Polymerase chain reaction (PCR) is an efficient and cost-effective way to copy or “amplify” small segments of DNA or RNA.
Using PCR, millions of copies of a section of DNA are made in just a few hours, yielding enough DNA required for analysis. This innovative yet simple method allows clinicians to diagnose and monitor diseases using a minimal amount of sample, such as blood or tissue.
What is PCR?
Sample preparation Though PCR occurs in vitro, or outside of the body in a laboratory, it is based on the natural process of DNA replication. In its simplest form, the reaction occurs when a DNA sample and a DNA polymerase, nucleotides, primers and other reagents (man-made chemical compounds) are added to a sample tube. The reagents facilitate the reaction needed to copy the DNA code.
In addition to detecting diseases in a sample, PCR enables the monitoring of the amount of a virus present, or viral load, in a person’s body. In diseases such as hepatitis C or human immunodeficiency virus (HIV) infections, viral load is a good indication of how sick a person may be or how well a person’s medicine and treatment is working. Armed with this information, physicians may determine when to start treatment and the person’s response to treatment, making treatment personalised to each individual.
There are three clear steps in each PCR cycle, and each cycle approximately doubles the amount of target DNA. This is an exponential reaction so more than one billion copies of the original or “target” DNA are generated in 30 to 40 PCR cycles.
Before initiating PCR, DNA must be isolated from a sample. DNA extraction is a multi-step process that may be done manually or with an instrument like the COBAS® AmpliPrep Instrument, the first instrument that prepared samples automatically without human intervention. Following sample preparation, the three-step PCR process is initiated.
1. Separating the target DNA—denaturation
During the first step of PCR, called denaturation, the tube containing the sample DNA is heated to more than 90 degrees Celsius (194 degrees Fahrenheit), which separates the double-stranded DNA into two separate strands. The high temperature breaks the relatively weak bonds between the nucleotides that form the DNA code.
2. Binding primers to the DNA sequence—annealing
PCR does not copy all of the DNA in the sample. It copies only a very specific sequence of genetic code, targeted by the PCR primers. For example, Chlamydia has a unique pattern of nucleotides specific to the bacteria. The PCR will copy only the specific DNA sequences that are present in Chlamydia and absent from other bacterial species. To do this, PCR uses primers, man-made oligonucleotides (short pieces of synthetic DNA) that bind, or anneal, only to sequences on either side of the target DNA region.Two primers are used in step two—one for each of the newly separated single DNA strands. The primers bind to the beginning of the sequence that will be copied, marking off the sequence for step three. During step two, the tube is cooled and primer binding occurs between 40 and 60 degrees Celsius (104 – 140 degrees Fahrenheit) . Step two yields two separate strands of DNA, with sequences marked off by primers. The two strands are ready to be copied.
3. Making a copy—extension
In the third phase of the reaction, called extension, the temperature is increased to approximately 72 degrees Celsius (161.5 degrees Fahrenheit). Beginning at the regions marked by the primers, nucleotides in the solution are added to the annealed primers by the DNA polymerase to create a new strand of DNA complementary to each of the single template strands. After completing the extension, two identical copies of the original DNA have been made.
After making two copies of the DNA through PCR, the cycle begins again, this time using the new duplicated DNA. Each duplicate creates two new copies and after approximately 30 or 40 PCR cycles, more than one billion copies of the original DNA segment have been made. Because the PCR process is automated, it can be completed in just a few hours. In a healthcare setting, PCR makes enough copies of target DNA from the clinical sample to allow analysis; the results of these diagnostic and monitoring tests provide clinicians and other healthcare providers with information to guide treatment.