Sequence untranslated (French : séquence non traduite) A region of mRNA that is not used in the synthesis of an amino acids sequence of a given peptide or protein. They usually appear at either end of the sequence that codes for the amino acid sequence.
Southern blotting (French : marquage Southern) Transfer by absorption of DNA fragments, separated in electrophoretic gels, to membrane filters for the detection of specific base sequences by radio labelled complementary probes.
Ribonucleic acid, RNA (French : acide ribonucléique, ARN) A chemical found in the nucleus and cytoplasm of cells; it plays an important role in protein synthesis and other chemical activities of the cell. The structure of RNA is similar to that of DNA. There are several classes of RNA molecules, including messenger RNA, transfer RNA, ribosomal RNA, and other small RNAs, each serving a different purpose.
Repressor (French : répresseur) Protein synthesised by a regulator gene, which, by binding to a specific site on DNA, the operator gene of an operon, prevents the formation of messenger RNA by the operon's other structural genes and hence stops protein -enzyme- synthesis.
Northern blot, RNA transfer (French : Northern blot, transfert d'ARN) A gel base procedure that locates mRNA sequences on a gel that are complementary to a piece of DNA used as a probe.
Ribosomes (French : ribosomes) Small cellular components composed of specialized ribosomal RNA and protein; site of protein synthesis. See .
(French : code génétique) The sequence of nucleotides, coded in triplets (codons) along the mRNA, that determines the sequence of amino acids in protein synthesis. The DNA sequence of a gene can be used to predict the mRNA sequence, and the genetic code can in turn be used to predict the amino acid sequence.
The level of sensitivity of theselabeled probes is purported to be 0.1 pg on a Southern blot but would be about 10 foldless sensitive for in situ hybridization.
One of the advantages of the DIG-System is the stability of the labeled probe. After hybridization against the blotted target, the hybridization solution still contains large amounts of unannealed DIG-labeled probe. Simply pour the solution into a plastic tube and store at –20° C for DNA probes and –70°C for RNA probes. DIG labeled probes are stable for at least 1 year when stored in this manner. For reuse, thaw and denature by heating to +95°C for 10 min. If the hybridization solution contains formamide or .
In addition, because they are synthetically designed, it ispossible to make a series of probes that have the same GC content; Since G/C base pairsbond more strongly than A/U base pairs, differences in GC content would require differenthybridization conditions, so with oligonucleotides protocols can be standardizedfor many different probes irrespective of the target genes being measured.
The DIG System is an effective system for the labeling and detection of DNA, RNA, and oligonucleotides. The protocols for labeling with digoxigenin (Figure 1) and subsequent detection are based on well established, widely used methods. DNA, RNA, and oligonucleotide probes are labeled according to the methods (usually enzymatic) used for preparing radioactive probes. Hybridization of digoxigenin-labeled probes (e.g., to target DNA or RNA on a Southern or Northern blot) is also carried out according to standard protocols, except that a special blocking reagent is used to eliminate background. The signal on the nucleic acid blot is detected according to the methods developed for western blots. The incorporation and spacing of digoxigenin in DNA, RNA, and oligonucleotides can be varied by using different labeling protocols.
In the biotin system biotin is incorporated in the probe by using biotinylated dNTPs during probe synthesis. The incorporated biotin is detected directly by avidin or streptavidin or an anti-biotin antibody conjugated to a fluorochrome or an enzyme such as alkaline phosphatase or horseradish peroxidase. Avidin is a 68 kD glycoprotein derived from egg white and streptavidin is a 60 kD protein from .
Interferon (French : interferon) A protein that is synthesized by animal cells in response to viral infection and non specifically inhibits replication of the viruses. It is found in serum almost at the onset of the infection and long before the production of antibody.
Nonisotopically labeled probes have become increasingly popular in recent years and replace radioactive probes in techniques such as in situ hybridization, Southern, Northern and Western blot analysis. The advantages of these techniques are: