Ramification amplification: A novel isothermal DNA amplification method

David Y. Zhang, Margaret Brandwein, Terence Hsuih, Hong Bo Li

Research output: Contribution to journalArticlepeer-review

67 Scopus citations

Abstract

We have developed a novel isothermal DNA amplification method with an amplification mechanism quite different from conventional PCR. This method uses a specially designed circular probe (C-probe) in which the 3′ and 5′ ends are brought together in juxtaposition by hybridization to a target. The two ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed DNA circle. In the presence of an excess of primers (forward and reverse primers), a DNA polymerase extends the bound forward primer along the C-probe and displaces the downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to the "rolling circle" replication of bacteriophages in vivo. 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. This ramification process continues until all ssDNAs become double-stranded, resulting in an exponential amplification that distinguishes itself from the previously described nonexponential rolling circle amplification. In this report, we prove the principle of ramification amplification. By using a unique bacteriophage DNA polymerase, Ø29 DNA Polymerase, that has an intrinsic high processivity, we are able to achieve significant amplification within 1 hour at 35°C. In addition, we applied this technique for in situ detection of Epstein-Barr viral sequences in Raji cells.

Original languageEnglish
Pages (from-to)141-150
Number of pages10
JournalMolecular Diagnosis
Volume6
Issue number2
DOIs
StatePublished - 2001
Externally publishedYes

Keywords

  • Circular probe
  • In situ amplification
  • PCR
  • Rolling circle

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