Procedure of creation of RMDD library

Procedure of creation of RMDD library

Restriction mediated differential display (RMDD) library is composed of the whole cDNA molecule, received from the cut fragments of respectful biology samples. It has been calculated that one cell approximately has 10 000 different mRNA molecules, i.e. the same cell has also 10 000 different types of cDNA. Seeking equal distribution within the gel and easy evaluation of the fragments, which were composed from 3′ end of cDNA molecules, this mixture of fragments must be subdivided into separate small parts, which would contain a small amount (for example, 50 – 100) of different fragment types.

mRNA synthesis is started from the synthetic oligonucleotide primer (oligo dT), using one-chained mRNA molecule as a matrix (extinguished from certain cells) and inverted transcriptasis, double-chained DNA molecule is synthesised. This synthesized DNA is called complementary DNA (cDNA), because it is synthesized on the bases of complementary to RNA matrix principle. cDNA molecule has no introns, because it has been synthesised from mature mRNA molecule. The received chain is cut and switchers are attached to the cut ends. The fragments amplified during PCR are marked with radioactive or non-radioactive markers and are further used for blotting.