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Differential Gene Expression: Mechanisms of Cell Differentiation
The polymerase chain reaction (PCR) is a method of in vitro gene cloning[i] that can generate enormous quantities of a specific DNA fragment from a small amount of starting material (Saiki et al. 1985). It can be used to clone a specific gene or to determine whether a specific gene is actively transcribing mRNA in a particular organ or cell type. Standard methods of gene cloning use living microorganisms to amplify recombinant DNA. PCR, however, can amplify a single segment of a DNA molecule several million times in a few hours, and can do it in a test tube. The techniques of PCR are extremely useful in cases where there is very little sample to study.
For instance, preimplantation mouse embryos have very tiny amounts of mRNA, and we cannot obtain millions of such embryos to study. If we wanted to know whether a single preimplantation mouse embryo contained the mRNA for a particular protein, it would be very difficult to find out using standard methods—we would have to lyse thousands of mouse embryos in order to obtain enough mRNA. However, by combining PCR techniques with the ability of the reverse transcriptase (RT) enzyme to make DNA out of mRNA, we can get around the problem of scarce messages. The RT-PCR technique allows us to convert the mRNA into DNA and to copy the specific DNA sequence of interest (Rappolee et al. 1988).
The use of RT-PCR to find rare mRNAs is illustrated in Figure 1. First, the mRNAs from a sample are purified and converted into complementary DNA (cDNA) using the RT enzyme. Next, a specific cDNA is targeted for amplification. Two small oligonucleotide primers that are complementary to a portion of the message being looked for are added to the population of cDNA. Oligonucleotides are relatively short stretches of DNA (about 20 bases). If the oligonucleotides bind to sequences in the cDNA, this means that the mRNA being sought was present in the original sample. The oligonucleotide primers are made so that they hybridize to opposite strands at opposite ends of the targeted sequence. (If we are trying to isolate the gene or mRNA for a specific protein of known sequence, we can synthesize oligonucleotides that are complementary to the sequences encoding the amino end and the carboxyl end of the protein.) The 3′ ends of the primers face each other, so that replication will run through the target DNA.
These strands are then denatured, and the primers are hybridized to them, starting the cycle again. In this way, the number of new strands having the sequence of interest between the two primers increases exponentially.
Once the first primer has hybridized with the cDNA, DNA polymerase can be used to synthesize a new strand. The DNA polymerase used in this process is extracted from thermophilic (heat-loving) bacteria such as Thermus aquaticus or Thermococcus littoralis[ii] and can withstand temperatures near boiling. Once the second strand of DNA is made, it is heat-denatured from its complement. The temperatures used would inactivate the more usual E. coli DNA polymerase, but the thermostable polymerases are not damaged.
The second primer is added, and now both strands can synthesize new DNA. Repeated cycles of denaturation and synthesis amplify the DNA sequence exponentially. After 20 such rounds, that specific sequence has been amplified 220 (a little more than a million) times. When the DNA is subjected to electrophoresis, the presence of such an amplified fragment is easily detected. Its presence shows that there was an mRNA with the sequence of interest present in the original sample.
[i] Gene cloning—making numerous copies of the same DNA sequence—should not be confused with organism cloning—making genetically identical copies of an organism.
[ii] These bacteria normally live in hot springs (such as those in Yellowstone National Park) or in submarine thermal vents, where the temperature reaches nearly 90°C. RT-PCR takes advantage of this evolutionary adaptation.