Capillary Fragment Analysis Genotyper

What is the Capillary Fragment Analysis Genotyper?

A capillary fragment analysis genotyper sizes fluorescently labelled PCR products to score short tandem repeat and microsatellite markers, and is the reference method for cell line authentication and forensic profiling. It reports allele lengths from 60 to 600 bases with high precision.

Overview

The genotyper amplifies marker regions with fluorescently labelled primers, then separates the products by capillary electrophoresis with an internal size standard in every sample. Software converts migration times to fragment lengths and assigns allele numbers by comparison with an allelic ladder, producing a genotype table and an electropherogram for review. Instruments usually carry 4 to 24 capillaries and process a 96-well plate in 30–90 minutes, with sizing precision better than 0.15 bases for repeat markers. Multiplex kits combine up to 24 markers with different dyes and size ranges so that a full profile comes from one reaction. Because results depend on consistent fragment lengths, the polymer lot, capillary condition and injection parameters should be controlled and monitored with an allelic ladder in every run.

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?

• 4 to 24 capillaries with internal size standard per sample
• Allelic ladder assignment for standardised allele numbering
• Multiplex kits covering up to 24 markers in one reaction
• Sizing precision better than 0.15 bases for repeat markers
• Automatic allele calling with manual review of electropherograms
• Audit trail and project-based sample tracking

What are the technical specifications?

A typical capillary fragment analysis genotyper configuration offers capillaries of 4, 8, 16 or 24 channels, allele size range of approx. 60–600 bp, sizing precision of within ±0.15 bp (typical), markers per multiplex of up to 24 with labelled primers, run time of approx. 30–90 min per plate and sample format of 96-well plate or 8-strip tubes. 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 Capillary Fragment Analysis Genotyper is used in cell line authentication and cross-contamination checks, human identity and forensic profiling, plant variety and seed purity testing and microsatellite marker-assisted breeding. 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, ISO 17025 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 Capillary Fragment Analysis Genotyper?

Select a capillary genotyper for short tandem repeat and microsatellite work where allele length precision and standardised nomenclature matter. Specify capillary count, allele size range, multiplex kit compatibility, ladder availability, analysis software and forensic or accreditation packages if applicable.

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.