Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Molecular Genetics and Regulatory Systems
Recombinant DNA Technology
Identification of Cloned DNA

Suppose that the recombinant Plasmids have been successfully introduced into an E. coli Cell population, and that the plasmid-bearing Cells have been identified on Agar plates containing a selective medium. In this section, we will briefly review the Methods for screening the clones obtained in this way and for conducting a detailed Analysis of the DNA insertions within the clones of interest.

First of all, we need to distinguish clones containing plasmids with foreign DNA insertions from those that harbor only the original vectors; this can be achieved through insertional inactivation during cloning. For example, suppose that DNA fragments are inserted into the PstI restriction site of the plasmid pBR322. Because an insertion at this site disrupts the ampicillin resistance Gene, cells containing recombinant plasmids will become sensitive to ampicillin while remaining resistant to tetracycline. Conversely, cells with non-recombinant plasmids will be resistant to both ampicillin and tetracycline.

In this case, clones carrying recombinant plasmids can be identified as follows. First, the cells are grown on plates in the presence of tetracycline, which effectively eliminates the significant Background of plasmid-free cells. Next, the replica plating technique is employed: a velvet pad or a nitrocellulose filter paper is pressed first against the master plate and then onto a second plate (the replica), thereby transferring an exact replica of the colony distribution from the master plate. When the replica is treated with ampicillin, cells containing plasmids with insertions in the ampicillin resistance gene will grow on the master plate but fail to grow on the replica.

The search for specific nucleotide sequences within a clone can be accomplished using a variety of techniques based on the Hybridization of denatured (i.e., single-stranded) clone DNA with a specialized nucleotide probe, usually labeled with the radioactive isotope ³²P. The nucleotide sequence of the probe (which may be either DNA or mRNA) is complementary to a portion of the target DNA segment. mRNA probes are obtained by isolating mRNA from cells enriched with the Genetic information of interest, whereas DNA probes are prepared during the Cytology/cytology/16.html">Early stages of cloning or through direct chemical synthesis.

In the colony in situ hybridization method, a replica of the clones is grown on nitrocellulose filter paper placed over a Petri dish containing nutrient medium. The cells are then lysed with Lysozyme, and the DNA is denatured by Treatment with NaOH. Afterward, the nitrocellulose paper is removed, treated with the probe, washed, and exposed to X-ray film. Dark spots appear on the film at the locations where the probe has hybridized with complementary DNA sequences; these clones are of primary interest for further study.

Plasmid DNA, which can be isolated via cesium chloride density gradient ultracentrifugation, is subsequently digested with a restriction endonuclease. The resulting DNA fragments generate a characteristic fingerprint for the given plasmid DNA. These fragments can be separated according to their molecular weights by agarose gel Electrophoresis (0.5–1.5%); ethidium bromide, a fluorescent dye specific for double-stranded Nucleic Acids, is frequently used to visualize the DNA fragments. By comparative analysis of fragment lengths generated by other restriction Enzymes and through double digests with multiple Restriction Endonucleases, a restriction map is constructed, illustrating the relative positions of various restriction sites. Such maps are extremely useful for verifying the correct incorporation of DNA into specific Regions of the plasmid and serve as a foundation for subsequent work with recombinant plasmids.

The radioactively labeled probes mentioned earlier can also be used to identify specific fragments generated by restriction enzyme Digestion. In the Southern blotting technique, DNA fragments within a gel are denatured with alkali, and a nitrocellulose membrane is placed on top of the gel. Filter paper is positioned above the membrane to draw up the buffer solution via capillary action, simultaneously transferring the DNA fragments onto the nitrocellulose membrane. The DNA is then covalently bound to the nitrocellulose at 80°C. Subsequently, the bound denatured DNA fragments are treated with the probe as described above, exposed to X-ray film, and used to identify the bands on the electrophoregram that correspond to The nucleotide sequences complementary to the probe.

Modern experimental methods in DNA biochemistry are so sensitive that a mere few nanograms of DNA isolated from a gel electrophoregram band are sufficient for cloning or further analysis. Obviously, the definitive identification of a DNA fragment must include the determination of its nucleotide sequence. During the 1970s, two rapid, reliable, and efficient Methods for determining nucleotide base sequences in DNA were developed: the Maxam–Gilbert and Sanger methods. These techniques have had a profound impact on the biological sciences in general and on biotechnology in particular.

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FIG. 6.24. Determination of nucleotide sequence by the Maxam–Gilbert method. Upon treating the sequence under study with four specific Reagents that selectively cleave different bases, a mixture of fragments is generated whose molecular weights depend on THE POSITION OF the cleaved base. The arrangement of bands across the four lanes of the gel electrophoregram allows the nucleotide sequence to be read directly from the gel; reading from top to bottom corresponds to the 5'-to-3' direction toward the labeled end of the original oligonucleotide.

In the Maxam–Gilbert method, the DNA double helix is first labeled by attaching a radioactive marker to one end of each strand. The DNA is then denatured, and the preparation of one of the two strands is divided into four aliquots, each treated with a specific reagent. Each of these treatments selectively destroys one (or two) bases, causing the DNA chains to break at those sites. For the success of this method, it is essential that this destruction be partial; ideally, each strand should be cleaved at only one site. The fragments resulting from these four parallel Cleavage reactions are then analyzed on a high-resolution polyacrylamide gel. The bands in the gel are visualized using an X-ray-sensitive photographic film. The target nucleotide sequence is read directly from the four parallel lanes of the gel electrophoregram (Fig. 6.24). Using this method, The sequence of approximately 200 nucleotide residues can be readily determined in a single experiment. The Sanger dideoxy-sequencing method is discussed in reference [21]; it is typically applied when studying larger DNA fragments.

It is frequently necessary to screen A large number of distinct clones to identify a single clone (or a few clones) capable of expressing a specific protein. In most cases, this is accomplished using an antibody specific to the protein of interest. The Antibodies are radiolabeled (e.g., with ¹²⁵I) and used to treat the colonies on the plate; the plate is then washed, and X-ray film is employed to detect the target colonies containing the protein. Alternatively, antibodies can be linked to an enzyme, allowing Antigens to be detected via the enzymatic activity. Colonies containing the target protein can be identified, for instance, by using a chromogenic substrate (i.e., a substrate that changes color or becomes colored upon enzymatic conversion). More sensitive immunochemical methods capable of detecting Proteins at concentrations of 1–5 molecules per E. coli cell are described in the literature cited at the end of the chapter.

The scope and focus of this book do not allow for a detailed Structure/133.html">Discussion of all methods used for clone identification and analysis; nevertheless, it is hoped that the information presented here provides a clear sense of how laborious, time-consuming, and tedious clone screening and the verification of each step in the construction of novel recombinant molecules can be. Although readers specialized in biochemical engineering will likely find the topic of the next section—dedicated to the Expression of cloned genes—more compelling, one must recognize that Gene cloning itself is likely to be the primary bottleneck in any program aimed at synthesizing a foreign protein in E. coli or another host cell. At the same time, methodological advancements and the ongoing automation of these processes will facilitate the Implementation of "standardized Procedures" in Molecular Genetics, which in turn will lay the groundwork for a substantial expansion of the repertoire of available and interesting genes and regulatory sequences.