PCR Machine- Principle, Parts, Steps, Types, Uses, Examples


PCR machines, also known as thermal cyclers or DNA amplifiers, are laboratory devices that are used to amplify segments of DNA or RNA chosen from the genome with a primer . PCR stands for polymerase chain reaction, a technique that was invented by Kary Mullis in 1983 and revolutionized molecular biology. PCR machines allow scientists to make millions of copies of a specific DNA or RNA sequence in a matter of hours, which enables them to study the genetic material in detail .

PCR machines work by cycling through three main steps: denaturation, annealing, and extension . In denaturation, the DNA template is heated to separate the two strands. In annealing, short synthetic oligonucleotides called primers bind to the complementary regions of the single-stranded DNA. In extension, a heat-stable enzyme called Taq polymerase adds nucleotides to the 3` end of the primers, synthesizing new strands of DNA that are identical to the template . These steps are repeated for 20 to 40 cycles, resulting in an exponential amplification of the target DNA or RNA sequence .

PCR machines require five essential reagents for the reaction: DNA template, Taq polymerase, primers, deoxyribonucleotide triphosphates (dNTPs), and PCR buffer. The DNA template is the source of the target sequence that needs to be amplified. The Taq polymerase is a thermostable enzyme that can withstand high temperatures and catalyze the DNA synthesis. The primers are short sequences of nucleotides that are designed to match the ends of the target sequence and provide a starting point for the Taq polymerase. The dNTPs are the building blocks of DNA that are added by the Taq polymerase to extend the primers. The PCR buffer provides the optimal conditions for the reaction, such as pH, salt concentration, and magnesium ions.

PCR machines consist of three main parts: a thermal block, a heated lid, and a control panel . The thermal block has holes where tubes or plates holding the reaction mixtures can be inserted. The thermal block can change the temperature rapidly and accurately according to a programmed protocol. The heated lid presses against the tops of the tubes or plates and prevents condensation of water from the reaction mixtures on the inside of the lids. The control panel has a graphical display and a key-pad that allow users to enter and monitor the parameters and status of the reaction .

PCR machines have many types and applications in various fields of science and medicine. Some examples of types of PCR are:

  • Real-time PCR or quantitative PCR (qPCR), which measures and quantifies the amount of amplified DNA or RNA in real-time using fluorescent dyes or probes .
  • Reverse-transcriptase PCR (RT-PCR), which converts RNA to DNA using an enzyme called reverse transcriptase and then amplifies the resulting complementary DNA (cDNA) .
  • Nested PCR, which reduces the chances of unwanted products by performing a second PCR using new primers that are nested within the first ones .
  • Hot start PCR, which uses antibodies or chemical modifications to inactivate Taq polymerase until a high temperature is reached, preventing nonspecific amplification at low temperatures .
  • Multiplex PCR, which amplifies several fragments in a single DNA sample by using multiple sets of primers .
  • Long-range PCR, which uses a combination of polymerases and additives to amplify larger fragments of DNA than conventional PCR .
  • In situ PCR, which performs PCR directly in cells or fixed tissue on a slide .
  • Asymmetric PCR, which amplifies one strand of the target DNA more than the other by using unequal amounts of primers .
  • Assembly PCR, which uses overlapping primers to create longer DNA fragments from shorter ones .

Some examples of applications of PCR are:

  • Gene expression analysis, which studies how genes are turned on or off in different cell types, tissues, or conditions using RT-PCR or qPCR .
  • Genotyping, which detects variations in alleles or genes among different cells or organisms using PCR with specific primers or probes .
  • Cloning and mutagenesis, which introduces new DNA sequences into bacteria or other hosts using PCR-generated fragments as inserts for vectors such as plasmids or phages .
  • Sequencing, which determines the order of nucleotides in DNA using PCR to prepare and process template DNA for various methods such as Sanger sequencing or next-generation sequencing (NGS).
  • Medicine and biomedical research, which uses PCR to diagnose genetic diseases, identify infectious agents, perform prenatal testing, screen embryos for IVF, and more.
  • Forensic science, which uses PCR to analyze DNA evidence from crime scenes or paternity tests.
  • Environmental microbiology and food safety, which uses PCR to detect pathogens or contaminants in water, soil, food, or other samples.

In summary, PCR machines are powerful and versatile tools that can amplify small amounts of DNA or RNA for various purposes. They work by cycling through three steps: denaturation, annealing, and extension. They require five reagents: template DNA/RNA , Taq polymerase , primers , dNTPs , and buffer . They consist of three parts: thermal block , heated lid , and control panel . They have many types and applications in different fields of science and medicine.