The procedure of cloning of fragmented cDNA based on PCR method

The procedure of cloning of fragmented cDNA based on PCR method

mRNA separated from tissues A and B is used for the synthesis of double-chain cDNA and the received products are affected by restriction endonucleases, which have identification sequences of 4 bp. Later two different collections of adapters (a1/a2 and b1/b2) are attached to two different cDNA samples, receiving A0 and B0. After hybridisation reaction the following collections are received: A0 - B0 and B0 - A0. In any of the cases, during PCR synthesis, the finer is marked with a small amount of [α32P]dCTP and the guide is marked with bio-11-dUTP. The finder and the guide are mixed at the ratio 1:20 and denaturated. Guide/ guide and finder/ guide hybrids are eliminated by affecting them with sreptavidin and extracting with phenol. The next cutting of the respectful cDNA is performed in the next amplification cycle. When the cutting is finished, cDNA is cloned into the respectful vectors for further analyses.

AFLP based on transcription grading


Figure 4. Principles of performing the grading of transcription based on polymorphism of amplified fragment length. Above marked poli(A)+RNA is with poli(A) tail at 3’ end. cDNA is marked by a double line. ds TaqI and MseI adapters are shown respectfully as grey and black boxes, attached to the cut end of the fragment. Bellow under “X” poorly amplified MseI – MseI fragments are shown.