MuPlex: multi-objective multiplex PCR assay design

Rachlin, John; Ding, Chunming; Cantor, Charles R.; Kasif, Simon

doi:10.1093/nar/gki377

Cited by 81 publications

(60 citation statements)

References 9 publications

(7 reference statements)

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“…We gave methodological information in detail to allow other investigators to use these panels, to use individual genotypes of the parental populations in other admixture studies, or to follow the same steps to design additional panels of AIMs more suitable for specific populations to obtain more accurate estimates of admixture. Specifically, we recommend the use of the Muplex resource (Rachlin et al, 2005) to design primers for multiplex amplification and subsequent genotyping. Muplex may also be used to design multiplex panels of insertiondeletion, that have proven to be cost-effective markers for admixture and population structure studies (Bastos-Rodrigues et al, 2006;Santos et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…These SNPs were pre-selected by avoiding physical proximity in the human genome. We assessed compatibility for multiplex polymerase chain reaction (PCR) amplification using the Muplex resource (Rachlin et al, 2005), which is a convenient webenabled system that, starting from a set of targeted sequences, automatically designs sub-sets of primers that will likely co-amplify in multiplex PCR assays under a number of conditions imposed by the investigator. After applying these criteria, we selected the following two SNP panels to be tested experimentally: AFR (Africans) (rs2697520, rs8035530, rs1372115, rs2789823, rs241679, rs7512316, rs9626698, rs1443985, rs6046024, rs735480) and AMR (Native Americans) (rs8058694, rs691968, rs2234636, rs3760657, rs2619681, rs2569190, rs800292, rs2518967, rs2088102, rs700518).…”

Section: Selection Of Aims For the Two Panelsmentioning

confidence: 99%

“…We imposed on the Muplex (Rachlin et al, 2005) the condition that the minimum difference between PCR product lengths within each panel had to be 10 bp. This allows us, during the set up period of the multiplex PCR, to better evaluate if all primers are properly working, using polyacrylamide gel electrophoresis.…”

Section: Enzymatic Purification Of Pcr and Mini-sequencing Productsmentioning

confidence: 99%

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Development of two multiplex mini-sequencing panels of ancestry informative SNPs for studies in Latin Americans: an application to populations of the State of Minas Gerais (Brazil)

et al. 2010

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ABSTRACT. Admixture occurs when individuals from parental populations that have been isolated for hundreds of generations form a new hybrid population. Currently, interest in measuring biogeographic ancestry has spread from anthropology to forensic sciences, direct-to-consumers personal genomics, and civil rights issues of minorities, and it is critical for genetic epidemiology studies of admixed populations. Markers with highly differentiated frequencies among human populations are informative of ancestry and are called ancestry informative markers (AIMs). For tri-hybrid Latin American populations, ancestry information is required for Africans, Europeans and Native Americans. We developed two multiplex panels of AIMs (for 14 SNPs) to be genotyped by two mini-sequencing reactions, suitable for investigators of medium-small laboratories to estimate admixture of Latin American populations. We tested the performance of these AIMs by comparing results obtained with our 14 AIMs with those obtained using 108 AIMs genotyped in the same individuals, for which DNA samples is available for other investigators. We emphasize that this type of comparison should be made when new admixture/population structure panels are developed. At the population level, our 14 AIMs were useful to estimate European admixture, though they overestimated African admixture and underestimated Native American admixture. Combined with more AIMs, our panel could be used to infer individual admixture. We used our panel to infer the pattern of admixture in two urban populations (Montes Claros and Manhuaçu) of the State of Minas Gerais (southeastern Brazil), obtaining a snapshot of their genetic structure in the context of their demographic history.

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Section: Discussionmentioning

confidence: 99%

Section: Selection Of Aims For the Two Panelsmentioning

confidence: 99%

Section: Enzymatic Purification Of Pcr and Mini-sequencing Productsmentioning

confidence: 99%

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Development of two multiplex mini-sequencing panels of ancestry informative SNPs for studies in Latin Americans: an application to populations of the State of Minas Gerais (Brazil)

et al. 2010

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“…A database of 18 CPS loci was created to identify unique regions of each serotype, and PCR primers were designed using the online software MuPlex MultiObjective multiplex PCR (40). Primers were designed with the following parameters: length between 18 and 30 residues, 20 to 50% GC content, and melting temperature ranging from 57 to 63°C.…”

Section: Methodsmentioning

confidence: 99%

Discrimination of Major Capsular Types of Campylobacter jejuni by Multiplex PCR

et al. 2011

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The polysaccharide capsule (CPS) of Campylobacter jejuni is the major serodeterminant of the Penner serotyping scheme. There are 47 Penner serotypes of C. jejuni, 22 of which fall into complexes of related serotypes. A multiplex PCR method for determination of capsule types of Campylobacter jejuni which is simpler and more affordable than classical Penner typing was developed. Primers specific for each capsule type were designed on the basis of a database of gene sequences from the variable capsule loci of 8 strains of major serotypes sequenced in this study and 10 published sequences of other serotypes. DNA sequence analysis revealed a mosaic nature of the capsule loci, suggesting reassortment of genes by horizontal transfer, and demonstrated a high degree of conservation of genes within Penner complexes. The multiplex PCR can distinguish 17 individual serotypes in two PCRs with sensitivities and specificities ranging from 90 to 100% using 244 strains of known Penner type.

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“…Although multiplex PCR requires that reactions be optimized for each set of targets, a variety of strategies exist to optimize multiplex PCR reactions (Elnifro et al, 2000;Schoske et al, 2003). In addition, programs to support the design of multiplex PCR reactions (Rachlin et al, 2005) have become available.…”

Section: The Challenge To Overcome Environmental Molecular Diversitymentioning

confidence: 99%

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

Baums

Goodwin

Kiesling

et al. 2007

Marine Pollution Bulletin

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Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex ® 100™ system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli & Shigella spp., B. distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.

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MuPlex: multi-objective multiplex PCR assay design

Cited by 81 publications

References 9 publications

Development of two multiplex mini-sequencing panels of ancestry informative SNPs for studies in Latin Americans: an application to populations of the State of Minas Gerais (Brazil)

Development of two multiplex mini-sequencing panels of ancestry informative SNPs for studies in Latin Americans: an application to populations of the State of Minas Gerais (Brazil)

Discrimination of Major Capsular Types of Campylobacter jejuni by Multiplex PCR

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

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