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.
Making DNA from RNA: Reversal of the Central Dogma
"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.
Discovering Reverse Transcription
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:
- The DNA polymerase only incorporated deoxyribonucleotides, not ribonucleotides, into its product.
- The product itself "behaved" like DNA--in other words, it was sensitive to treatment by deoxyribonucleases but not ribonucleases.
- The RNA itself was the template, as shown by the fact that treatment of virions with ribonucleases destroyed the ability of the polymerase to incorporate radioactively labeled nucleotides.
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.
How Reverse Transcriptase Works
So how exactly does reverse transcriptase work? As shown in Figure 1, the production of cDNA involves two basic steps: separating mRNA molecules from other cellular RNA, and then using reverse transcriptase to copy the mRNA molecules into cDNA.
Figure 1: The production of cDNA from mRNA.
cDNA is produced in two basic steps: (a) first, mRNA is isolated from other cellular RNA using an elution column. (b) Second, the enzyme reverse transcriptase synthesizes strands of DNA using the mRNA molecules as templates.
© 2014 Nature Education Adapted from Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed. All rights reserved. 
The first step relies on the fact that most eukaryotic mRNAs have poly(A) tails at their 3′ ends. The poly(A) tails serve as "hooks" during the separation process. This process involves pouring all of the cellular RNA (tRNA, rRNA, snRNA, mRNA, etc.) into what is known as an elution column of short DNA pieces, mostly thymine nucleotides. As the RNA mixture moves downward through the column, the poly(A) tails of the mRNA molecules bind to the thymine nucleotides. The rest of the RNA molecules--those without poly(A) tails, in other words--run right through. Afterward, the column is washed with a solution that breaks the hydrogen A-T bonds, and the released mRNA molecules are collected.
The second step, the copying of the now-isolated mRNA molecules into cDNA, involves adding oligo(dT) primers to the mRNA collection. These primers pair with the poly(A) tails of the mRNA molecules, again at the 3′ end, providing the exposed 3′-OH group required for the initiation of DNA synthesis. At this point, the reverse transcriptase enzyme is added, and this enzyme proceeds to utilize the mRNA strand as a template for the synthesis of a complementary DNA strand. The enzyme adds nucleotides, one by one, to the 3′ end of the new strand, with each newly added nucleotide a complement to its template pair just as in DNA replication, with the exception that RNA contains Us in place of Ts. Thus, Cs are paired with Gs and vice versa, Ts are paired with As, and As are paired with Us. Scientists then use several different methods to convert the RNA-DNA hybrids into double-stranded cDNA molecules, such as enzymatic digestion of the RNA strand followed by DNA synthesis utilizing, this time, the cDNA strand as the template.
Using Reverse Transcriptase to Clone Expressed Genes
Following the development of this method, the use of reverse transcriptase to clone expressed genes grew for several decades. However, there were limits to this practice. For example, most cDNA molecules that were synthesized in a single reaction were incomplete, with the 5′ end of the mRNA not represented in the final cDNA. Therefore, the cDNA did not contain a complete copy of the amino acid-encoding region of the gene.
Eventually, in the late 1990s, Piero Carninci and his colleagues at the Genome Science Laboratory in Ibaraki, Japan, devised a series of methods to get around this and other problems. In particular, these researchers developed a new technique for selecting full-length cDNA molecules. This process is known as biotin capping, and it involves capping the 5′ end of the mRNA with a biotin group and then washing the cDNAs with an RNA digestion enzyme, like RNAse I. When washed with a solution containing a cap-binding protein, all of the cDNA-mRNA hybrids with only partial cDNA copies are carried away, thus only leaving behind the hybrids with full-length cDNA molecules. In fact, the researchers demonstrated that biotin capping yielded about 95% full-length cDNA clones (Carninci et al., 1996).
Today, scientists continue to build and utilize what are known as cDNA libraries, or collections of cDNAs from particular tissues gathered at particular times during an organism's life cycle. The synthesis of cDNA molecules is referred to as cloning, because the cDNA molecules are matching copies of the DNA responsible for encoding the mRNA template.
Scientists often generate cDNA libraries as a way to find genes of interest. They screen these libraries using what are known as probes--complementary pieces of DNA that hybridize to the cDNA molecules. They also use cDNA libraries to identify genes that are expressed differently in different types of tissues or at different developmental stages. Libraries of cDNA molecules provide snapshots of gene activity, because only those genes that are actually expressed and transcribed into mRNA molecules can be cloned. For example, one would expect a cDNA library compiled from mRNA isolated during a stage of prenatal development to be very different from a cDNA library generated from sequences transcribed during adulthood.
