Journal Article

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

Yusa, Sei-ichi, Zhong, Huayan, Hu, Lin, Weiss, Tristen C., Romaniuk, Paul J., You, Qimin, Xu, Gaolian, Wang, Hongying, Pickerill, Sam

2012

Overview

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5′ ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism – single crossing CPA – and provide details on alternative mechanisms.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/1513/2216 / 631/208/721 / 631/208/727 / 631/337/151 / Base Sequence / Denaturation / Deoxyribonucleic acid / DNA / DNA, Bacterial - genetics / DNA-directed DNA polymerase / Humanities and Social Sciences / Molecular Sequence Data / multidisciplinary / Mycobacterium tuberculosis - genetics / Nicking endonuclease / Nucleic Acid Amplification Techniques - methods / Primers / Science / Temperature effects / Templates, Genetic