which is used in molecular biology to drive gene expression on a plasmid. lacUV5 is very similar to the classical lac promoter, containing just 2 base pair mutations in the -10 hexamer region, compared to the lac promoter. LacUV5 is among the most commonly used promoters in molecular biology because it requires no additional activators and it drives high levels of gene expression.
The G-less cassette transcription assay is a method used in molecular biology to determine promoter strength in vitro. The technique involves quantification of an mRNA product with the use of a plasmid. The G-less cassette is part of a pre-constructed vector, usually containing a multiple cloning site (MCS) upstream of the cassette. For this reason, promoters of interest can be inserted directly into the MCS to ultimately measure the accuracy and efficiency of a promoter in recruiting transcription machinery.
Long-range restriction mapping is an alternative genomic mapping technique to short-range, also called fine-scale mapping. Both forms utilize restriction enzymes in order to decipher the previously unknown order of DNA segments; the main difference between the two being the amount of DNA that comprises the final map. The unknown DNA is broken into many smaller fragments by these restriction enzymes at specific sites on the molecule, and then the fragments can later be analyzed by their individual sizes. A final long-range map can span hundreds to thousands of kilobytes of genetic data at many different loci.
