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DNA analysis with multiplex microarray-enhanced PCR (2005)
| Content Provider | CiteSeerX |
|---|---|
| Author | Modi, A. Pemov H. |
| Abstract | 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 30 end of a universal amplification primer. The complex primer pair is covalently tethered through its 50 end to the polyacrylamide 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 solutionphase 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. |
| File Format | |
| Journal | Nucleic Acids Res |
| Language | English |
| Publisher Date | 2005-01-01 |
| Access Restriction | Open |
| Subject Keyword | Multiplex Microarray-enhanced Pcr Dna Analysis Gel Element Array Bulk Solution Gel Element Single Reaction Chamber Conventional Multiplex Pcr 6-fold Amplification Bacillus Subtilis Unattached Universal Primer Polyacrylamide Backbone Multiple Complex Primer Independent Amplification Reaction Sensitive Method Primer Interference Specific On-chip Amplification Bacterial Genomic Dna Pseudo-monoplex Pcr Average Amplification Efficiency Universal Amplification Primer Complex Primer Pair Solution Accelerates Gel Element Microarrays Amplification Strategy Single Pair Reaction Chamber Normal Solutionphase Pcr Single Universal Pcr Primer Pair |
| Content Type | Text |
| Resource Type | Article |
