L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

Qi, Xinxin

doi:10.1093/nar/29.22.e116

Cited by 104 publications

(41 citation statements)

References 30 publications

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“…Importantly, the ease of performance, rapid turnaround, and multiplex capacity indicate its potential for high-throughput routine screening of AFB-positive specimens or M. tuberculosis isolates, with significant clinical benefits. Formation of the circular probe molecule, mediated by DNA ligase, confers very high allele discriminatory ability (22,23). As a result, the signal generated is highly specific and easy to interpret, in contrast to many other probebased methods in which the result depends on a subjective assessment of spot intensity.…”

Section: Discussionmentioning

confidence: 99%

Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

et al. 2014

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e Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources. With one-third of the world's population infected, tuberculosis (TB) is a major public health threat worldwide (1). The two most important drugs used to treat TB are isoniazid (INH) and rifampin (RIF). Inadequate or inappropriate therapy, together with long and costly treatment courses, often result in the emergence of drug resistance (2). Multidrug-resistant (MDR) Mycobacterium tuberculosis strains-defined as bacillary resistance to at least INH and RIF-have spread rapidly and become a global health emergency (3). Identification of these strains by drug susceptibility testing (DST) allows optimization of therapeutic efficacy and improved treatment outcomes while minimizing transmission of drug-resistant strains. However, phenotypic DST involving subculture is costly, it is too slow to guide optimal management of MDR-TB, and it lacks high-throughput capability. Commercial systems for rapid molecular identification of RIF resistance are available but expensive.RIF resistance in M. tuberculosis is associated with amino acid changes in the ␤-subunit of RNA polymerase, encoded by rpoB. The majority (90 to 95%) of mutations responsible for resistance are located in the 81-bp RIF resistance determining region (RRDR) of rpoB (4-6), and ca. 60 to 70% are found within two codons, 531 and 526. The fact that most mutations known to confer resistance are localized to a specific region facilitates the development of molecular methods for their detection. Sequencing and hybridization-based assays have been commonly used and can define the precise mutations involved (4, 7). Hybridization-based assays also provide a rapid...

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Section: Discussionmentioning

confidence: 99%

Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

et al. 2014

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“…As a result of this worldwide intensive effort, more than 2.8 million SNPs have been identified and a high-density map has been constructed in some cases (Iida et al 2001a-d;Iwasaki et al 2001;Saito et al 2001;Osier et al 2001). Although several mapping methods (Table 2), such as single-strand conformational polymorphism (Orita et al 1989), denaturing gradient gel electrophoresis (DGGE), enzymatic mutation detection (Youil et al 1995), microarray or variant detector arrays (Wang et al 1998;Marshall and Hodgson 1998;Ramsay 1998;Dong et al 2001;Qi et al 2001;Yoshino et al 2001), and heteroduplex analysis (Lichten and Fox 1983) are available, so far none of them has supplanted DNA sequencing as the method of choice. SNP mapping requires a tremendous amount of time and resources (hundreds and thousands of individuals must be studied to eliminate false-positive and false-negative results).…”

Section: Mapping and Characterization Of Snpsmentioning

confidence: 99%

SNP alleles in human disease and evolution

Shastry¹

2002

J Hum Genet

330

176

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information and how to take advantage of this effort. The majority of them believe that one possible benefit of this information is to use it to understand the genetic basis of the most common familial traits, evolutionary processes, and complex and common diseases such as hypertension, diabetes, obesity, and psychiatric disorders. These common diseases are likely to be caused by multiple genes and multiple nongenetic factors (environmental factors), each contributing a modest effect. Their cumulative effect results in the condition or trait. The traditional methods of identifying disease-related genes are not readily applicable to the detection of genes responsible for these multifactorial diseases. The availability of the entire genome sequence, therefore, may speed up the gene-hunting efforts in the near future, but what approaches are to be taken to accomplish this task and what are their limitations?The human genome and the discovery of singlenucleotide polymorphisms (SNPs) as genetic markersIn two randomly selected human genomes, 99.9% of the DNA sequence is identical. The remaining 0.1% is thought to include some differences or variations in the genome between individuals. This variation, called polymorphism, arises because of mutations. The simplest form of these variations is the substitution of one single nucleotide for another (Fig. 1A), termed SNP. SNPs are more common than other types of polymorphisms and occur at a frequency of approximately 1 in 1000 base pairs (Brookes 1999) throughout the genome (promoter region, coding sequences, and intronic sequences). These simple changes in DNA sequence, most of which are probably located in intergenic spacers, are believed to be stable and not deleterious to organisms. SNPs that do not change encoded amino acids are called synonymous and are not subject to natural selection (Kimura 1983, snp.cshl.org). On the other hand, nonsynonymous SNPs alter amino acids and might be subject to natural selection. SNPs can be observed between individuals in a population, may influence promoter activity B.S. Shastry (*) Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA Tel. ϩ1-248-370-3577; Fax ϩ1-248-370-4225 e-mail: Barkur@aol.com Abstract In two randomly selected human genomes, 99.9% of the DNA sequence is identical. The remaining 0.1% of DNA contains sequence variations. The most common type of such variation is called a single-nucleotide polymorphism, or SNP. SNPs are highly abundant, stable, and distributed throughout the genome. These variations are associated with diversity in the population, individuality, susceptibility to diseases, and individual response to medicine. Recently, it has been suggested that SNPs can be used for homogeneity testing and pharmacogenetic studies and to identify and map complex, common diseases such as high blood pressure, diabetes, and heart disease. Consistent with this proposal is the identification of the patterns of SNPs in conditions such as diabetes, schizophrenia, and bloodpressure homeostasis. Alt...

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“…Almost all techniques use the polymerase chain reaction (PCR) for amplification, albeit other strategies could be used. 8,9 In brief, PCR works by using heat to separate a duplex DNA molecule into two single-stranded molecules and then copying each of the two single-stranded molecules with thermostable DNA polymerases and an oligonucleotide primer. When the DNA polymerases finish copying the single stranded molecules, each becomes a new duplex DNA molecule.…”

Section: Target Dna Amplificationsmentioning

confidence: 99%

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

2003

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The large number of single nucleotide polymorphism (SNP) markers available in the public databases makes studies of association and fine mapping of disease loci very practical. To provide information for researchers who do not follow SNP genotyping technologies but need to use them for their research, we review here recent developments in the fields. We start with a general description of SNP typing protocols and follow this with a summary of current methods for each step of the protocol and point out the unique features and weaknesses of these techniques as well as comparing the cost and throughput structures of the technologies. Finally, we describe some popular techniques and the applications that are suitable for these techniques.

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L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

Cited by 104 publications

References 30 publications

Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

SNP alleles in human disease and evolution

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

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