Circle-to-circle amplification for precise and sensitive DNA analysis

Abstract

We present a tightly controlled process for strand-specific amplification of circularized DNA molecules. Tandem repeated complements of DNA circles are generated by rolling-circle replication, and converted to monomer circles of opposite polarity to that of the starting material. These circles are then subjected to one more round of rolling-circle replication and circularization, and the process can be further repeated. The method can be directed to produce singlestranded circular or linear monomers, or linear concatemers of the desired polarity. The reaction is not product inhibited, and can yield Ϸ100-fold higher concentrations of monomer products than PCR. Each generation of the amplification process proceeds in a linear fashion, ensuring precise quantification. The procedure is suitable for parallel amplification of large numbers of DNA circles, because the few cycles and the robust reaction mechanism preserves the proportion of amplified molecules. We demonstrate the utility of the method for multiplexed genotyping of polymorphic loci and for quantitative DNA analysis. Circularized DNA strands can be formed in highly specific detection reactions using so-called padlock probes (1). These unimolecular, dual-recognition ligation probes allow analysis of single-nucleotide variation directly in unamplified genomic DNA samples (2, 3), and the probe design permits multiplexed genotyping of thousands of loci in individual reactions (4, 5). Reacted probes can be amplified by PCR, or the DNA circles can be replicated by rolling-circle replication (RCR) (2, 6-8). RCR proceeds in a linear fashion, and the highly processive ⌽29 DNA polymerase (9) can copy a 100-nt circular probe into a DNA strand containing Ϸ1,000 complements of the circularized molecule in 1 h (8). The mechanism is promising for detection of circularized DNA probes, but the limited amplification rate has proven insufficient for many applications. By including a second primer, complementary to the RCR product, these products are in turn replicated by a strand-displacement mechanism, and the process can yield a billionfold amplification in an isothermal reaction of Ͻ1 h (2). The amplification kinetics of this so-called hyperbranched rolling-circle amplification technique is difficult to control and often produces circle-independent amplification artifacts (10-12). The reaction has not proven useful for sensitive quantitative analysis or for multiplex amplification.We demonstrate here that a billionfold or greater amplification of reacted probes is possible in a highly controlled RCR-based process that we call circle-to-circle amplification (C2CA). We characterize the mechanism and investigate its suitability for applications like parallel genotyping of sets of loci and for quantification of specific DNA sequences. Materials and MethodsOligonucleotides. Oligonucleotide sequences are shown in Table 1. The polarity of the padlock probes is referred to as (ϩ), and that of their complements as (Ϫ), and we identify the polarities of molecules by the appropriat...

Cytokine detection by antibody-based proximity ligation

“…The precision of proximity ligation is currently at the level of real-time PCR detection, but improved quantitative detection strategies for nucleic acids may offer a further increase in precision (16)(17)(18). Multiplexed detection is the goal for many technologies under development, especially for antibody-based microarrays.…”

DNA Memory and Input/Output

“…Processing of the resulting double-stranded DNA concatemers into monomeric DNA circles occurs by homologous recombination at Lox sites catalyzed by Cre recombinase (Sauer, 2002). This approach has advantages over existing rolling-circle (Dahl et al, 2004) or PCR (Mitra and Church, 1999) replication methods since it requires neither solid phase oligo synthesis nor changes in temperature, and is far simpler than natural DNA replication systems (Khan, 1997).…”

“…First, a simpler version could be tested in which the T7 RNA polymerase and RNA processing are substituted by addition of short RNA primers. The efficiency of synthesis of monomeric DNA circles would be followed by gel electrophoresis (Dahl et al, 2004), and replication fidelity at the base pair and whole genome levels should be tested with different polymerases. (Lewin, 2004) or the single polypeptide enzyme encoded by coliphage T7 (Studier et al, 1990) seem best, with the choice influenced by several considerations that also determine possible modes of regulation.…”

Microchip Electrophoresis for Detection of Circle-to-Circle Amplification Products towards Sensitive and Rapid DNA Analysis

“…5 Circle-to-circle amplification (C2CA) is a precise and sensitive technique for signal amplification of circularized padlock probes (Figure 1b). 6 Techniques for detection of C2CA have far been limited to microarrays, real time monitoring using molecular beacons, denaturing acrylamide-gel electrophoresis and single-molecule detection. 6,7 Although all those techniques are quite accurate, they are not ideally suited for multiplex medical diagnostics.…”

“…6 Techniques for detection of C2CA have far been limited to microarrays, real time monitoring using molecular beacons, denaturing acrylamide-gel electrophoresis and single-molecule detection. 6,7 Although all those techniques are quite accurate, they are not ideally suited for multiplex medical diagnostics. Although detection based on molecular beacons and singlemolecule detection is quite rapid and no post-amplification process is required, both strategies require design and synthesis of additional probes for labeling, and the multiplexing capacity is limited by the number of spectrally separable fluorophores.…”