What are the steps involved in multiplex PCR?

Multiplex assays run on the Luminex platform typically consist of three major steps: nucleic acid amplification by PCR, target-specific extension, and liquid bead array decoding (Merante et al., 2007). After PCR amplification, the amplicons are mixed with a second set of tagged primers specific for each target.

What is a multiplex PCR assay?

Multiplex PCR is the simultaneous detection of multiple targets in a single reaction well, with a different pair of primers for each target. This technique requires two or more probes that can be distinguished from each other and detected simultaneously.

How do you do multiplex?

Multiplexing is achieved by using a device called Multiplexer (MUX) that combines n input lines to generate a single output line. Multiplexing follows many-to-one, i.e., n input lines and one output line. Demultiplexing is achieved by using a device called Demultiplexer (DEMUX) available at the receiving end.

How do you make primers for multiplex PCR?

Key primer features

1. PCR primers are generally designed to be 18 – 30 bp in length.
2. The melting temperature (Tm) of the primers should be between 58°C – 60°C, and all primers in the reaction should have a Tm within 0.5 – 1°C of each other.
3. The GC content of the primers should be between 40% and 60%.

How is multiplex PCR different from standard PCR?

In conventional singleplex PCR, a single target is amplified in a single reaction tube. In contrast, multiplex PCR allows for simultaneous amplification of multiple target sequences in a single tube using specific primer sets in combination with probes labeled with spectrally distinct fluorophores.

How do you make a multiplex PCR primer?

During multiplexing, more than one target sequence is amplified by using multiple primer sets in a single PCR reaction….How to Design PCR Primers for Multiplex Assays.

How does multiplex PCR differ from standard PCR?

What is the process of PCR test?

PCR tests work by: Taking a sample of blood, saliva, mucus, or tissue. The sample will contain your own DNA and possibly the DNA of a pathogen or cancer cell. The sample is put in a special machine.

Is multiplex PCR quantitative?

Multiplex and real-time PCR are molecular techniques designed to amplify nucleic acid sequences in a quantitative manner.

What is multiplex PCR?

Multiplex PCR: critical parameters and step-by-step protocol By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory.

What is the required length of primer for multiplex PCR assay?

Multiplex PCR assays involve designing of large number of primers, hence it is required that the designed primer should be of appropriate length. Usually, primers of short length, in the range of 18-22 bases are used.

What is multiplexing assay?

In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment. 1.

What are the important primer design considerations for a successful multiplex reaction?

Design of specific primer sets is essential for a successful multiplex reaction. The important primer design considerations described below are a key to specific amplification with high yield. 1. Primer Length