How can we identify microbes by PCR

Laboratory results: polymerase chain reaction (PCR)

The method of the PCR was developed in 1983 by the American biochemist Kary B. Mullis and is essentially based on two principles:

  • Multiplication ("amplification") of a small part of the genetic material (DNA or RNA) as well
  • Detection and identification of the multiplied ("amplified") products of the amplification.

Accordingly, the following components are required to carry out a PCR:

  • a test material (e.g. blood, cell or tissue samples),
  • certain reagents ("polymerase", "primer", "nucleotides" [these are the DNA building blocks] etc.) and laboratory equipment for the amplification of the genetic material as well
  • a detection system for the PCR products.

In terms of the reagents and laboratory equipment for performing a PCR, the following enzyme is this The heart of the entire PCR process:

This is an enzyme that is obtained from the microorganism "Thermus aquarius" (Taq) - a bacterial strain that exists in the vicinity of hot, volcanic springs and can survive adapted to the high temperatures of its environment.

The peculiarity of Taq polymerase is that this enzyme even at high temperatures above 50 ° Celsius (C) can fulfill its function - namely

  • the multiplication ("amplification") of DNA.

In addition to the Taq polymerase, it must be precisely defined in advance of the PCR analysis which part of the DNA is to be examined. For this purpose, short DNA fragments are required, the fine structure of which corresponds to the DNA target region with regard to the respective genetic code (“DNA sequence). These DNA fragments are called

The actual amplification process in the context of a PCR finally comprises the following steps:

  • Extraction of the genetic material (DNA or RNA) from the test material.
    • When analyzing RNA, the code sequence of the ribonucleic acid must first be converted into a DNA code sequence, which must be done with the enzyme "reverse transcriptase" in an analytical step before the actual amplification.
  • Amplification of the genome in several steps ("cycles"):
    • Step one - “DNA denaturation”: by heating (95 ° C) the double-stranded DNA, it is melted into single strands.
    • Step two - "Annealing": at a lower temperature (approx. 55 ° C) the "primers" can now attach to their DNA target sequences.
    • Step three - "Elongation": at a temperature of 72 ° C, the enzyme Taq polymerase now extends ("elongates") the primer start sequence using so-called "nucleotides" (these are the DNA building blocks) and builds it in this way a new, elongated DNA molecule.

Since steps one to two are repeated in several cycles, longer and longer newly formed DNA chains are created, which is where the name of this laboratory process “polymerase chain reaction” derives.

  • Analysis of the newly formed DNA chains (so-called "PCR products"):
    • At the end of the chain reaction (cycles), the PCR products - i.e. the newly created DNA molecules - are examined and evaluated qualitatively or quantitatively. There are numerous possibilities for this evaluation itself, mostly based on staining (especially fluorescent dyes) of the DNA and making it visible (by means of gel electrophoresis or photometric fluorescence detection).