High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Alsmadi, Osama; Bornarth, Carole J.; Song, Wanmin; Wisniewski, Michele; Du, Jing; Brockman, Joel; Faruqi, A. Fawad; Hosono, Seiyu; Sun, Zhenyu; Du, Yunpeng; Wu, Xiaohong; Egholm, Michael; Abarzúa, P; Lasken, Roger S.; Driscoll, Mark D.

doi:10.1186/1471-2164-4-21

Cited by 50 publications

(34 citation statements)

References 18 publications

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“…Rolling circle amplification (RCA) was introduced as a rapid and specific tool for molecular diagnostics [4][5][6][7] of microbial agents including fungi. 8,9 The RCA method is discussed in this article using the genus Fonsecaea.…”

Section: Introductionmentioning

confidence: 99%

Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model

Najafzadeh

Sun

Vicente

et al. 2011

Mycoses

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We aimed to describe a rapid and sensitive assay for identification of pathogenic fungi without sequencing. The method of rolling circle amplification (RCA) is presented with species of Fonsecaea, agents of human chromoblastomycosis, as a model. The internal transcribed spacer (ITS) rDNA region of 103 Fonsecaea strains was sequenced and aligned in view of designing three specific padlock probes to be used for the detection of single nucleotide polymorphisms in three Fonsecaea species. The 38 strains included for testing the specificity of RCA comprised 17 isolates of Fonsecaea pedrosoi, 13 of Fonsecaea monophora and eight of Fonsecaea nubica. The assay successfully amplified DNA of the target fungi at the level of species, while no cross reactivity was observed. The amplification product was visualised on a 1% agarose gel to verify the specificity of probe-template binding. Amounts of reagents were minimised to avoid the generation of false-positive results. The simplicity, sensitivity, robustness and low costs provide RCA a distinct position among isothermal techniques for DNA diagnostics as a very practical identification method.

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Section: Introductionmentioning

confidence: 99%

Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model

Najafzadeh

Sun

Vicente

et al. 2011

Mycoses

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“…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)(11)(12). The reaction has not proven useful for sensitive quantitative analysis or for multiplex amplification.…”

mentioning

confidence: 99%

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

Dahl¹,

Banér²,

Gullberg³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

191

163

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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...

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“…When they hybridize, head to tail, to the target, the 5'-and 3'-terminals of the probe are juxtaposed, forming a closed, circular molecule following incubation with a DNA ligase [17]. The ability of padlock probes to accurately identify SNPs has been demonstrated [18][19][20], and the intensity of the signal generated by the circularized probe can be increased, exponentially, by hyperbranched rolling circle amplification (HRCA) (Fig. 1) [21].…”

Section: Introductionmentioning

confidence: 99%

“…1) [21]. This technology has been used for SNP genotyping in human populations [18][19][20], as well as for the detection of human pathogenic viruses [22] and bacteria [23]. In this study, we investigate the use of padlock probes used in combination with HRCA and real-time PCR for highthroughput rapid identification and differentiation of isolates of the Cryptococcus species complex.…”

Section: Introductionmentioning

confidence: 99%

Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex

et al. 2008

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The Cryptococcus species complex contains two closely related basidiomycetous yeasts: Cryptococcus neoformans and C. gattii, which cause cryptococcosis in humans and other animals. The species and varieties are characterized, by different clinical, epidemiological, biochemical and molecular features. The currently used identification methods are either time-consuming or not anymore commercially available. However, a rapid, sensitive and robust assay for the detection of these pathogens is vital for early diagnosis and appropriate treatment decisions. To overcome those limitations, four padlock probes targeting speciesspecific single nucleotide polymorphisms at the internal transcribed spacers (ITSs) of the RNA gene locus were developed and applied during isothermal hyperbranched rolling circle amplification (HRCA). The probes were tested against 99 samples, including 94 clinical cryptococcal cultures, three closely related Cryptococcus species, and two clinical specimens. The use of the padlock probes and the combination of probe signal amplification by HRCA provided a quick and sensitive assay for the accurate identification of C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. HRCA was also useful to detect hybrids, when they were heterozygous at the ITS locus. The HRCA results were in agreement with previous genotyping data based on PCR fingerprinting, amplified fragment length polymorphism and ITS sequencing.

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High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Cited by 50 publications

References 18 publications

Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model

Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model

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

Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex

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