Did You Ever Want to Know… What is PCR? Ever heard of TMA?
Polymerase Chain Reaction (PCR)
PCR is the acronym with the greatest fame. The process became famous as a DNA study tool because one can amplify (aka replicate or copy) DNA millions of times. If a technician uses PCR to replicate DNA millions of times, that DNA in that technician’s hands is a magnitude that could be seen with the naked eye when dyed. Add something called restriction enzymes and put the DNA in a gel electrophoresis… you get that funny little dot pattern that they use for paternity tests and CSI work.
All organisms use nucleic acids as the “blueprint” for their genetics. When a cell is dividing and the nucleic acids in that cell are replicating, you start with the double-stranded nucleic acids that must separate during the replication. Now you have two strands of single-stranded nucleic acids, in an overly-simplified explanation. Unlinked nucleic acids that compliment the original strands will then anneal – or bind – to the original strands. Polymerase is the biochemical that zips all the unlinked nucleic acids together to make another strand and what you end up with is two double-stranded nucleic acid chains.
Another common nucleic acid is RNA, but it’s not our genetic material. Ours is DNA. The only differences are: DNA is missing an oxygen on the saccharide backbone (geek talk), and DNA uses Thymine where RNA uses Uracil. Turns out, that’s a big difference! DNA is more stable and is therefore the genetic material used in most organisms.
So what’s the big deal with polymerase in regards to PCR? Polymerase is used for nucleic acid replication. If you want to amplify human DNA, you have to break apart the two strands so unlinked nucleic acids can fit in there and you need polymerase to link them all together. The problem is that to break apart the two strands in a test tube, you need to heat the sample up to a temperature that destroys polymerase. Shit! Turns out, some genius was studying micro-organisms in the hot pots of Yellowstone and found that those organisms use a form of polymerase that is functional at high temperatures. That guy got a Nobel Prize and rightfully so.
How does it work? You inject a sample of DNA into your test tube and add unlinked nucleic acids and high-temperature polymerase to the sample. In a process of repeated heating and cooling one can copy the nucleic acid strands. Each heating and cooling cycle has the ability to double the strands of nucleic acids.
PCR has been around for a couple decades now and has been well-studied. Many other amplification techniques rely on PCR as the trusted confirmatory technique. Therefore when I say TMA is better, PCR has the reputation that counts. So now you know PCR. Ever heard of TMA?
PCR in a Nutshell
Transcriptase-Mediated Amplification (TMA)
What is it? Most people have never heard of Transcriptase-Mediated Amplification. Turns out most scientists haven’t either. TMA is another process of nucleic acid amplification. I’m a fan not just because I’ve used it on countless occasions, but because I believe the throughput is far faster and insanely brilliant.
I must introduce a concept to you that I eluded to previously: some organisms use RNA as their genetic material. HIV is one of them (technically viruses aren’t organisms as they don’t have a metabolism.). Transcriptase is a protein that converts a DNA code into an RNA code while performing the linking functions similar to polymerase. Turns out, HIV uses a protein called reverse transcriptase to convert its RNA code into a DNA code. Isolate the protein away from the virus and you’ve got yourself a useful tool there.
How does it all fit together? Pretend you’re holding a sample of DNA and you want it amplified. Add a mixture of unlinked nucleic acids of both the RNA and DNA types. Then add both transcriptase and reverse transcriptase. What happens is that the transcriptase will make hundreds of copies of the DNA while converting the code to RNA. Reverse transcriptase will make hundreds of copies of the RNA while converting the code back to DNA. The transcriptase will then make tens of thousands of copies of the DNA while converting the code to RNA. Then the reverse transcriptase makes tens of thousands of copies right back to DNA. Now this all sounds like a cycle of back and forth, when in actuality it’s a continuous and smooth reaction pathway. You can’t pinpoint an exact number of copies made because the amplification is not a fixed increase. One just knows that the amplification is lots of copies. In just an hour, you will literally run out of unlinked nucleic acids to feed the reaction. It is estimated that you will have about a billion times the starting count in just an hour of amplification.
A billion, you say, Science Guru?
That’s right. A billion. It would take almost thirty cycles of PCR heating and cooling to reach the same level of amplification.