Real-Time PCR Genotyping System

What is the Real-Time PCR Genotyping System?

A real-time PCR genotyping system scores known alleles with fluorescently labelled allele-specific probes across 96 or 384 wells, delivering results in about two hours with minimal set-up. It is the practical choice for laboratories genotyping a small number of markers in many samples.

Overview

The system uses hydrolysis or hybridisation probes whose fluorescence increases only when the probe matches the template, so each allele gives a distinct colour. After cycling, software plots the signal from two channels against each other and assigns samples to homozygous, heterozygous or no-call clusters. A typical plate runs in 60–120 minutes and 384-well formats allow several thousand data points per day. Multiplexing usually covers 2–6 targets per well, and melt-curve analysis with high-resolution dyes can add genotype discrimination without labelled probes. Instruments include four to six optical channels, calibrated LED excitation and interchangeable filter blocks. Because chemistry is inexpensive and consumables are standard plates and tubes, cost per data point is low, but each new marker needs validated primers and a known-genotype reference set.

How does it work?

Genotyping always compares a measured signal with a known allele pattern. Allele-specific real-time PCR uses two probes labelled with different fluorophores that match each allele, and the ratio of signal from the two channels, plotted against a reference, classifies the sample as homozygous, heterozygous or no call. SNP arrays work by hybridising fragmented, amplified DNA to immobilised probes and reading fluorescence intensity for each allele. Capillary fragment analysis amplifies short tandem repeats or microsatellites and sizes the products, so allele lengths are scored from the electropherogram. Targeted sequencing reads the variant positions directly, which allows discovery of unexpected variants and detection of low-frequency alleles. Across all methods, the quality metrics of cluster separation or coverage depth determine which calls are accepted, and laboratories usually include positive and negative controls in each batch.

What are the key features?

• Four to six optical channels for multiplexed allelic discrimination
• 96-well and 384-well block options with fast cycling modes
• Automatic genotype clustering with editable call boundaries
• High-resolution melt analysis for probe-free genotyping
• Positive and no-template control wells defined per plate
• Export of call tables and cluster plots for records

What are the technical specifications?

A typical real-time pcr genotyping system configuration offers optical channels of 4–6, block formats of 96-well and 384-well, run time of approx. 60–120 min, multiplex level of 2–6 targets per reaction, reaction volume of 10–30 µL and temperature uniformity of within ±0.3 °C (typical). Values are indicative of the product class; the exact figures for each configuration are confirmed on the quotation and the calibration documents supplied with the instrument.

Which applications is it used for?

The Real-Time PCR Genotyping System is used in clinical single nucleotide polymorphism testing, crop and livestock marker-assisted selection, genetically modified organism detection and pathogen strain discrimination. Across the wider Genotyping Systems class, laboratories also deploy it for clinical variant and pharmacogenomic testing, crop and livestock marker-assisted selection and cell line authentication and STR profiling.

Which standards and certifications apply?

Relevant standards and methods include ISO 20395, ISO 15189, CLSI MM01, ISO 17025 and IEC 61010-1. Certification candidates for this model are CE, ISO 9001 manufacturing and IEC 61010-1 safety; each is to be confirmed for the specific configuration before shipment, and certificates are supplied only where they have been issued.

Genotyping results depend heavily on DNA quality, so extract samples with a consistent method and check concentration and purity before starting. Real-time PCR genotyping needs validated primers and probes and a thermocycler with calibrated optics; arrays and sequencers need controlled temperature and humidity plus a scanner with consistent gain settings. Run positive controls for each genotype and a no-template control in every batch, and re-run ambiguous calls on an independent method rather than re-reading the same data. Cell line authentication should be repeated whenever a line is re-established or a stock is replaced. In clinical use, genotyping assays fall under ISO 15189 and require documented validation of limit of detection, precision and specificity for each reported genotype.

How do I select the right Real-Time PCR Genotyping System?

Select a real-time PCR genotyping system when you score a limited number of validated markers in many samples and want the lowest cost per data point. Specify optical channel count, block format, multiplex level, melt capability, analysis software and whether the assay design tools include validated primer and probe sets.

How does it compare with other Genotyping Systems variants?

Start with the number of variants and samples. If you score a few known markers in many samples, real-time PCR with allele-specific probes gives the lowest cost per data point and a simple workflow. For hundreds of thousands of markers across a population, arrays provide the highest call rate per sample but need a scanner, and custom arrays have long lead times. Capillary fragment analysis remains the reference for short tandem repeat markers in cell line authentication, forensic work and plant breeding. Targeted sequencing suits panels where you want both known and unexpected variants, or where alleles occur at low frequency in mixed samples. Check multiplexing limits, DNA input, turnaround time, analysis software, genotype-calling algorithm transparency and the ability to re-analyse raw data under your own rules.