All About PCR - Beta

Each time a cell divides, it must first copy its DNA—a process called DNA replication. PCR piggybacks on this natural process.

In a cell, many proteins work together to replicate DNA. One key player is an enzyme called DNA polymerase—the same enzyme that's used in PCR.

DNA polymerase can’t start building from scratch. It can only attach new nucleotides to an existing string of nucleotides. A cell and PCR have different ways of getting started. In a cell, an enzyme called primase builds a primer out of RNA. DNA polymerase then extends the primer, adding complementary nucleotides as it goes. In PCR, human-engineered primers steer DNA polymerase to the desired target sequence.

The innovation with PCR is in having two primers flanking the target sequence.

If you were to set up a reaction with one primer, you could make one DNA copy at a time. But with two primers, copy numbers grow exponentially with each cycle. One copy becomes two, two become four, four become eight, and so on. That’s the “chain reaction” in P-C-R.

The orientation of the primers is important, too. The two primers attach to opposite DNA strands, on either end of the target sequence. DNA polymerase can copy DNA in just one direction (from 5-prime to 3-prime)—and the primers are set up for polymerase to extend them toward each other.

Primers are manufactured, and they are designed to be specific to each target. So if you know the DNA sequence around your target, you can amplify just about any section of DNA. The main limit is length: targets that work best are between about a hundred and a few thousand nucleotides long.

When things are working well, PCR can amplify a single copy of the target sequence—as in, DNA from one cell. But it’s easy to get many more than one copy to start with, and with more target sequences PCR is more reliable. A swab swiped across the inside of a cheek can pick up more than 100 cells. This is one way samples are collected for medical testing and in law enforcement.

For forensic analysis, DNA is often collected from hair or blood left at a crime scene. There’s enough DNA for PCR in one hair root or in a spot of blood smaller than a pinhead.

Sometimes DNA is purified first, but this step isn’t always needed.

To copy DNA, you must first separate its strands. But heating to 95° C, just 5 degrees below boiling, kills most cells. Fortunately, you don’t have to boil DNA to separate its strands. Cells have other ways of doing this, and at much lower temperatures.

Your cells carry out DNA replication at 37° C. They do it with the help of a few other proteins. DNA helicase pries DNA strands apart, and DNA binding proteins hold onto them while DNA polymerase makes a copy.

And yes, high heat inactivates most proteins—including most types of DNA polymerase. As for the DNA polymerase in PCR, its heat tolerance is unusual. It comes from a type of bacteria that lives in near-boiling hot springs. The bacteria is called Thermus aquaticus, and its DNA polymerase is known as Taq polymerase.

You could use non-heat-tolerant polymerase for PCR. And that’s what the scientists who first performed PCR did. But, as these scientists did, you would need to add a fresh dose of enzyme with each cycle.

RNA is a single-stranded molecule similar to DNA. With an extra step, PCR can amplify RNA, too. This method is called RT-PCR. The 'RT' stands for reverse transcriptase. That’s an enzyme that reads an RNA sequence and builds a complementary DNA copy.

RT-PCR is used to diagnose infections with viruses (like the coronavirus SARS-CoV-2) that use RNA as their genetic material. It can also amplify messenger RNA (mRNA). mRNA is the intermediate molecule that cells make when they read the information in their genes to build proteins. By looking at a cell’s mRNA, you can tell which of its genes are active, or switched ‘on.’