The polymerase chain reaction, or PCR, has become one of the most widely used techniques in all of science. Millions and millions of copies of a specific DNA fragment can be produced enzymatically in a matter of hours. This technique can also be used to analyze messenger RNA (mRNA) molecules, but first a complementary DNA (cDNA) copy of the mRNA needs to be made in a process known as reverse transcription (RT) polymerase chain reaction or RT-PCR.
The Basics of PCR
Every PCR reaction needs a specific mixture of components including:
- A template or target DNA that is to be copied
- Short oligonucleotide primers engineered to be complementary to specific regions of the “sense” and “antisense” strands of the target
- Buffer mixture containing the necessary precursors required for DNA synthesis (deoxynucleotide triphosphates or dNTPS)
- A thermostable DNA polymerase (capable of still being enzymatically active after repeated exposures to temperatures near 100 degrees Celsius)
Denature, Anneal, Extend, Repeat
Because of the development of electronically controlled heating blocks capable of rapid temperature changes with accuracies of better than 0.5 degree Celsius, the necessary reaction steps for PCR can be readily accomplished.
- First, heating to high temperatures (95 degrees Celsius) to “melt out” or denature all hybridized DNA strands
- Second, lowering of the temperature to allow primer oligonucleotides to anneal to their target sequences
- Third, increasing the temperature to the optimum (typically around 72 degrees Celsius) so that the thermostable DNA polymerase can synthesize the targeted DNA strands
- Repeat these three steps over and over again
Making DNA copies of messenger RNA
The ability to use PCR to characterize mRNA molecules depends upon making a complementary DNA (cDNA) copy of the mRNA molecule. This is done using an enzyme known as reverse transcriptase or RT. These enzymes were first isolated from cells infected with viruses from the class known as “retroviruses”, which normally carry their genetic instructions as RNA molecules and when they infect cells they direct the production of an enzyme that can make a DNA copy from RNA molecules. Used in the laboratory, these enzymes can be used to make complementary DNA copies of any RNA molecule.
For most mRNA molecules in eukaryotic cells the process is also helped by the presence of a “tail” of adenine nucleotides that are almost always added to the end of mRNAs when they are synthesized, what is known as a “poly(A) tail”. Thus, a synthetic oligonucleotide known as “oligo(dT)” can be used as the primer for reverse transcriptase to start making the DNA copy of the mRNA. This is referred to as “oligo(dT)-primed cDNA” synthesis.
Once cDNA has been synthesized from mRNA, it can serve as the template for any PCR reaction and thus is now known as RT-PCR. The different types of PCR reactions that can be performed are numerous but they are generally doing the exact same thing, making more DNA copies of the original cDNA. Some of the more usual examples are:
- Targeted cloning: where a copy of an mRNA molecule is synthesized to have sites for specific restriction enzymes on its ends to allow for more rapid “directed” cloning into an expression plasmid.
- Site-directed mutagenesis: where a copy of an mRNA is made to contain a particular type of mutation in its nucleotide sequence so that the effects of this mutation can later be tested.
- Quantitative Real-Time PCR (Q-PCR): the levels of a particular mRNA in a sample can be rapidly measured using fluorescent nucleotides or fluorescent nucleic acid dyes to detect the synthesis of a specific cDNA copy of an mRNA. Q-PCR is used everyday in tens of thousands of labs around the world and has, for the most part, become the method of choice to determine how active a particular gene is by measuring how much mRNA has been produced from it.
Read a wonderful description of the development of PCR and its applications in the web article by Peter Gwynne and Gary Heebner at Science magazine.
Kenneth Rosen - I am a medical research doctor with more than 20 years of experience studying how the nervous system and skeletal muscle develop and ...
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