Laboratory Methods

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Polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) is a laboratory method used for making a very large number of copies of short sections of DNA from a very small sample of genetic material. This process is called "amplifying" the DNA and it enables specific genes of interest to be detected or measured. 

DNA is made up of repeating sequences of four bases – adenine, thymine, guanine, and cytosine. These sequences form two strands that are bound together in a double helix structure by hydrogen bonds (like a spiral staircase). Each half of the helix is a complement of the other. In humans, it is the difference in the sequence of these bases on each strand of DNA that leads to the uniqueness of each person's genetic makeup. The arrangement of the bases in each gene is used to produce RNA, which in turn is used to code the amino acid sequence of a protein. There are about 25,000 genes in a human genome, and expression of these genes leads to the production of a large number of proteins that make up our bodies. The DNA of other organisms such as bacteria and viruses is also composed of thousands of different genes that code for their proteins.

How is the method performed?

 The PCR method uses cycles of repeated heating and cooling to enable separation and subsequent replication of the DNA strands. There are several components that are required for amplification of DNA by the polymerase chain reaction, each of which is described below:

1. Template DNA. This is double stranded DNA that contains the DNA sequence of interest (i.e. the region which will be amplified in the PCR reaction).

2. Primers: Primers are short sequences of bases made specifically to recognise and bind to the section of DNA to be amplified. Each PCR reaction mixture will contain two primers; one for each of the strands of the double stranded DNA template. The primers are called "forward" and "reverse" in reference to the direction that the bases within the section of DNA are copied.

3. DNA Polymerase (frequently Taq polymerase): This is a DNA enzyme which copies DNA to make new strands. Taq polymerase is an enzyme found in a bacterium (Thermues aquaticus) that grows in very hot water, such as hot springs. The Taq polymerase is especially helpful for laboratory testing because (unlike many other enzymes) it does not break down at very high temperatures needed to do PCR.

4. DNA bases: These are the individual DNA bases (adenine, thymine, guanine, and cytosine) that are incorporated by the DNA polymerase into the new DNA strands.

5. A thermo cycler: PCR is carried out in several steps or "cycles" in an instrument called a thermo cycler. Within each of cycles, the instrument will increase and decrease the temperature of the specimen at defined intervals.

Each cycle of PCR cycle involves 1) a denaturing step at 95 ºC, 2) an annealing stage at approximately 55 ºC and 3) an amplification step at 72 ºC. These steps are described further below:

Step 1: The first step of PCR is to separate the strands of template DNA into two single strands by increasing the temperature of the sample to 95 ºC. This is called "denaturing" the DNA.

Step 2: Once the strands separate, the sample is cooled slightly (to approximately 55 ºC). At this temperature the forward and reverse primers are allowed to anneal (bind) to the single DNA strands. This step is referred to as the annealing stage.

Step 3: The temperature of the sample is then increased to 72 ºC. This is the optimum temperature for the DNA Taq polymerase . The polymerase works by incorporating DNA bases on to the end of the reverse and forward primers in a sequence that is complement to the template strand. The result is the formation of two double stranded sections of DNA each with one original strand and one new strand of DNA. This newly formed double stranded DNA can then act as a template for the next cycle. This step is referred to as the amplification stage.

The above cycle of steps is then repeated many times within a single PCR reaction. In the second cycle, each of the two double strands separate to make four single strands and, when cooled, the primers and polymerase act to make four double strand sections. The four strands becomes eight in the third cycle, eight become sixteen, and so on. Within 30 to 40 cycles, as many as a billion copies of the original DNA section can be produced and are then available to be used in numerous molecular diagnostic tests. This process has been automated so that it can be completed within just a few hours. How is it used?

This method can be used, for example, to detect certain genes in a person's DNA, such as those associated with cancer or genetic disorders, or it may be used to detect genetic material of bacteria or viruses that are causing an infection.

These are just a few examples of laboratory tests that use PCR:

  • Huntington’s disease
  • Pertussis
  • Chicken Pox and shingles: Varicella Zoster Virus
  • CMV
  • Gonorrhoea
  • Chlamydia
  • DNA fingerprinting
  • Screening of donated blood for viruses
  • Often PCR is used as a preparatory step to provide sufficient DNA for use in further diagnostic tests.

    Real-time PCR

    Real-time PCR (also known as quantitative PCR or qPCR) is similar to PCR except that data are obtained as the amplification process is taking place (i.e., "real time") rather than at a prescribed endpoint. This shortens the time for the test from overnight to just a few hours. The method requires incorporation of a fluorescent marker into the PCR products. The level of fluorescence is then measured by a detector, enabling quantification of the PCR products after each PCR cycle.

    RT – PCR (Reverse Transcriptase PCR)

    This method uses PCR to amplify RNA. RNA is a single stranded nucleic acid molecule and needs to be made into DNA before it can be amplified. The addition of a new strand that is the complement of RNA is achieved by the enzyme called Reverse Transcriptase (RT). A reverse primer binds to the single stranded RNA and the enzyme RT copies the RNA strand to make a single stranded DNA, which it then copies to make a double stranded DNA molecule. The double stranded molecule can then be amplified by PCR. Detection can also be by real-time methods.

    Here are two examples of laboratory tests that use RT-PCR:

  • HIV viral load
  • HCV
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