Nucleic Acid Amplification Techniques

Abstract

Nucleic acid amplification techniques (NATs), more commonly but imprecisely called polymerase chain reaction (PCR), have evolved, among others, as central tools for pathogen identification and strain typing. The huge field of applications of the technique in all its modifications already marks the centerpiece of diagnostic approaches and has not only opened the door to a completely new understanding of elementary and sophisticated metabolic processes but also is the basis of the most important trends in life science industries. In a sense, it acts as the lens of a molecular microscope which visualizes the code from the genomic book, while its meanings have to be deciphered by a further armory of laboratory avenues.In 1983, PCR was invented by Kary Mullis, for which he later (1993) was awarded with the Nobel Prize in Chemistry along with Michael Smith [1]. The protocol is based on targeting specific DNA or rDNA sequences with more or less specific short tracer sequences (primers/oligonucleotides, colloquially oligos) binding to flanking regions of the core sequence to be proliferated. A succession of thermal incubation steps defined by length and temperature are cycled and conduct double-stranded DNA (dsDNA) melting, that is, separation of the target DNA into antiparallel, complementary, single-stranded DNA (ssDNA) strands, specific binding of primers (annealing), and enzymatic amplification out of the basic set of four nucleotide monomers of the sequence embedded. In continuation, the newly synthesized dsDNA copies, selected in length by the specific primer binding sites (PBSs), themselves serve as templates for further replication rounds, setting in motion a chain reaction in which the DNA template is exponentially amplified. The core enzyme is a DNA polymerase, which was heat-sensitive at time of invention but subsequently was destroyed at the melting temperature. To gain useful numbers of target copies, an expensive and time-consuming replacement of the enzyme was needed at the beginning of each of the usually 20-40 replication cycles. The discovery of an extraordinarily thermostable DNA polymerase purified from the thermophilic bacterium Thermophilus aquaticus, which naturally lives in hot (50-80 ∘ C) environments such as hot springs [2], Modern Techniques for Pathogen Detection, First Edition. Edited by Jürgen Popp and Michael Bauer.

Assessment of pathogenic bacteria using periodic actuation

“…In the few lab on a chip methods available, manipulation of the magnetic beads is done using on-chip micro-structures, linked with external electromagnets, which are rather complicated and difficult to manufacture. [10][11][12] Optical (e.g. surface plasmon resonance) or electrochemical sensing methods, in direct assays, or in conjunction with magnetic beads within flux injection assays are currently based on the assessment of a sensitive sensor surface (immobilized with a specific biorecognition element e.g.…”