PCR primers: what defines the amplified region
PCR (polymerase chain reaction) is a laboratory method that makes millions of copies of one chosen stretch of DNA, and the primers decide which stretch.
Remember
To change the region that PCR amplifies, change the primers: the same polymerase, nucleotides and magnesium are used for any target.
Memory aid
the primers are the address label, and the polymerase is the courier that copies whatever the label points to
Use it to recall that the primers decide where the copying happens, while the polymerase carries out the copying.
PCR (polymerase chain reaction) is a laboratory method that makes millions of copies of one chosen stretch of DNA. The choice is made by the primers: short pieces of DNA made to match the sequences on either side of the target, which is the region to be copied. The forward primer binds one strand and the reverse primer binds the opposite strand, so together they mark the start and end of that region.
Each cycle has three steps. Heating denatures the DNA, which means it separates the two strands by breaking the hydrogen bonds between them. Cooling allows the primers to anneal, which means they attach by pairing with the complementary sequence on each strand. Then a heat-stable DNA polymerase (Taq, the enzyme that builds new DNA) extends from each attached primer.
The polymerase can only start from a primer, so the stretch between the two primer sites is the one that is copied. Repeating the cycle makes the number of copies of that stretch grow, and the primers are what let PCR pick out one gene, or the region around one mutation, from a whole genome.
The other ingredients that make the reaction work are the same for any target, although they are adjusted to improve yield and efficiency, and none of them chooses the target. The polymerase, the free nucleotides (the building blocks it joins together) and the magnesium ions (a helper the polymerase needs) are used whatever sequence is copied. To change the region, you change the primers.
Key facts
- Primer pair
- the forward primer binds one strand and the reverse primer binds the opposite strand, marking the start and end of the region copied.
- Starting point
- the polymerase can only start copying from a primer.
- Same for any target
- the same polymerase, free nucleotides and magnesium ions are used whatever the target.
- Medical use
- PCR can investigate single-base mutations, such as sickle cell disease.
Exam traps
- A heat-stable polymerase does not choose which region is copied, because it extends from whichever primer has attached.
- Repeated temperature cycling does not select the region that is copied: it raises the number of copies of whatever the primers selected.
- Free nucleotides (deoxynucleotide triphosphates, abbreviated dNTPs) do not define the region copied, because they are the building blocks that the polymerase joins together for any target.
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