Sequence-specific primers (SSP) are assays that bind to and amplify polymorphic regions. SSPs are broken down into individual loci (A, B, C, DP, DQ, and DR). SSPs are sold in kits and usually come embedded in individual 24 to 96 well plates including controls. Primers, buffer, and Taq polymerase are pooled together and added to the negative control for QC.
General Procedure:
- DNA is extracted from whole blood or another applicable body source via manual or automated methods.
- The DNA is quantitated and assessed for quality with OD ratio assessment.
- A DNA aliquot from the patient is added to the cocktail mix of primers, buffers, and Taq polymerase.
- The cocktail mix is aliquoted into the individual wells on a microtiter plate and placed on a thermocycler for polymerase chain reaction (PCR).
- Typically, SSPs are analyzed via electrophoresis as DNA products migrate through the gel based on size and charge.
- Bands are detected and compared to a panel of expected reactions to determine HLA typing as provided by the manufacturer.
Advantages of SSPs include:
- Commercially available
- Easy to setup
- Reduced turnaround time (TAT) for STAT orders
Disadvantages of SSPs include:
- New alleles may not be amplified due to the stringency of the assay
- Primer dimer formation (primers that are used for PCR fold and anneal to each other, which leads to no amplification and detectable products in the gel electrophoresis)
