The Polymerase Chain Reaction (PCR) is a well-known approach to amplify a specific DNA sequence. PCR involves the reiterative cycling of a reaction cocktail between different temperatures to achieve amplification. As routine as PCR is in the molecular biology and molecular diagnostic laboratory, there are other methods of sequence-specific DNA amplification. These alternative approaches often do not require changing the reaction temperature and are, therefore, often referred to as sequence-specific isothermal amplification protocols. Isothermal amplification protocols are varied and have varied advantages. However, some common advantages are that isothermal techniques are extremely fast and they do not require thermocyclers. Four examples of sequence-specific isothermal DNA amplification technologies include:
Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA. A strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification. LAMP is rapid, sensitive, and amplification is so extensive that the magnesium pyrophosphate produced during the reaction can be seen by eye, making LAMP well-suited for field diagnostics.
Strand displacement amplification (SDA) relies on a strand-displacing DNA polymerase, typically Bst DNA Polymerase, Large Fragment or Klenow Fragment (3’-5’ exo–), to initiate at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. SDA is typically used in clinical diagnostics.
Helicase-dependent amplification (HDA) employs the double-stranded DNA unwinding activity of a helicase to separate strands, enabling primer annealing and extension by a strand-displacing DNA polymerase. Like PCR, this system requires only two primers. HDA has been employed in several diagnostic devices and FDA-approved tests.
Nicking enzyme amplification reaction (NEAR) employs a strand-displacing DNA polymerase initiating at a nick created by a nicking enzyme, rapidly producing many short nucleic acids from the target sequence. This process is extremely rapid and sensitive, enabling detection of small target amounts in minutes. NEAR is commonly used for pathogen detection in clinical and biosafety applications.