Gene cloning and PCR allow scientists to make a large amount of DNA from only a small fragment. How do these technologies work?
The cloning of expressed genes and the polymerase chain reaction (PCR), two biotechnological breakthroughs of the 1970s and 1980s, continue to play significant roles in science today. Both technologies give researchers the means to make more DNA , but they do so in different ways. In particular, cloning involves the synthesis of DNA from mRNA using an enzyme called reverse transcriptase . Although this method reverses the flow of genetic information as described by the central dogma , it effectively mimics the process by which RNA viruses "flip" the direction of transcription in their host cells, thereby causing these cells to manufacture viral DNA even though the viruses themselves contain only RNA. In contrast, the polymerase chain reaction does not involve the use of an initial mRNA template to manufacture DNA. Rather, PCR involves the synthesis of multiple copies of specific DNA fragments using an enzyme known as DNA polymerase . This method allows for the creation of literally billions of DNA molecules within a matter of hours, making it much more efficient than the cloning of expressed genes. However, cloning remains the go-to method for researchers when only the mRNA template (and not the DNA template) of a sequence of interest is available.
"The central dogma, enunciated by Crick in 1958 and the keystone of molecular biology ever since, is likely to prove a considerable over-simplification. That is the heretical but inescapable conclusion stemming from experiments done in the past few months in two laboratories in the United States."
--"Central Dogma Reversed," Nature, June 27, 1970
The so-called central dogma of molecular biology states that all genetic information flows in one direction: from DNA to RNA through the process of transcription, and then from RNA to protein through the process of translation (Crick, 1958). For over a decade, the central dogma was thought to be a universal truth--in other words, researchers believed that genetic information always flowed in this order, otherwise it could not be passed along. In 1970, however, the two experiments mentioned in the Nature quote--one conducted by David Baltimore, then of the California Institute of Technology in Pasadena, and the other by Howard Temin and Satoshi Mizutani, then of the University of Wisconsin in Madison--called this belief into question. Specifically, these researchers independently published scientific papers demonstrating that RNA tumor viruses contain enzymes that use viral RNA as a template for the synthesis of DNA, thereby reversing the direction of transcription (Baltimore, 1970; Temin & Mizutani, 1970). Not only did these two experiments challenge the validity of the central dogma, but they also laid the foundation for a series of technological developments that eventually earned reverse transcription and the synthesis of complementary DNA, or cDNA , central places in the molecular biologist's toolbox.
During the late 1960s, Baltimore, Temin, and Mizutani were each driven by unanswered questions about how RNA viruses transformed healthy cells into tumor cells. They knew that transformation ensued when healthy cells incorporated DNA from the external environment (in this case, RNA tumor virus DNA) into their genomes. But how could a eukaryotic cell incorporate DNA from a virus that didn't have any DNA?
Howard Temin had hypothesized the existence of an enzyme capable of making DNA from RNA as early as 1964 ("Central Dogma Reversed," 1970). But, as is the case with all scientific hypotheses, the research community remained skeptical of this proposal until the 1970 publications wiped that skepticism away. At that point, the race was on to identify the enzyme responsible for the creation of DNA from RNA. Today, that enzyme is known as reverse transcriptase.
Interestingly, in their groundbreaking papers, the two sets of scientists didn't actually identify reverse transcriptase, but they did provide clear and conclusive evidence of the existence of an enzyme that utilized viral RNA as a template for DNA synthesis. The experiments supporting the existence of this DNA polymerase produced data that revealed the following:
Although the motivation for both studies was to better understand the role of viruses in some cancers, there is also some suggestion in the papers that the scientists were aware, at least on an intuitive level, that there were far greater implications to their findings. As Temin and Mizutani (1970) wrote, "This result would have strong implications for theories of viral carcinogenesis and, possibly, for theories of information transfer in other biological systems."
It did not take long for scientists to isolate the reverse transcriptase responsible for Baltimore's findings (Verma et al., 1972). Another team (Bank et al., 1972) then used the enzyme to synthesize DNA from mRNA in a test tube for the first time. (The so-called complementary DNA that results is referred to as cDNA.) Both teams used globin mRNAs, or mRNAs that encode blood hemoglobin polypeptides, to demonstrate that reverse transcriptase does in fact synthesize DNA from mRNA templates. Moreover, the teams also found that the reaction works best in the abundance of short sequences composed entirely of thymine nucleotides known as oligo(dT) primers. Knowing that most eukaryotic mRNAs have a string of adenine nucleotides--also known as a poly(A) tail--at their 3′ end, the scientists had predicted that cDNA synthesis would require oligo(dT) primers, or that it would at least be made more efficient by the presence of these primers.
