Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs.
In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RNA or DNA that are complementary to the sequence in the probe. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ.
In molecular cloning, a vector is any particle (e.g., plasmids, cosmids, Lambda phages) used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
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. The resulting digested DNA is very often selectively amplified using polymerase chain reaction (PCR), making it more suitable for analytical techniques such as agarose gel electrophoresis, and chromatography.
Les souris knock-out sont des souris domestiques (Mus musculus) qui ont été génétiquement modifiées pour inactiver un ou plusieurs gènes dans les cellules souches embryonnaires dont elles sont issues. Ces cellules souches étant pluripotentes, elles sont donc toutes initialement identiques mais une fois réimplantées dans un blastocyste, peuvent former l'ensemble des types cellulaires d'une souris qui porteront alors la mutation introduite. Il faut distinguer le knock-out d'une simple recherche de mutants.
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.
Un transgène est la séquence isolée d'un gène, transférée d'un organisme à un autre, lors de la mise en œuvre de la transgenèse. Cette modification peut altérer le comportement génétique de l'organisme (p. ex. production d'une nouvelle protéine). En agronomie, on parle de gène d’intérêt permettant de faire exprimer à un organisme un caractère choisi ; par exemple, on peut chercher à rendre une plante résistante à un antibiotique ou à augmenter sa teneur en vitamines.
La mutagénèse dirigée est l'induction d'une ou plusieurs mutations dans un génome, de façon précise et volontaire. Cette méthode est employée pour modifier les structures de l'ADN, l'ARN et des protéines. Cette technique de biologie moléculaire a été mise au point par Michael Smith en 1978. Apparemment, l’idée de la mutagénèse dirigée lui serait venue au cours d’une conversation avec Clyde Hutchison en 1976 à l’institut de Cambridge en Angleterre, alors qu’il travaillait sur la préparation d’oligonucléotides pour purifier des fragments d’ADN.
The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal.
In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries (formed from reverse-transcribed RNA), genomic libraries (formed from genomic DNA) and randomized mutant libraries (formed by de novo gene synthesis where alternative nucleotides or codons are incorporated).
