HLA Sequencing Workflow
A key to achieving 100% resolution, 100% of the time, is a comprehensive human leukocyte antigen sequence-based typing (HLA SBT) workflow, powered by one of the most accurate methodologies, Sanger sequencing, utilizing Life Technologies SeCore® Kits.
One of the tools many HLA labs depend upon for easy, fast, and accurate DNA isolation is the iPrep™ Purification instrument. Designed with a streamlined, easy-to-automate workflow, the iPrep™ instrument can help improve the overall yield, concentration, and purity of DNA isolated from both routine and challenging samples. Employing the iPrep™ instrument in this important first step helps labs achieve excellent data quality and more accurate analysis of the results.
Compared to other HLA typing methodologies, DNA input requirements for SBT are significantly lower.
The iPrep™ Purification Instrument can make your sample processing faster and easier, helping to significantly increase reliability
- Easy to use—just load the reagent cartridges, add samples, and select a protocol
- Fast—process up to 12 samples, typically in less than 30 minutes
- Accurate—enables sample integrity with prefilled, sealed reagent cartridges
- Flexible—works well with both RNA and DNA samples
Amplify with SeCore® HLA SBT Kits and Veriti® Thermal Cycler
SeCore® HLA SBT kits are available for Class I (loci: A, B, and C) and Class II (loci: DRB1, DQB1, and DPB1). Each kit supplies all the necessary reagents and protocols for achieving high-resolution results utilizing a capillary-electrophoresis (CE) instrument.
- Pipette Amp Mix with FastStart™ Taq (included in SeCore® kit) and DNA sample into tubes or strip tubes (according to Class I or Class II protocol)
- Load into thermal cycler (Note: all loci use the same thermal cycling program)
This step removes excess PCR primer and dNTPs to help ensure clean sequence data.
- Run a gel to confirm amplification
- Add ExoSAP-IT® enzyme to all of PCR products (all are included in the SeCore® kit)
Cycle sequencing with Veriti® Thermal Cycler
Fast ramp rates in the Veriti® Thermal Cycler shorten the time it takes to the instrument to achieve set temperatures, reducing the overall time needed for this step.
Veriti® Thermal Cycler Features
- Reduced PCR reaction time when using faster ramp rates
- Easy-to-use graphical interface
- Precise temperature control is provided by 6 separate Peltier blocks—VeriFlex™ Blocks—for convenient PCR optimization that can be utilized in other protocolsStandard and Fast-enabled cycling to address current and future PCR needs
- Standard and Fast-enabled cycling to address current and future PCR needs
- Using ethanol, precipitate the DNA to remove residual dye
The Applied Biosystems® 3500xL Genetic Analyzer is designed to provide the highest level of performance to deliver superior data quality. For HLA typing, Sanger sequencing is the gold-standard technology.
3500xL Series Genetic Analyzer Features
- Excellent data quality with long-read sequence (300 bases) in approximately 30 minutes
- Powerful, integrated data collection and primary analysis software provides real-time assessment of data quality
- Easy-to-run, easy-to-own sequencer
- Intuitive dashboard design simplifies setup, operation, and maintenance for all users
- New single-line, 505 nm solid-state long-life laser utilizes a standard power supply and requires no heat-removal ducting
- RFID-tagged, snap-in-and-run consumables pouches give you trackable and auditable reagent status and usage
Designed to make SBT HLA analysis easy and fast, uTYPE® HLA Software utilizes proprietary algorithms to translate sequencing data into an intuitively guided analysis. The feature-rich software provides a multitude of options for customized viewing along with built-in databases and codes. These features and flexibility, along with a layered review system, allow for a more streamlined analysis process with the ability to track analysis by technician and supervisor. The software is updated on a quarterly basis so labs are continually typing to the most current IMGT® database release.
Webinars for HLA Labs
For Research Use Only. Not for Use in Diagnostic Procedures