Molecular Biotechnology: Principles and Applications - Glick, B. R., & Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Recombinant DNA Technology
Cloning of Eukaryotic Structural Genes

Special techniques are required for the cloning of eukaryotic structural genes. Prokaryotes are unable to remove introns from primary RNA transcripts; therefore, the correct Translation of eukaryotic mRNAs in a bacterial Cell is impossible. Furthermore, the Expression of Eukaryotic DNA can only occur in the presence of prokaryotic signal sequences that regulate METABOLISM/31.html">Transcription and translation. The terminal regions of eukaryotic mRNAs are modified in a specific manner: their 5' ends are capped (containing a «cap» consisting of a G residue, often methylated), and their 3' ends are polyadenylated (containing a poly(A) «tail» of approximately 200 adenosine residues).

The presence of the poly(A) tail makes it possible to separate mRNA from ribosomal and Transfer RNA. To achieve this, total eukaryotic RNA is passed through a Column packed with Cellulose, to which short oligonucleotide chains of thymidine residues approximately 15 units in length, oligo(dT), are attached. The poly(A) tails of the mRNA molecules pair with the oligo(dT) and are retained in the column, whereas tRNA and rRNA molecules pass freely through it. The column is then washed with a buffer that disrupts the Hydrogen Bonds between A and T, releasing the mRNA.

mRNA itself cannot be inserted directly into a DNA vector; double-stranded DNA must first be synthesized using the mRNA as a template. To accomplish this, two different polymerases are used sequentially: Reverse Transcriptase and the Klenow fragment of DNA polymerase I (Fig. 4.15). First, short oligo(dT), reverse transcriptase, and four dNTPs (dATP, dTTP, dGTP, dCTP) are added to the reaction mixture containing purified mRNA. The poly(A) tail of the mRNA pairs with the oligo(dT), which bears a free 3'-OH group that initiates the Synthesis of the complementary strand. The mRNA molecule serves as the template for this synthesis, which is catalyzed by reverse transcriptase, an enzyme produced by certain RNA Viruses. It sequentially adds T, C, G, or A residues to the growing chain, complementary to the A, G, C, or U of the mRNA. In vitro, DNA Synthesis does not run to completion; instead, before stopping, reverse transcriptase typically «turns back» and adds a few NUCLEOTIDES in the reverse direction (Fig. 4.15), resulting in The formation of a «hairpin» Structure.

A Klenow fragment of DNA polymerase I from E. coli is added to the reaction mixture to synthesize the second DNA strand using the first strand as a template. It incorporates deoxynucleotides into the growing chain, starting from the 3'-OH end of the hairpin. Following synthesis, the preparation is treated with RNase H, which degrades mRNA molecules, and S1 nuclease, which cleaves off single-stranded DNA ends. The resulting product is a mixture of partially and fully double-stranded complementary DNA (cDNA) copies of the mRNA predominantly found in the initial sample. These various cDNAs can be inserted into a plasmid vector to generate a cDNA library. To screen the cDNA library and identify clones carrying specific hybrid Plasmids, Hybridization or immunological Methods can be employed. In the latter case, the cDNA must be inserted into a site controlled by a bacterial promoter that drives transcription. However, virtually no vector guarantees that the inserted cDNA will maintain the correct reading frame and synthesize the proper polypeptide chain. Nevertheless, all positive clones identified by either method must undergo further validation to isolate those carrying the full-length nucleotide sequence that encodes the target protein.

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Fig. 4.15. cDNA synthesis. An oligo(dT) primer is added to the purified mRNA preparation. Reverse transcriptase and four dNTPs are used to synthesize DNA on the RNA template. In vitro, reverse transcriptase fails to synthesize full-length cDNA copies on all templates, forming a hairpin structure with a free 3'-OH group at the end of the growing chain. This group initiates the synthesis of the second DNA strand mediated by the Klenow fragment. Upon completion of synthesis, the mRNA molecules are hydrolyzed with RNase H, and the DNA is treated with S1 nuclease, yielding linear, blunt-ended DNA molecules lacking hairpins.