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.
Zhang
D Y
DY
Department of Pathology, Mount Sinai School of Medicine, New York University, New York, NY 10029, USA. david.zhang@mountsinai.org
Zhang
W
W
Li
X
X
Konomi
Y
Y
D015139:Blotting, Southern; D004247:DNA / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics; D004279:DNA, Viral / Q000187:drug effects; D005784:Gene Amplification; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D020298:U937 Cells
D015342:DNA Probes; D004270:DNA, Circular; D004279:DNA, Viral; C099537:Epstein-Barr virus encoded RNA 1; D012367:RNA, Viral; D004247:DNA