14 So, let’s do the experiment!
In the PCR tests that you will be performing, you will be obtaining data on your control samples and on 90 biobank DNA samples.
- Go to the PCR laboratory and click on "Enter experiment" [Tip: hold Ctrl and click a link to open it in a new tab. (Hide tip)] (open this link in a new window, so you can get back to the instructions easily, you may wish to sign up for this site)
- Read the ‘Welcome’ tab (you can ignore the link to ‘Instructions’ and instead follow the steps given here)
- Choose population A, click on ‘Start’ and then click on the ‘Master Mix’ tab
- Answer the following questions as you work through the onscreen instructions, being careful to choose the correct primer for the test you want to perform. The first test you should do is for the CYP2D6 *4 variant.
(Note that the word ‘aliquot’ just means to divide up into portions).
Identify the four components to be added to the master mix – what is the purpose of each?
Answer
PCR water – This is guaranteed free of any contaminating DNA.
Taq polymerase enzyme – This is a heat-tolerant DNA polymerase which bonds nucleotides to the ends of the primers, building the replicate DNA strands. It needs to be heat-tolerant because of the high temperatures used to separate the DNA strands at the beginning of each PCR cycle. (It comes from a species of bacterium that lives in hot springs.)
Nucleotide solution –This contains a supply of the four types of nucleotide that will be incorporated into the growing DNA strands by the Taq polymerase enzyme.
Reaction buffer – This provides the right conditions for the Taq polymerase to function efficiently. It also contains the light sensitive dye that fluoresces.
What is a primer and why do they come in pairs?
Answer
Primers are short chains of single-stranded DNA. They bind (anneal) to a specific target sequence of complementary bases on the DNA strands and so are able to ‘find’ a specific section of the DNA code.
The primer pairs have complementary bases. They are needed to bind to each of the complementary strands of DNA that are separated in the first heating step of the PCR cycle. The chains of new DNA that are made will include the target site for the other primer for the next copying cycle. This means that the number of strands doubles with each cycle leading to millions or billions of copies, so amplifying the specific target section of DNA.
There are two different pairs of primers used in this PCR laboratory which bind to different target sequences of DNA – one which contains the CYP2D6 *4 variant and one which belongs to all variants of the CYP2D6 gene.
- As you set up the PCR machine on the ‘PCR setup’ tab answer the following:
What is the heating block for?
Answer
This controls the temperature of the mixture in each tube. Each of the three steps in each PCR cycle requires a different temperature. This process of thermal cycling is computerised.
13 Using control samples