Ramification amplification: a novel isothermal DNA amplification method.
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 degrees C. In addition, we applied this technique for in situ detection of Epstein-Barr viral sequences in Raji cells.
Zhang
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Department of Pathology, Mount Sinai School of Medicine, New York University, One Gustave Levy Place, New York, NY 10021, USA. David.Zhang@Mountsinai.org
Brandwein
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Hsuih
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Li
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D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005821:Genetic Techniques; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D008280:Magnetics; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
D004247:DNA; D004259:DNA-Directed DNA Polymerase