PCR multiplex

Lekowska-Kochaniak, A. Czajkowska, D. Popowski, J. Detection of Escherichia coli 0157 H7 in raw meat by immunomagnetic separation and multiplex PCR. Acta Microbiol. Polon. 2002,51, 327-337. 

Microsatellite markers have been extensively used in family studies. These are regions of variation, mostly (CA) repeats, which are distributed throughout the genome. There are potentially as many as 105 loci, each of which has many alleles and these markers are, therefore, highly informative. Microsatellites can be typed by automated multiplex PCR. 

Multiplex PCR a PCR reaction where more than one primer set is included in the reaction pool allowing 2 or more different DNA targets to be amplified by PCR in a single reaction tube. 

Connil, N., Prevost, H., and Dousset, X. (2002). Production of biogenic amines and in cold smoked salmon inoculated with Carnobacterium divergens V41, and specific detection of this strain by multiplex-pcr. J. Appl. Microbiol., 92(4), 611-617. 

Genetic methods Polymerase chain reaction (PCR) and its variants, e.g., multiplex PCR, are widely used for C. botulinum detection. Reverse-transcriptase PCR is recommended as an alternative to biological tests using mice (McGrath et al., 2000). 

Multiplex PCR amplification can apparently tolerate degraded DNA because the size of PCR products generated for subsequent hybridization is 100 50 base pairs. However, the 10 K mapping array and the 100 K and 500 K arrays use a specific restriction enzyme to digest the genomic DNA for subsequent ligation with a specific DNA linker. The DNA linkers then act as binding sites for the specific primer to initiate PCR amplification... 

Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR Critical parameters and step-by-step protocol. Biotechniques 1997 23(3) 504 511. 

For complex samples containing several different DNAs, multiplex PCR has been carried out. For instance, on-chip multiplex PCR was achieved on four DNAs representing regions in the bacteriophage A.DNA (199 and 500 bp), Escherichia coli genomic DNA (346 bp), and E. coli plasmid DNA (410 bp). After PCR, the fluorescent intercalating dye (TO-PRO) was added to the PCR reservoir, and CGE separation was performed downstream [929]. 

Multiplex PCR of the muscular dystrophin gene was performed on-chip. The method of degenerate oligonucleotide-primed PCR (DOP-PCR) was first used to increase the number of DNA templates before multiplex PCR. Therefore, DOP-PCR can provide the template DNA from the whole human genome, but this procedure is feasible only when the amplicon size is less than 250 bp [931]. 

Waters, L.C., Jacobson, S.C., Kroutchinina, N., Khandurina, J., Foote, R.S., Ramsey, J.M., Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. Anal. Chem. 1998, 70, 158-162. 

Jacob RM, Johnstone EC, Neville MJ, Walton RT. Identification of CYP2B6 sequence variants by use of multiplex PCR with allele-specific genotyping. Clin Chem 2004. 

Figure 2 Minisequencing reaction on with extension primers immobilized on the microarray surface. The multiplex PCR products of the regions containing the SNPs are allowed to hybridize to the oligonucleotides immobilized on the microarray (A). The primers are extended with fluorescently labelled ddNTPs complementary to the nucleotide at the SNP position. Four different fluorophores, one for each nucleotide is used allowing for a simultaneous detection of the four nucleotides in one single reaction (B). After washing the slide with sodium hydroxide and salt buffers, only the extended primers covalently attached to the surface remains and the fluorescence is measured (C) and the genotypes assigned.

Shuber AP, Grondin VJ, Klinger KW. A simplified procedure for developing multiplex PCRs. Genome Res 1995 5 488-493. 

Duponchel C, Di Rocco C, Cicardi M, Tosi M. Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the Cl inhibitor gene of hereditary angioedema patients. Hum Mutat 2001 17(1) 61 70. 

Casilli F, Di Rocco ZC, Gad S, Tournier I, Stoppa-Lyonnet D, Frebourg T, Tosi M. Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments. Hum Mutat 2002 20(3) 218-226. 

There are however difficulties in running multiplex PCR, and it is difficult to differentiate PCR products of approximately the same size in a multiplex PCR or PCR-RFLP gel. However DNA microarrays do not have these problems, and have been used to serotype pathogenic strains and proved to be rapid, reliable and sensitive. The approach involves the immobilization of numerous oligonucleotide DNA probes on a solid support to which fluorescence-labeled amplified target DNA is hybridized, and is a powerful tool for the detection of pathogens by virtue of high throughput, speed and sensitivity. 

DNA microarrays have many advantages. The whole operation is easier and more efficient than conventional serological methods or multiplex PCR. In our experience the results are repeatable and give good identification. A drawback is that it takes time to generate the necessary probes, and of course one needs to have the necessary... 

While these examples illustrate diagnosis by identification of a single PCR-amplification product, microdevices have also been used for separation of clinically relevant multiplex amplifications. Duchenne muscular dystrophy (DMD) is caused by a number of mutations within the very large dystrophyn gene. These are identified by PCR amplification of several exon regions of the gene in two multiplex PCR reactions. Separation of these products on microchips has... 

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