Introduction to PCR

Materials

No materials are needed for this guide.

Equipment

  • Bento Lab

Abstract

This guide is the second chapter in the Biotechnology 101 kit, that teaches you the basics of hands-on molecular biology. The pipette is an indispensable tool when working with DNA and analysing genes, so you will need to be able to pipette with skill and confidence, before starting the research projects in this kit.

There are many different kind of pipettes. This is an introduction to variable micropipettes.

Guide

  1. What is PCR

    PCR (polymerase chain reaction) is a method used in molecular biology to make millions of physical copies of a specific DNA sequence, for example, a gene.

    It has several key ingredients: a DNA template to copy, short DNA sequences called “primers”, and a master mix containing the rest of necessary molecules.

    What is the template?

    The DNA template is double-stranded genomic DNA from the sample.

    What is a primer?

    A primer is a short single strand of DNA that serves as a starting point for DNA synthesis of a new DNA strand. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing fragment of DNA.

    How does PCR work?

    First of all, you need a DNA template (1), the physical DNA sample, which contains the sequence to be copied. It is a double stranded piece of DNA. You also need primers (2). These are short stretches of single-stranded DNA, which are designed to match the particular sequence of DNA you want to copy. Overall, you need four different primers that flank each side of the DNA you are trying to copy.

    First Step: Denaturing

    First, the DNA template is heated, which causes the double-stranded DNA to separate into two single strands.

    Second Step: Annealing

    Then the temperature is lowered to a temperature between 46-60 °C. The exact temperature depends on the experiment, and is known as the annealing temperature. At this point, the primers (2) will attach, or anneal, to their binding positions on the single strands of the template DNA.

    Third Step: Extending

    In the next step, the reaction moves to an optimal temperature for the polymerase enzyme (1), from which PCR takes its name, to start working. The polymerase enzyme builds DNA strands, and it will extend the DNA from the primer along the DNA template, creating a new DNA strand, which combines with the single-stranded template to form a double strand. The polymerase enzyme uses dNTPs (2), free DNA nucleotide bases as the building blocks for the new strand.

    Repeated cycles for exponential growth

    This process is repeated about 30-35 times. Each cycle doubles the copies of the DNA targeted by the primers.

    Why PCR is so useful for analysis

    Note that PCR will only work, if the template DNA contains the sequence you are targeting with the primers. This allows for the design of identification experiments. If some primers do not work, it is possible that the DNA sample did not contain the right DNA for the targeted sequence.

  2. PCR on Bento Lab

    Bento Lab has a thermocycler module on the right side. This is a programmable heat block, which fits 0.2mL tubes. This size tube is the standard tube to mix the ingredients for PCR.

    To use the thermocycler on Bento Lab, navigate to the module from the home screen.

    By default, Bento Lab has two standard protocols: One for PCR, and a simpler heat block program, which is useful for incubation and heating samples. For example, the latter is used in the DNA Extraction method of this kit.

    The protocol editor shows a graphic representation of the program.

    Here you can edit and change all the parameters of the protocol (1), give the custom protocol a name and save it (2), and start the thermocycler program (3).

    From the graphical representation, you can see the three stages that are repeated 30 times. In this protocol, denaturing has been set at 95°C for 30 sec, annealing to 55°C, and extending to 72°C.

    Additionally, there is an initial 95°C step for 5 min, which ensures that the initial template DNA is sufficiently denatured, i.e. become single-stranded. There is also an additional 5 min at 72°C after all cycles have completed, to ensure extension is complete.

    Finally, a hold step at the end cools the sample down to 15°C for storage.
    You can find out more about how to edit a protocol, and use the interface in this detailed chapter of the Bento Lab manual, which has animations demonstrating changing temperatures and durations, cycles, etc.

    A note on the heated lid

    Bento Lab’s thermocycler has a heated lid, which is enabled by default. During PCR, the heated lid is heated to a higher temperature that the heat block where the tubes are placed, so the top of the tube is hotter than the bottom.

    If there was no heated lid, the reaction mix inside the tube would soon evaporate and condensate inside the tube.

  3. PCR with Biotechnology 101 Kit

    For all experiments using PCR in the Biotechnology 101 Kit, you will be provided with a specific set of primers. (1)

    You also have PCR tubes already prepared with a freeze-dried PCR master mix bead (2). This bead contains:

    1. Taq Polymerase, the enzyme which performs the reaction.
    2. dNTPs (DNA nucleotide bases). These are the DNA bases (A, C, G and T) that are the building blocks of DNA. They are used by the polymerase to construct the new DNA copies.
    3. Buffer to ensure the right conditions for the reaction.

    Finally, you will prepare, as part of each experiment, specific template DNA (3). These need to be mixed in precise proportions, for which you will need the micropipette (4).