Complementary DNA and Complementary DNA Libraries

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18.8— Complementary DNA and Complementary DNA Libraries
The insertion of specific functional eukaryotic genes into vectors that can be expressed in a prokaryotic cell could produce large amounts of ''genetically engineered" proteins with significant medical, agricultural, and experimental potential. Hormones and enzymes are currently produced by these methods, including insulin, erythropoietin, thrombopoietin, interleukins, interferons, and tissue plasminogen activator. Unfortunately, it is impossible, except in rare instances, to clone functional genes from genomic DNA. One reason for this is that most genes within the mammalian genome yield transcripts that contain introns that must be spliced out of the primary mRNA transcript. Prokaryotic systems cannot splice out the introns to yield functional mRNA transcripts. This problem can be circumvented by synthesizing complementary DNA (cDNA) from functional eukaryotic mRNA.
mRNA Is Used As a Template for DNA Synthesis Using Reverse Transcriptase
Messenger RNA can be reverse transcribed to cDNA and the cDNA inserted into a vector for amplification, identification, and expression. Mammalian cells normally contain 10,000–30,000 different species of mRNA molecules at any time during the cell cycle. In some cases, however, a specific mRNA species may approach 90% of the total mRNA, such as mRNA for globin in reticulocytes. Many mRNAs are normally present at only a few (1–14) copies per cell. A cDNA library can be constructed from the total cellular mRNA but if only a few copies per cell of mRNA of interest are present, the cDNA may be very difficult to identify. Methods that enrich the population of mRNAs or their corresponding cDNAs permit reduction of the number of different cDNA species within a cDNA library and greatly enhance the probability of identifying the clone of interest.
Desired mRNA in a Sample Can Be Enriched by Separation Techniques
Messenger RNA can be separated by size by gel electrophoresis or centrifugation. Utilization of mRNA in a specific molecular size range will enrich several­fold an mRNA of interest. Knowledge of the molecular weight of the protein encoded by the gene of interest gives a clue to the approximate size of the mRNA transcript or its cDNA; variability in the predicted size, however, will arise from differences in the length of the untranslated regions of the mRNAs.
Enrichment of a specific mRNA molecule can also be accomplished by immunological procedures but requires the availability of antibodies against the protein encoded by the gene of interest. Antibodies added to an in vitro protein synthesis mixture will react with the growing polypeptide chain associated with the polysome and precipitate it. The mRNA can be purified from the immunoprecipitated polysomal fraction.
Complementary DNA Synthesis
Figure 18.14 Synthesis of cDNA from mRNA. The 3 poly(A) tail of mRNA is hybridized with an oligomer of dT [oligo(dT)12–18] that serves as a primer for reverse transcriptase, which catalyzes the synthesis of the complementary DNA (cDNA) strand in the presence of all four deoxynucleotide triphosphates (dNTPs). The resulting cDNA–mRNA hybrid is separated into single­stranded cDNA by melting with heat or hydrolyzing the mRNA with alkali. The 3 end of the cDNA molecule forms a hairpin loop that serves as a primer for the synthesis of the second DNA strand catalyzed by the Klenow fragment of E. coli DNA polymerase. The single­stranded unpaired DNA loop is hydrolyzed by S nuclease to 1
yield a double­stranded DNA molecule.
An isolated mRNA mixture is used as a template to synthesize a complementary strand of DNA using RNA­dependent DNA polymerase, reverse transcriptase (Figure 18.14). A primer is required for the reaction; advantage is taken of the poly(A) tail at the 3 terminus of eukaryotic mRNA. An oligo(dT) with 12–18 bases is employed as the primer that will hybridize with the poly(A) sequence. After cDNA synthesis, the hybrid is denatured or the mRNA hydrolyzed in alkali in order to obtain the single­stranded cDNA. The 3 termini of single­stranded cDNAs form a hairpin loop that serves as a primer for the synthesis of the second strand of the cDNA. Either the Klenow fragment or a reverse transcriptase can be used for this step. The resulting double­stranded cDNA contains a single­