2. Polymerase chain reaction (PCR)

PCR is an in vitro technique which allows the amplification of a specific DNA region that lies between two region of known DNA sequence. PCR is a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment or sequence of interest of DNA, via enzymatic replication, without using a living organism. It can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations. PCR was invented in 1983 by Kary Mullis. PCR is now a common technique used in medical and biological research labs for a variety of tasks, such as the sequencing of genes and the diagnosis of hereditary diseases, the identification of genetic fingerprints (used in forensics and paternity testing), the detection and diagnosis of infectious diseases, and the creation of transgenic organisms. PCR is being also used for biosystematics, population biology, conservation biology, ecology, developmental biology, and genetics.

The PCR process is similar to that of a Xerox machine in that it makes copies of specific regions of DNA. This process is known as amplification and the resulting product is referred to as an ‘amplicon’. Most commonly, PCR is carried out in three steps, often preceded by one temperature hold at the start and followed by one hold at the end. Initialization step. Prior to the first cycle the PCR reaction is often heated to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), and this temperature is then held for 1-9 minutes. This first hold is employed to ensure that most of the DNA template and primers are denatured, i.e., that the DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding two single strands of DNA. Also, some PCR polymerases require this step for activation (hot-start PCR). Following this hold, cycling begins, with one step at 94-98°C for 20-30 seconds (denaturation step). Annealing step. In this step the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template. Brownian motion causes the primers to move around, and DNA-DNA hydrogen bonds are constantly formed and broken between primer and template. Stable bonds are only formed when the primer sequence very closely matches the template sequence, and to this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis. The temperature at this step depends on the melting temperature of the primers, and is usually between 50-64°C for 20-40 seconds. The annealing step is followed by an extension/elongation step during which the DNA polymerase synthesizes new DNA strands complementary to the DNA template strands. The temperature at this step depends on the DNA polymerase used. Taq polymerase has a temperature optimum of 70-74°C; thus, in most cases a temperature of 72°C is used.

The DNA polymerase condenses the 5\\'-phosphate group of the dNTPs with the 3\\'-hydroxyl group at the end of the nascent (extending) DNA strand, i.e., the polymerase adds dNTP\\'s that are complementary to the template in 5\\' to 3\\' direction, thus reading the template in 3\\' to 5\\' direction. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. A final elongation step of 5-15 minutes (depending on the length of the DNA template) after the last cycle may be used to ensure that any remaining single-stranded DNA is fully extended. A final hold of 4-15°C for an indefinite time may be employed for short-term storage of the reaction, e.g., if reactions are run overnight.

Collaborators

  • Smithsonian
  • UNIPA
  • UB
  • IBRC

Fundings:

  • USAID