DESIGNING RSCA.® REFERENCE MOLECULES TO IMPROVE A LOCUS ALLELE RESOLUTION

7.4
TITLE: DESIGNING RSCA.® REFERENCE MOLECULES TO IMPROVE A LOCUS ALLELE RESOLUTION

Paula Larsen,¹ Daniel Ramon, Mary Parlow,¹ Xiangjun Liu,¹ Lu Wang,¹ Inta Kalve.²

¹Reserach and Development, Pel–Freez Clinical Systems, Brown Deer, WI; ²Sales and Marketing, Pel–Freez Clinical Systems, Brown Deer, WI

RSCA®, a medium–high resolution HLA typing method, detects DNA conformational differences of labeled heteroduplexes separated by native gel electrophoresis. RSCA is not based upon direct nucleotide recognition of allele sequence like SBT, SSP, and SSOP. Parental alleles are independently identified, eliminating cis/trans ambiguities. RSCA ambiguities are encountered when alleles that differ by a single base and the surrounding bases have a similar DNA thermal stability (DG). The number of ambiguous combinations can be reduced significantly by using multiple reference strands. Based upon thermal stability, the least destabilizing reference strand mismatches are G–T, G–G and G–A; whereas C–C and C–T are the most destabilizing. The adjacent nucleotide position, or nearest neighbor, least destabilizing base is G and the most destabilizing base is C. Reference strand candidates with allele specific destabilizing mismatches in addition to a C nearest neighbor would result in the highest DG. This theory was applied when a 3rd reference strand was chosen to resolve the ambiguous combinations with the current HLA–A RSCA kit.

The SSOP SCORE™ program design was applied, using 30bp theoretical probes to identify an HLA A reference strand. The probes surrounded the mismatch between the ambiguous alleles. The best reference candidates were those that were most similar to one of the ambiguous allele sequences. Five alleles were chosen as reference candidates and tested with 11 ambiguous allele pairs. The references were chosen based upon their best overall electrophoretic separation of the ambiguous heteroduplexes. Nine of the 11 pairs resulted in successful separation with two of the five reference candidates. Using this method we demonstrate that one mismatch surrounded by 30bp is sufficient to create a DNA conformation change. In addition, we are able to design reference molecules to resolve RSCA ambiguous combinations and predict which alleles may result in similar DNA thermal stability, DG.

RSCA® is a registered trademark of Pel–Freez Clinical Systems, LLC®, uses patent pending technology and is sold under license from the Anthony Nolan Bone Marrow Trust.