2010 Annual Report
Initially, 94 classical U.K. BSE cases and 98 controls were targeted for complete PRNP sequencing (25.2 kb). These data could be used to test PRNP haplotypes for an association with classical BSE incidence. The approach used 24 overlapping PRNP amplicons and a considerable number of forward- and reverse-sequencing primers to cover virtually all of PRNP (25.2 kb). This work produced 33,567 PRNP trace files. However, analysis of the sequenced trace file data revealed two problems. The first was that the quality of DNA, which was extracted from blood samples collected in the field in two batches (the first in the early 1990’s and the second in the mid 1990’s), were of different quality. The second problem was that the performance of some of the oligonucleotides used for either PRNP amplification or sequencing were more robust to DNA quality than others. These two problems resulted in incomplete PRNP sequence coverage for many of the 192 targeted samples.
To circumvent these problems, a high-quality DNA sample set was identified, and PRNP primers were optimized for performance. Within the last year, PRNP sequence coverage gaps for the original 192 samples were filled in. This involved the generation of 1,158 new PRNP amplicons and a total of 8,837 new trace files. Additionally, a complete PRNP sequence was produced for an additional 47 unrelated U.K. animals with different BSE status. A total of 18,528 trace files were produced for the 47 new samples.
The ADODR has maintained contact with collaborators via phone and email.