AAT Bioquest

What are the alternative methods of polymerase chain reaction (PCR)?

Posted May 20, 2023


PCR is the preferred nucleic amplification technique but it has a few limitations that make it unfeasible for all applications. Several alternative methods of polymerase chain reaction (PCR) have been developed that overcome these limitations. Most widely used alternative methods of PCR include: 

 Loop Mediated Isothermal Amplification (LAMP) – LAMP is a simple, quick, and cost-effective isothermal nucleic acid amplification technique. This one-step amplification reaction is highly sensitive and capable of detecting minute amounts of DNA. It also eliminates the need for extensive DNA purification as it is highly resistant to a number of inhibitory substances found in clinical substances, 

Helicase Dependent Amplification (HAD) – An isothermal nucleic amplification technique, HAD allows multiple replication cycles to be performed at a single incubation temperature using the replication fork mechanism. This completely eliminates the need for thermal cycling equipment that is essential in PCR. 

Nucleic Acid Sequence Based Amplification (NASBA) – NASBA is an isothermal transcription-based amplification system that is designed specifically to amplify RNA, although DNA can also be amplified in some systems. The amplification reaction is performed at the predefined temperature of 41°C from start to finish. Maintaining a constant temperature throughout the reaction allows each step to proceed seamlessly as soon as the amplification intermediate is formed.  

Multiple Displacement Amplification (MDA) – MDA is an isothermal technique that is capable of amplifying the entire genome with high fidelity from a small amount of starting material. It is widely used to produce DNA of very high purity, generate capture probes for microarrays, and amplify stored DNA. Sensitivity and yields are high using MDA, making it the preferred technique when limited amounts of samples are available. 

Ligase Chain Reaction (LCR) – A cyclic DNA template dependent amplification reaction, LCR works by amplifying the probe molecule. This differentiates it from PCR, which produces amplicon through polymerization of nucleotides. The reaction is driven by a DNA ligase enzyme and a DNA polymerase enzyme.  

Ramification Amplification Method (RAM) – RAM is an isothermal nucleic acid amplification technique that derives its amplification power from strand displacement, primer extension and multiple branching or ramification points. 

Strand Displacement Amplification (SDA) – An isothermal amplification technique, SDA uses 4 different primers. One primer, which contains a restriction site, is annealed to the DNA template. Its inability to amplify long target sequences efficiently is a major limitation of this method. 

Rolling Circle Amplification (RCA) – RCA is an isothermal nucleic acid amplification method that amplifies the probe DNA sequences more than 109 times at a single temperature. It is capable of detecting down to a few target-specific circularized probes in a test sample. Its high resistance to contamination and its capacity to yield surface-bound amplification products make RCA the method of choice for in situ or microarray hybridization assays.

Additional resources

Nucleic acid amplification: Alternative methods of polymerase chain reaction

Polymerase Chain Reaction (PCR)

Helixyte™ Green *20X Aqueous PCR Solution*