Detection of rare DNA targets by isothermal ramification amplification

We described previously a novel DNA amplification technique, termed ramification amplification (RAM) (Zhang et al., Gene 211 (1998) 277). This method was designed to utilize a circular probe (C-probe) that is covalently linked by a DNA ligase when it hybridizes to a target. Then, a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to in vivo 'rolling circle' replication of bacteriophage. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex, and resulting in an exponential amplification. Previously, we were able to achieve a significant amplification using phi29 DNA polymerase that has a high processivity and strong displacement activity. However, due to the intrinsic limitations of the polymerase, we only achieved a sensitivity of 10,000 target molecules, which is insufficient for most practical uses. Therefore, we tested several DNA polymerases and found that exo^- Bst DNA polymerase meets the requirement for high sensitivity. By further improving the assay condition and format, we are able to detect fewer than ten targets in 1 h and to apply successfully this method for detection of Epstein-Barr virus in human lymphoma specimens.