Gene structure and any of the processes involved in DNA expression including transcription, mRNA processing and translation and protein synthesis can all be examined by molecular techniques. In toxicology this may include toxic effectson these processes or the role of the processes in the mechanism of toxic action.
Molecular cloning: The basic principle of molecular cloning is the insertion of a DNA segment into a suitable vector. The vector is an autonomously replicating DNA molecule and the inserted DNA segment may be as large as a gene or a small as a few nucleotides.The vector containing the DNA is inserted into a cell such as yeast, where it can be replicated many times, and either the DNA or the expressed protein subsequently isolated.
cDNA & Genomic Libraries:DNA library is a collection of cloned DNA fragments. There are two types of DNA library:The genomic library contains DNA fragments representing the entire genome of an organism. The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. These libraries are used in many screening procedures and many transgenic proteins now routinely available were obtained by their use.
Northern Blotting and southern Blot Analyses: The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence. The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane, however the entire process is commonly referred to as northern blotting.
Southern blot is a method to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization .
Polymerase Chain Reaction (PCR) PCR is a powerful technique that can, starting with amounts of DNA as small as those found in single cells, amplify the DNA until large amounts are available for many different kinds of research. Twenty to 40 cycles of can provide up to 105 times the original DNA sample. The PCR technique has been used for many types of toxicological investigation including;uncovering polymorphisms in xenobiotic-metabolizing enzymes, isolating genes from cDNA and genomic libraries and for mutational analysis, to name only a few.
