Polymerase chain reaction (PCR) is a revolutionary method used to analyze and amplify short DNA (or RNA) sequences in a sample with small quantities of RNA or DNA. PCR amplifies the selected sequence of DNA segment producing millions of copies within a few hours. Initially, DNA or RNA amplification involved cloning the selected segment using bacteria. This took weeks to amplify. But with PCR, it only takes few hours to produce millions of DNA sequence of interested.
Polymerase chain reaction requires the following components
- Two primers: These are short pieces of single-stranded DNA sequence that are complimentary from beginning to the end of the targeted DNA sequence to be copied.
- Polymerase: This is a type of enzyme that moves along starting from the end of the primers synthesizing new strand of DNA complimentary to the targeted DNA sequence. A polymerase commonly used is Taq DNA polymerase
- DNA template: This is the sample DNA that has the DNA sequence of interest.
- Nucleotides: These are single units of bases C, T, G and A that polymerase needs to make the new DNA strands.
How is PCR done?
Three major steps are involved in polymerase chain reaction. Done in an automated thermal cycler, also called a PCR machine, these three steps are repeated for 30 to 40 cycles with each cycle producing a new strand of DNA. The automated cycler is a device that heats and cools the test tubes contain the sample mixture in a repeated cycle. The cooling and heating process is essential since the three steps takes place at a different temperature.
These three steps are
- Denaturation: A temperature of 94 C (201.2 F) is applied to the original double-stranded DNA molecule so as to open it into two pieces of single-stranded DNA
- Annealing: At a temperature of 54 C (129.2 F), the primers in the reaction mixture anneal or pairs up with the single-stranded DNA template (the sequence to be copied). Starting from the primers, the polymerase attaches and starts copying the template.
- Extension: At a temperature of 72 C (161.6 F), the DNA building blocks or nucleotides which are complimentary to the DNA template of interest are coupled to the primers. This produces a double-stranded DNA molecule.
Within one amplification cycle, a single double-stranded DNA template is copied and amplified resulting into two separate double-stranded DNA segment. The two new DNA segments now will be available for the next amplification cycle and as the cycles continue, more and more copies of double-stranded DNA segment will be generated thus increasing the number of interested template.
How was PCR discovered?
Polymerase chain reaction technique was invented by Kary Mullis in 1985 when he was working in Emeryville, California for a biotechnology company known as Cetus Corporation. The PCR technique allowed scientist to make millions of DNA or RNA copies from a sample that has minute quantities of DNA or RNA.
Since then, the PCR has revolutionized many aspects of scientific research including;
- Criminology where it is used to link specific individuals to samples of hair and blood obtained from a crime scene using DNA comparison.
- Detection of HIV virus in human cells and diagnosis of genetic defects.
- Affects evolutionary studies since small quantities of DNA in fossils can be amplified to large quantities allowing further studies.
- Establishing paternity or biological identity
The widespread and innumerable uses of PCR have made it to become an important tool for use in DNA forensic laboratories among other laboratories that analyze, detect and study genetic materials either in animals or human.
Reverse Transcriptase-Polymerase Chain Reaction
Reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique that is highly sensitive and used to detect and quantify the messenger RNA (mRNA). It consists of two main parts;
- Reverse transcription: Involve Synthesis of complimentary DNA (cDNA) from RNA
- Polymerase chain reaction: Amplification of specific cDNA
This technique is used to measure viral load in HIV patients and to detect other RNA viruses such as mumps and measles.