Pentaplex-PCR development with different Reporters & Dyes combination

Introduction

Pentaplex-PCR refers to the detection of five gene target sequences in a single-tube PCR reaction setup. To achieve an efficient pentaplex detection system, oligonucleotides are chemically modified at 5’ and 3’ end with fluorescent dyes and quenchers.  In the multiplexing PCR development program more than one small gene targets can be identified in a single tube efficiently. The diverse dyes and quenchers combination help to quantify specific genes by selection of 5’ Fluorophore Absorbance (nm) and the emission wavelength (nm) and should be kept a minimum enough distance to detect a single gene in real-time PCR.

The most commonly used 5’Flurophore dyes are FAM (6-carboxyfluorescein), HEX (hexachloro-fluorescein), JOE, (4-5-Dichloro carboxy fluorescein) ROX (carboxy-X-rhodamine), Texas Red & Cynine-5 (CY5). The oligonucleotide dye combination appropriately tags with 3’ Black Hole Quenchers to bring the diversity and get an effective signal to differentiate 5 different gene targets while running qRT-PCR.

        (Table: Refer to dye combination for selection of dye and quencher for pentaplex-multiplexing PCR development)

Eurofins Genomics India Pvt Ltd. has > 15 years experience in probe synthesis, synthesizing various scales of pentaplex probes from 5nmole to 100nmole or greater as per the customer requirement. These probes are produced at our Bangalore facility and will be delivered within 5-7WDs. The short Turn Around Time (TAT) will help companies or R&D centers expedite their R&D as well as Kit manufacturing.

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