POLYMERASE CHAIN REACTION (PCR) Introduction

Adapted from work by: John Urbance, Ph.D. by Doug Luckie, Ph.D., and Mike Haenisch

Experimental Objectives:

1. To learn how to set up and run PCR reactions and the role of the reaction ingredients.
2. Learn how to mix a PCR reaction cocktail with correct concentrations diluted from stock solutions.
3. To gain expertise in working with DNA laboratory equipment (e.g. micropipettors, thermocycler, agarose gels).

Introduction

Have you ever wondered how forensic scientists get enough DNA from a single drop of dried blood or from a single hair to conduct investigations? Or how, out of the millions of base pairs that make up an organism’s genome, scientists isolate a particular gene, or set of genes, for analysis?

They can accomplish these things (and many others) by “amplifying” the targeted region of the genome using a technique called the Polymerase Chain Reaction (PCR). In PCR we essentially replicate the desired target region of the DNA in a test tube using the same enzyme that the cell uses for DNA replication (DNA polymerase). These days, there are very few genetic analyses that don’t include a PCR step somewhere in the process. It is arguably the most important technique in the molecular biologist’s repertoire (important enough to win its inventor, Kary Mullis, the Nobel Prize) and has become a ubiquitous and powerful tool in diagnostics, forensics and research biology. PCR is a method of synthesizing (“amplifying”) large quantities of a targeted region of DNA in vitro (in the test tube). The DNA is synthesized the same way that cells do it—using a DNA polymerase (the enzyme that cells use to replicate their DNA). Once amplified, PCR products can simply be visualized by agarose gel electrophoresis or can be further analyzed by subsequent enzymatic digestion for DNA fingerprinting, by cloning, or by DNA sequencing.

PCR works by using a thermostable DNA polymerase (Taq polymerase) and short DNA fragments, called ‘primers’, to direct the synthesis of a specifically-targeted region of the genomic DNA. The synthesis reaction is repeated numerous times called ‘cycles’. The products of previous synthesis cycles serve as template for the next cycle. This results in an exponential amplification of the targeted region of DNA—every cycle will double the copy number of the target region. This repeated cycling is made possible by the use of Taq polymerase, a thermostable (heat-tolerant) DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus, originally isolated from a hot spring in Yellowstone National Park (ambient temperature 80 °C!). Because it comes from a heat-adapted bacterium, Taq polymerase can withstand the repeated, high-temperature DNA denaturation steps that are part of the PCR procedure.

The Primers

All DNA polymerases require a short segment of double-stranded nucleic acid to initiate DNA synthesis. During DNA replication, cells use short stretches of complementary RNA—synthesized by enzymes called ‘primases’—to initiate polymerization. In the laboratory, short, complementary single-stranded DNA primers are also used in PCR to initiate DNA synthesis and to designate the specific target region to be amplified. The primers (also called oligonucleotides—meaning small number of nucleotides) are easily synthesized and can be designed to be complementary to any known DNA sequence. They can range in size from 10 to 100 nucleotides in length, but typically they range from 15 to 30 bases for PCR. The primers determine the target specificity (i.e. which segment of the template DNA will get amplified) of the PCR reaction.

The Cycles

1. Denaturation (HOT). During the denaturation step, the reaction cocktail is exposed to high temperature, usually 95 °C. This high temperature will denature the DNA– meaning the two complementary strands of the DNA molecule unravel, exposing the nucleotide bases. The high temperature of the denaturing step has the added advantage of denaturing proteins and disrupting cells so you don’t have to always start with purified DNA as your amplification template; you can often amplify DNA directly from cell lysates—or even whole cells.
2. Primer Annealing (COOL). During the second step of each cycle, the temperature is lowered to an annealing temperature, allow annealing of the primers to their complementary targets on the DNA template (one for each DNA strand). These are designed to flank the desired target region of your DNA template and serve as the starting points for DNA synthesis by the Taq polymerase. Each pair of primers will have a particular annealing temperature determined by the length of the primers and their nucleotide content. Using an annealing temperature that is too low can result in non-specific amplification (amplifying the wrong region of the DNA). Using an annealing temperature that is too high can result in no amplification at all.
3. Extension. The reaction cocktail is now brought to the optimum reaction temperature for Taq polymerase (68 to 72° C). During this step, the Taq will bind to each DNA strand and “extend” from the priming sites (synthesize a complementary strand of the targeted DNA).

Notice that these three steps are accomplished simply by varying the incubation temperature of the reaction tubes. Typically, PCR reactions are run for 30 to 40 cycles, which are performed by a specialized machine called a thermocycler designed to rapidly heat and cool the reaction tubes to the desired temperatures. Each cycle DOUBLES the number of copies of the target DNA sequence. In the figure below, observe that after step 3 the first PCR cycle is complete and each parent DNA strand (the template) is now basepaired with newly a synthesized DNA strand. When steps 1-3 are repeated in the second cycle, both DNA amplicons (the products of PCR) are copied to give you 4 copies of the desired sequence. This doubling will continue with each cycle, which allows you to generate a huge quantity of your desired DNA sequence.  PCR allows exponential amplification: If n= the number of PCR cycles you obtain 2ⁿ copies of your original DNA (e.g., if you start with 10 molecules that contain your target sequence, after 4 cycles you would have 10 molecules * 2⁴ = 160 copies). In reality, PCR is not always 100% efficient, but it is the primary way scientists rapidly obtain large quantities of a desired DNA section that can be used for sequencing, gel electrophoresis, gene cloning, protein production, and more.

Schematic drawing of the PCR cycle by Enzoklop; CC-BYSA

What’s in the reaction cocktail?

You must create a reaction cocktail in your tube that provides everything the enzyme (in this case, Taq polymerase) needs to function as it would in the cell during DNA Replication. Below is an inventory of what is required.

• Taq Buffer and MgCl₂: Each cellular enzyme has a specific salt concentration, pH, and temperature required for its optimum performance. At 1X your PCR reaction buffer provides the proper salt concentration and pH (~8.5) for Taq polymerase.
• dNTPs: (i.e. nucleotide bases) DNA is a polymer of these four nucleotides [A,T,G,C]. They are the building blocks polymerase uses to synthesize new DNA.
• Oligonucleotide primers: Their roles in DNA replication and in PCR were described above. PCR usually requires two primers, one targeted to each DNA strand so that BOTH strands are copied. Each primer is a short single-stranded DNA molecule so it can to a complementary DNA strand.
• DNA Template: In the cell, DNA polymerases use denatured, genomic DNA as a template upon which to synthesize complementary DNA strands.
• Taq polymerase: You can’t carry out an enzymatic reaction without the enzyme.

How do we prepare this reaction mixture?

You typically perform many PCR reactions at one time. To set up these reactions efficiently, you will prepare a master mix, which is a single tube that contains all the reagents that each reaction with a given primer set requires (The buffer, dNTPs, MgCl₂, primers, polymerase, and water). After prepping the master mix, you dispense aliquots into individual reaction tubes, where you can add the unique component (usually the DNA template). This ensures that all of your reactions received the same concentration of reagents.You will perform your PCR reactions in small, 0.2 mL strip tubes (small tubes connected to each other by plastic).

What else do I need to know to successfully perform PCR?

• Accurate pipetting is essential to PCR success. Do not contaminate the PCR reagents. PCR is very sensitive!
• Keep your reactions COLD (on ice) before running them in the PCR machine