Polymerase Chain Reaction

The polymerase chain reaction, or PCR, is an incredibly useful and powerful technique for copying DNA. In Bio 6B, you'll use PCR in several different experiments, and there are several related pages:

This page gives the general background for PCR in general, while the pages linked above give the details for the Bio 6B PCR experiments and some more general concepts.

Why PCR is so useful

PCR, the Polymerase Chain Reaction, is a technique for copying specific fragments of DNA. Everything in biology is somehow connected to the nucleic acids DNA and RNA, and everyone who works with nucleic acids will end up needing to copy, detect, or alter specific nucleotide sequences. PCR does that quickly and efficiently.

PCR has two main characteristics that make it useful:

  • Sensitivity: PCR can amplify DNA starting from a tiny quantity of template – as little as a single molecule. This is true because PCR is a chain reaction, making DNA copies and then making copies of the copies.
  • Specificity: PCR can be used to copy exactly the DNA segment you want, even if you start with a complex mixture of template DNA. This is true because each primer is complementary to only one stretch of nucleotides on the template. PCR conditions can be controlled so that the primer will bind only where it is exactly complementary to the template.

DNA replication basics

PCR copies DNA. The basic concept is simple: PCR copies a specific target region of DNA and then copies the copies, over and over. The name Polymerase Chain Reaction comes from the fact that a DNA polymerase is used to copy fragments of DNA over and over in a chain reaction. The end result is that the reaction will double the number of copies of a particular DNA fragment over and over; in principle, you could start with as little as a single molecule of DNA and end up with millions of copies.

pcr expansion yourgenome

[Image credit: yourgenome.org]

First, the two strands of  the DNA template are separated, then each strand is copied. This process is repeated through many cycles, and the number of DNA copies doubles with each cycle.

This diagram is much too simple, though; it leaves out the actual steps of producing DNA. In order to understand the process well, you need to start by understanding DNA replication in general.

DNA replication basics

You may find it helpful to review the structure of nucleic acids in Campbell, Chapter 5. Here are some basic concepts you should understand for DNA synthesis, whether it's happening in a cell or in a test tube in a PCR machine.

DNA strands are complementary and antiparallel

The DNA we're working with is normally double-stranded. Each strand is a chain of nucleotides, covalently bonded together. The two strands are stuck to each other by a combination of hydrogen bonds (base pairing) and other interactions (base stacking). "Complementary" means that A (adenine) on one strand pairs with T (thymine) on the other, and G (guanine) pairs with C (cytosine). "Antiparallel" means that the 5' end of one strand is matched with the 3' end of the other strand.

Each strand serves as a template for a new strand

In DNA replication, whether it's happening in a cell or in a PCR tube, the strands of DNA must be separated so that a new strand can be assembled by base pairing new nucleotides with the original strand and joining them into a polymer. The original strand is called the template.

DNA is synthesized by DNA polymerase

The enzyme that catalyzes the formation of a new DNA polymer is called DNA polymerase. In cells, there are several kinds of polymerase; a specific type is used for PCR.

Nucleotides can only be added to the 3' end of the new strand

The nucleotides are the monomers for forming the new DNA polymer, and they also provide the energy needed to form the covalent bonds of the polymer. Each nucleotide starts out with three phosphate groups. (As you may recall, ATP, the commonly used energy transfer molecule, is also an RNA nucleotide.) As new DNA is formed, two of the the high-energy phosphate groups on the nucleotide are removed, and the remaining phosphate of the new nucleotide is bonded to the 3' end of the existing DNA strand. Thus, new nucleotides can only be added to the 3' end of a strand of DNA, because that's the part that can bond to the phosphate of the incoming nucleotide. This fact has important implications for the design of a PCR experiment, as you'll see.

DNA polymerase requires a primer

Since DNA polymerase can only catalyze the addition of new nucleotides to the 3' end of existing DNA molecules, it needs an existing DNA molecule to start with. It can't insert the first nucleotide of a new strand. Thus, DNA synthesis requires primers — short stretches of nucleic acid that give the polymerase a place to start.

When a cell copies its entire genome, it will synthesize numerous primers, so DNA replication can start in many places at once. These primers are made of RNA, which is later removed and replaced with DNA. The primer is not part of the finished DNA product.

In PCR, the goal is different. Rather than copying the entire genome, PCR is usually used to copy a specific target sequence within a larger DNA molecule. In designing  PCR protocol, we can determine which DNA will be copied by using specific primers matched to a specific target sequence. Look at the diagram below to see how the primers fit in PCR.

PCR: a more detailed look

This diagram shows the orientation of the template strands and the primers. When copying DNA, it's important to keep track of the 5' and 3' ends.

PCR diagram showing template & primers

PCR requires two primers (one for each direction; shown in different colors in this diagram). The nucleotide sequences of the primers determine where they will bind to the DNA template. Each primer is complementary to one of the two template strands, which also means that the nucleotide sequence of the primer  is identical to the sequence of other template strand.

When a new strand of DNA is synthesized it must eventually contain the binding site for the other primer, so the DNA that starts from one primer can later be copied using the other primer. Thus, the 3' ends of the two primers must face toward each other.

Each PCR experiment requires a set of carefully designed primers to ensure that the desired DNA will be copied. (You'll learn how to design PCR primers in an exercise later this quarter. In the diagrams on this page, the nucleotides are represented by little zippered lines; later, we'll replace the lines with actual nucleotide sequences.)

Steps of PCR

PCR happens in a series of steps, each occurring at a specific temperature.

Denature
The two strands of the DNA double helix are separated. In PCR, the two strands are separated by simply heating the DNA to 94° or so. This breaks the weak interactions joining the two strands, but leaves the covalent bonds of each strand intact.
Anneal
The reaction mix is cooled to 45-60° to allow the primers bind to the DNA. Primer annealing is controlled by allowing the PCR reaction tube to cool from 90° to 45-60°. At this temperature, the weak interactions that join two strands of DNA together can re-form. A high primer concentration is used to favor primer annealing rather than the re-annealing of the two original strands of DNA.
Extend
The polymerase copies the DNA. The polymerase used in PCR, like any DNA polymerase, can only work by adding new nucleotides to the 3’ end of an existing DNA strand. The polymerase copies DNA, starting by adding nucleotides to the 3’ end of the primer. Extension happens fastest at the enzyme’s optimal temperature. In PCR, the enzyme that’s normally used works at very high temperatures: 72° is its optimum, and it can survive 90° long enough to allow the denaturing step to occur. The DNA polymerase most commonly used in PCR is called Taq, which is short for Thermus aquaticus, the heat-loving bacterium from which the enzyme was first isolated.
Repeat
The denaturing, annealing, and extension steps constitute one cycle. These steps are repeated over and over, typically for 20-30 cycles. The temperature steps are controlled by a thermal cycler, or PCR machine, which can be programmed to put a set of PCR tubes through the appropriate set of temperature cycles. The exact temperature of each step is critical, and must be optimized for each set of primers and template.

If PCR works perfectly, the number of copies of the target DNA doubles with every cycle. If you started with 1 copy of the target sequence, after 25 cycles you could have 225, or 33 million copies. You would normally start with an extremely small amount of template DNA, and end up with plenty to use for further analysis.

Ingredients

The tools used for copying DNA in PCR are the same tools used by cells in copying their own DNA: a DNA polymerase, some nucleotides, primers, and the DNA to be copied (called the template), along with a few other components. Here are the ingredients needed for PCR:

Water
Added to adjust the volume and concentration of the other ingredients. Must be pure and sterile.
Reaction buffer
Controls the pH and salt concentrations to allow the enzyme to do its job. The buffer is normally included in a separate tube when you buy the enzyme. It comes as a concentrate (usually 10x); you dilute it to make the buffer concentration 1x in the PCR reaction tube.
MgCl2
Mg2+ interacts with both nucleic acids (primers & template) and with individual nucleotides. If the concentration of Mg2+ is too low, amplification may not occur; if the concentration is too high, the wrong products might be produced. In many cases, MgCl2 concentration must be optimized experimentally for a specific reaction.
Nucleotides 
The monomers that will be used by the polymerase to make new DNA strands. In the lab, these are often referred to as dNTPs, for Deoxy Nucleoside Triphosphates.
Primers
The oligonucleotides (short stretches of single-stranded DNA) that give the polymerase a place to start. Must be present in high concentration. Each PCR reaction uses primers specific for a particular target sequence.
Polymerase
The enzyme that makes the new DNA. Taq polymerase is commonly used in PCR.
Template DNA
The starting DNA, part of which will be copied in the PCR reaction.

All these components must be present in the proper concentrations to allow PCR to work. In a later lab, you'll see how to calculate the amounts for all these ingredients.

Where to go next

Once you understand the PCR basics on this page, there are a few more things to consider.

PCR primer sequences

Primers determine where DNA polymerase starts copying DNA. In PCR, specific primers are used to target the specific region of DNA that is to be copied (called the target sequence). This is different from cellular DNA replication, in which numerous primers are used to start copying DNA in many places simultaneously. Another difference is that the PCR primers are DNA, and will be part of the completed PCR product, unlike the RNA primers in cellular DNA replication.

For a detailed example of how primer sequences are determined, see PCR Primer Design.

How to set it up

See PCR setup for how to do the calculations and pipetting.

How to view the results

If your PCR is successful, you'll have some newly synthesized DNA in your tubes. You'll use electrophoresis to view the results. See electrophoresis and DNA gel method.

The Bio 6B PCR experiments

The 6B lab uses PCR for several different experiments:

Review

Terms & concepts

  • 5' & 3'
  • Annealing
  • Antiparallel
  • Complementary
  • Denaturing DNA (contrast this with denaturing protein)
  • Taq DNA polymerase
  • Extension
  • Template
  • Primer
  • Target sequence

Review questions

  1. Why does DNA synthesis go in only one direction? (For a detailed explanation of DNA synthesis, see Campbell, Chapter 16: The Molecular Basis of Inheritance).
  2. The synthesis of a DNA polymer requires energy. How is this energy supplied?
  3. Draw a simple diagram of the steps of PCR, including template, target sequence, primers, and PCR products.
  4. Explain the temperature steps for a PCR program.
  5. What does PCR do and why is it so useful?
  6. Why is it called PCR? What does it stand for, and what does it mean?
  7. What makes PCR so sensitive (that is, why can it work with such a tiny amount of template)?
  8. What makes PCR specific about which fragment of DNA it amplifies?
  9. What happens at each of the temperature steps of PCR?
  10. When the DNA is heated in the denaturing step of PCR, why do the strands get separated but the individual strands don't break?
  11. Explain the purpose of each ingredient in the PCR reaction.
  12. Could PCR work starting from a single-stranded DNA template? Could it work with only one kind of primer?
  13. Are the two primers complementary to each other?
  14. Theoretically, if you started with one molecule of DNA template and performed 25 cycles of PCR with 100% efficiency, how many new DNA copies would you make? Realistically, why wouldn’t your PCR be 100% efficient?
  15. In the annealing step of PCR, it is possible for the two complementary template strands to anneal to each other, instead of a primer molecule annealing to each template strand. How can you encourage primer-template annealing instead of template-template annealing?

Compare & contrast: PCR vs. chromosomal DNA replication

  1. How many different primers are there in chromosomal DNA replication? How many in PCR?
  2. What are the primers made of in chromosomal DNA replication and in PCR?
  3. How are the strands separated in chromosomal DNA replication and in PCR?
  4. Is there an end-replication problem in PCR? Why or why not?
  5. Which enzymes that are part of chromosomal DNA replication are not part of PCR? Why?
  6. In chromosomal DNA replication, telomerase synthesizes DNA. What does it use as a template? What does it use as a primer?

References

Overview

 Polymerase chain reaction (PCR) from Khan Academy.

Videos

PCR Education from ThermoFisher. Watch the video, "Introduction to PCR."

PCR - Polymerase Chain Reaction Simplified from MedSimplified.

PCR (Polymerase Chain Reaction) Tutorial - An Introduction from Applied Biological Materials. a manufacturer of PCR products. 8:50. Starts with a good basic overview, then goes into specifics of planning PCR experiments (maybe more than you want to know on Day 1).

PCR - Polymerase Chain Reaction (IQOG-CSIC) from CanalDivulgación. Visualization of PCR steps with dramatic music and no narration.

PCR Method Video from LabXchange. Walks you through the steps of performing PCR in the lab, along with the basic concepts.

Deep background

Role of MgCl2 in PCR Reaction. Genetic Education. Magnesium ions are important in PCR for several reasons, as explained in this article.

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