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Synthesize a new strand of DNA

Heat to separate strands, re-anneal with primers

Synthesize new DNA strands

FIGURE 15.5 Polymerase chain reaction. After the first several rounds of DNA synthesis, PCR generates the simple double-stranded structure at the bottom of the figure, which can now be copied over and over through the "repeat" step that separates the DNA into single strands, attaches the primers, and synthesizes DNA again. The number of copies you can obtain is limited mostly by how many reagents (primers, individual nucleotides, buffers, and DNA polymerase) you want to expend, and frankly more often the limiting issue is how many copies you even need to make. Usually, one PCR reaction gives all the DNA needed for a sequencing reaction to read the DNA sequence of the PCR-generated DNA fragment.

FIGURE 15.5 Polymerase chain reaction. After the first several rounds of DNA synthesis, PCR generates the simple double-stranded structure at the bottom of the figure, which can now be copied over and over through the "repeat" step that separates the DNA into single strands, attaches the primers, and synthesizes DNA again. The number of copies you can obtain is limited mostly by how many reagents (primers, individual nucleotides, buffers, and DNA polymerase) you want to expend, and frankly more often the limiting issue is how many copies you even need to make. Usually, one PCR reaction gives all the DNA needed for a sequencing reaction to read the DNA sequence of the PCR-generated DNA fragment.

two copies. This gains us almost nothing. We need a way to make a lot of copies. This is where the chain reaction part of PCR comes in. The real secret to PCR is not one primer that tags the spot you want to copy, but rather two primers that flank the place you want to copy that let you repeatedly copy exactly the same spot in the genome. So at the end of Step 1 in Figure 15.4, we had one new strand. If we have a second primer also present that can bind to the new strand, it can now copy back across the region containing M, but now it is copying on the other strand (Figure 15.5).

There is a second secret to PCR: the kind of DNA polymerase used in the reaction is stable at very high temperatures. In the procedure, every round of PCR calls for heating the DNA to separate the double strands back into single strands that can bind the primer. There is a problem here, which is that if you heat the reaction mixture to separate the DNA strands, you will kill most types of DNA polymerase, exactly the same enzyme that is needed in the next step to carry out the DNA synthesis step. A very clever solution to this problem was to go in search of organisms that live at very, very high temperatures. The logic is that, if an organism can live in a hot springs or along the edges of the hot thermal currents of the deep ocean vents, their enzymes must all be capable of surviving at temperatures close to boiling. By isolating DNA polymerase from organisms living in hot environments, scientists made PCR something practical and useful instead of a theoretical curiosity.

If you spend the time to follow Figure 15.5 through its steps, you can see how the use of two primers flanking a gene can rapidly isolate that gene (or other sequence of interest) onto a double-stranded fragment that has the primer sequences at its ends. Once this double-stranded sequence exists, it can go through the same loop—separate strands, bind to primers, synthesize new DNA, separate strands, bind to primers, synthesize new DNA—over and over.

You don't need to spend a lot of time contemplating the mechanisms in Figure 15.5 to be able to get the main point here: if you can make primers that flank the piece of DNA you want, you can make vast numbers of copies of the piece of DNA that lies in between the sequences where the primers bind based on their complementary base pairing (Figure 15.6). One double-stranded structure of this kind is copied to become two copies of the double-stranded structure after one round of PCR. After two rounds it has become four copies, after three rounds it has become eight copies, then sixteen copies, and so on. After twenty rounds of PCR, we have more than a million copies.

To truly appreciate the power of PCR, consider several things: After thirty rounds of PCR we have more than a billion copies of that one little double-stranded piece of DNA we started with. (In contrast, if we had used only one primer, we would have thirty new copies at the end of thirty rounds of DNA synthesis!) Usually, when we do a PCR reaction, we start out with more than one copy of the genome sitting in the test tube. Depending on how much DNA you need to end up with, a PCR reaction may often cost less than a dollar to carry out. Also, PCR is fast, with some reactions taking less than a minute per round (although sometimes it takes longer). It's no wonder that PCR is felt to have revolutionized modern biology and genetics!

30 rounds of DNA synthesis

30 rounds of PCR

30 copies of the target DNA

More than a billion copies of the target DNA

30 rounds of PCR

30 rounds of DNA synthesis

FIGURE 15.6 PCR can generate billions of copies of a DNA fragment in the time it takes a single-primer reaction to make tens of copies.

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