Multiplex reaction

In the original ASA protocols, the mutant and normal PCR primers were separated into two reactions, so that lack of amplification could occur in one PCR reaction depending on the sequence present in a test sample. This is not ideal for a diagnostic test due to the possible misinterpretation of a false negative result, and the ASA protocol is usually modified to be a multiplex reaction that includes a positive internal control in each PCR reaction. For example, an ASA assay has been developed, which detects 12 common CFTR mutations simultaneously (17). However in this assay, two reactions must still be run in parallel for every sample to be analyzed, since the mutant and normal products produced are the same size and so must be physically separated in order to be distinguished. 

The advantage of the ASA method is that multiplex reactions to detect several mutations simultaneously can be developed. Multiplex reactions... 

III) Allele specific hybridisation (Figure 2) Fragments amplified in an allele unspecific PCR are measured with allele specific probes (Molecular Beacons, TaqMan probes or Scorpion), which can either be used in a single PCR or in multiplex reactions for detection of both alleles in one reaction [13]. This method has minor background problems, however the dynamic range may be limited due to competition of the probes. The Molecular Beacon proach has been developed for B. graminis and M. fijiensis [13]. 

In the Injection area sel ct Injection Type ALL. This injection type gives better intensity. In the Default Injection Volume enter 8 pL for both single and multiplex reactions. For pooled samples, enter 16 pL. In the Feed Volume, that is, the volnme of syringe wash solution injected into the flow path, enter 25 pL when the Injection Type is ALL. 

The PCR is carried out in tv o reactions vith one FP and RP or as multiplex reaction with all three primers. The size of the generated DNA fragments varies depending on the location of the breakpoint and each neoplastic clone generates a different DNA fragment (usually shorter than 500bp in MBR and more than 600 bp in MCR). 

The PCR is carried out in two reactions with one FP and RP or in one multiplex reaction with all three primers. 

BIOMED-2 primers for the detection of the lambda light chain (Ig X) gene rearrangement. The multiplex reaction is carried out with both V X foreword primers (FR) and the J X reverse primer (RP) ... 

In an earlier section, some consideration was given to the criteria for balancing a reversible pair of reactions. One of the simplifying effects of working with high-temperature flames (1800 K) is that the multiplex reaction mechanisms proposed to account for oxidation kinetics around 400 K are reduced to a few, balanced, bimolecular exchanges, which form the propagation steps of the chain reaction at low temperature. 

The ability to multiplex reactions is of great importance for reducing the cost per SNP genotype and analysis time. Multiplexing at the PCR level and of the primer extension reaction is currently under investigation. The quality of the enzymatic reactions in a multiplex assay depends on the SNPs that have to be combined. The surrounding DNA sequences have an profound influence on the quality of the PCR reaction. 

Minimze the total amount of time and handling required to perform a multiplexed reaction. 

The miniaturization of microwell plates has been an important design objective because of the limited availability of candidate compounds, which are often archival. Until recently, 96-well micro plates containing a two dimensional 12 x 8 matrix of multiplexed reaction... 

---