DNA Manipulations and their Applications

A. Episomal DNA and Recombinant DNA Technology
Extrachromosomal or episomal DNA, present in prokaryotes and lower eukaryotes, is distinct from the genome of organelles such as mitochondria or chloroplasts and serves many purposes. In bacteria, plasmid DNA can be transmitted to progeny cells, and the genes in these plasmids encode distinct proteins which provide growth advantage or survival to the host bacteria. For example, many proteins which confer drug resistance by a variety of mechanisms are encoded by the plasmids, which are invariably present as double-stranded circular DNA containing several to hundreds of kilobase pairs.

The plasmid DNAs are self-replicating genomic units which are completely dependent on the host bacteria or yeast for their replication. These are also critical vehicles for recombinant DNA technology based on cutting and rejoining DNA fragments. Its invention, some three decades ago, revolutionized molecular biology and is at the root of nearly all modern breakthroughs in biology. Restriction endonucleases, which are enzymes characterized by stringent recognition of specific DNA sequences, cleave DNA duplexes and often leave identical terminal sequences in both plasmid DNA and a gene or segment of a genome. The fragments can then be joined by a DNA ligase. Joining heterologous fragments generates recombinant DNA, for example, a circular plasmid molecule containing foreign genes. These DNA molecules can then be introduced into living cells which allow their reproduction, so that a large amount of recombinant plasmid can then be generated.

Recombinant plasmids specific for bacteria, yeast, and even mammalian cells have been generated in the laboratory and exploited for a variety of basic and applied research applications. Specifically, recombinant expression plasmids can be constructed in order to express the ectopic protein encoded by the foreign (trans) gene in the appropriate host cell. Recombinant plasmids of mammalian cells are based on viruses, rather than on episomal DNA. Only the DNA replication function of the virus is incorporated into the plasmid, so that the plasmid is replicated without producing the active virus. In the case of human cells, simian virus 40 (SV40) is commonly used to generate recombinant DNA.

The circularity of the plasmid is essential for E. coli, but not mammalian or yeast cells. This may be consistent with the circular genome of the bacteria vs linear genomes of eukaryotes. However, plasmid vectors specific for mammalian cells must be propagated, preferably in E. coli. Such “shuttle” vectors are therefore required to have a circular configuration.

B. Polymerase Chain Reaction (PCR)
A critical advance in molecular biology came with the invention of PCR, based on a remarkably simple principle, and revolutionized many important aspects of biomedical research and medical jurisprudence. The method is based on the rationale that each strand of a piece of DNA sequence can be replicated repeatedly by using an oligonucleotide primer and a DNA polymerase (Fig. 7). After a duplex DNA molecule is generated, the next cycle is carried out by separating the two strands by heating and then starting the next cycle of synthesis after annealing oligonucleotide primers to each template strand. Thus, the repeated cycles of synthesis, denaturation, and primer annealing to both strands allow synthesis of a specific DNA sequence at an exponential rate. Thus, a tiny piece of a DNA molecule could be amplified about a millionfold after 20 cycles of this chain reaction (assuming 100% efficiency of the process; Fig. 7).

The PCR technology became viable after discovery of thermostable DNA polymerases derived from bacteria, such as Thermobacillus aqualyticus (Taq), which grow at high temperature. The cycles of PCR could then be automatically set in a thermal cycler. PCR does have some limitations. The most important of these are: (1) errors in DNA replication; (2) less than complete efficiency in each step of the reaction; and (3) improper primer annealing when complex DNA is used. Thus, when amplification of a segment of DNA in a complex genome is desired, the first requirement is the sequence information for the termini of the segment, based on which the oligonucleotides will be designed for each terminus and then synthesized. However, errors of replication cannot be completely eliminated. Any error in DNA synthesis that occurs early will be perpetuated. Furthermore, if replication is initiated by primers annealed to an incorrect DNA sequence, the wrong PCR product will be generated.

Primarily, because it has both sensitivity and specificity, PCR technology has revolutionized many aspects of biomedical research. Several modifications of the basic methodology have provided additional powerful tools. For example, a trace amount of RNA can be quantitated by reverse transcriptase PCR (RTPCR), where a reverse transcriptase synthesizes the complementary DNA strand of the RNA, which then serves as the template for regular PCR.

FIGURE 7 Principle of polymerase chain reaction (PCR). A copy of a relatively short fragment of DNA (0.1–20 kilobase pairs) can be specifically amplified from genomic DNA by PCR. A typical PCR reaction mixture contains genomic DNA; two oligonucleotide (∼20 bp) primers, which have same sequences as the two ends of the DNA fragment to be amplified; and a thermostable DNA polymerase. A cycle of PCR reaction consists of three steps, starting with denaturing the genomic DNA at high temperature (e.g., 95°C), followed by primer annealing at near Tm (melting temperature for primer-DNA hybridization), followed by DNA synthesis from the primers by the DNA polymerase. Theoretically, the copy number of the DNA of interest (N) can be amplified to 2C×NO, where NO is the original copy number and C is the number of PCR cycles.

DNA in a very small amount of biological samples can be amplified by PCR. This technique has been exploited in criminal investigations to identify suspects by “fingerprinting” their DNA, which involves determining a characteristic pattern of repeat sequences in the genome after PCR amplification of the total DNA. PCR has also been utilized in the identification of pathogens and other microorganisms, based on certain unique sequences of each organism. PCR has been exploited for a variety of in vitro manipulations of DNA sequences in plasmids, viruses, and synthetic DNA by generating site-specific mutations and a variety of recombinant DNA plasmids.