High-throughput SNP genotyping by single-tube PCR with Tm-shift primers.

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.

Successful autologous stem cell collection in patients with chronic myeloid leukemia in complete cytogenetic response, with quantitative measurement of BCR-ABL expression in blood, marrow, and apheresis products.

Imatinib mesylate is the initial therapy of choice for chronic myeloid leukemia in chronic phase (CML-CP), but in some patients, the disease becomes resistant to imatinib. Autologous stem cell transplantation using cells collected while in complete cytogenetic response (CCyR) may represent a therapeutic option for these patients. We mobilized and collected autologous CD34(+) stem cells from 20 CML-CP patients in CCyR, 19 of whom were taking imatinib, and measured BCR-ABL expression in the apheresis products, blood and bone marrow using real-time quantitative PCR (RQ-PCR). Stem cells were mobilized with G-CSF 10 microg/kg daily for 5 days. In patients whose initial collection was <2x10(6) CD34(+) cells/kg, G-CSF dose was increased to 10 microg/kg twice daily on the second attempt, and imatinib was held for 14 days if a third attempt was necessary. All 20 patients successfully mobilized the target yield of 2 to 5x10(6) CD34(+) cells/kg; 16 reached target yield with the first mobilization. The median number of CD34(+) cells collected was 4.4 (range, 2.0 - 8.4)x10(6)/kg in a median of 3 (range, 2 - 6) apheresis days. Of 17 patients whose stem cell products were evaluable by RQ-PCR, 11 (65%) had >or=1 daily product with undetectable BCR-ABL; 4 of these (24%) had no detectable BCR-ABL in any apheresis products. BCR-ABL expression in apheresis products was correlated with levels of expression in the blood and marrow prior to mobilization. No patient has yet required transplantation. With median follow-up of 18 months, all patients remain in CCyR and 9 of 16 (54%) have undetectable BCR-ABL in the most recent blood and marrow sample.

Parallel screening and activity profiling with HIV protease inhibitor pharmacophore models.

Parallel Screening has been introduced as an in silico method to predict the potential biological activities of compounds by screening them with a multitude of pharmacophore models. This study presents an early application example employing a Pipeline Pilot-based screening platform for automatic large-scale virtual activity profiling. An extensive set of HIV protease inhibitor pharmacophore models was used to screen a selection of active and inactive compounds. Furthermore, we aimed to address the usually critically eyed point, whether it is possible in a parallel screening system to differentiate between similar molecules/molecules acting on closely related proteins, and therefore we incorporated a collection of other protease inhibitors including aspartic protease inhibitors. The results of the screening experiments show a clear trend toward most extensive retrieval of known active ligands, followed by the general protease inhibitors and lowest recovery of inactive compounds.