DNA analysis with multiplex microarray-enhanced PCR
We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3' end of a universal amplification primer. The complex primer pair is covalently tethered through its 5' end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 10(6)-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 10(3) genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.
Detection of genetically modified crops by combination of multiplex PCR and low-density DNA microarray.
OBJECTIVE To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. METHODS Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to t...متن کامل
An intelligent primer design system for multiplex reverse transcription polymerase chain reaction and complementary DNA microarray
Various biological and clinical applications require the quantification of the messenger RNA (mRNA) abundance of the biological materials under investigation. Reverse transcription-polymerase chain reaction (RT-PCR) is a popular technique for mRNA quantification. However, in these applications, multiple quantifications of mRNA are required from the same sample. Multiplex RT-PCR uses multiple pr...متن کامل
Detection of sexually transmitted infection and human papillomavirus in negative cytology by multiplex-PCR
BACKGROUND The aim of this study was to determine the prevalence of human papillomavirus (HPV) and 15 species that cause sexually transmitted infections (STIs) in negative cytology. In addition, we compared the diagnostic performance of multiplex polymerase chain reaction (PCR) with widely available techniques used to detect HPV. METHODS We recruited 235 women of reproductive age who had nega...متن کامل
Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray.
In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for scr...متن کامل
Development and validation of a multiplex PCR-based DNA microarray hybridisation method for detecting bacterial antibiotic resistance genes in cheese
18 19 The aim of this study was to develop a method for detecting antibiotic resistance 20 (AR) genes in cheese based on a combination of multiplex PCR and a DNA microarray 21 hybridisation system. Twenty oligonucleotide probes were designed targeting 10 22 common AR genes, namely aac(6’)-Ie-aph(2’’)-Ia, aadE, aphA-3, ermB, tet(L), tet(M), 23 tet(O), tet(S), vanA and vanB. Specificity of the pr...متن کامل