October 11-15, 2003
59th Annual Meeting of the American Society for Reproductive Medicine (ASRM)
San Antonio, Texas
Improving the Accuracy of Genetic Diagnosis from Single Cells through Kinetic Analysis of Real-Time PCR Signals
Kenneth E. Pierce, John E. Rice, J. Aquiles Sanchez, and Lawrence J. Wangh
Design: Alleles of cystic fibrosis were analyzed in the Cepheid Smart Cycler, a lower cost real-time thermal cycler. Single human lymphoblasts homozygous/heterozygous for the normal and/or ∆F508 allele of cystic fibrosis were lysed using an optimized proteinase K treatment and the relevant sequences were amplified using a novel real-time PCR method, called Linear-After-The-Exponential (LATE) PCR. This amplification design reduces variation among replicate assays and increases the intensity of fluorescent signals obtained with allele-specific molecular beacons.
Materials and Methods: PCR primers were used at unequal concentrations, the limiting primer having a concentration-adjusted melting temperature greater than that of the excess primer. Under this condition, each reaction makes an abrupt transition from exponential to linear amplification close to the cycle of earliest signal detection, thereafter amplifying the strand detected by the molecular beacons. The resulting signals were analyzed for their time of detection (CT value), rate of fluorescence increase, and final fluorescence. Samples with atypical kinetics were objectively identified as outliers using the ESD statistic.
Results: Analysis of the signals generated by lymphoblasts homozygous for either the normal CF allele or the ∆F508 allele confirmed the specificity of each molecular beacon. We analyzed 106 samples with single lymphoblasts heterozygous for ∆F508, 98 (92%) of which generated at least one fluorescent signal above threshold. In the absence of kinetic analysis 11.2% (11/98) of these heterozygous samples would be scored as homozygous because of allele dropout (ADO). However, 4 of the 11 apparent ADO samples plus 5 heterozygote samples with both signals displayed atypical kinetics as determined by statistical analysis and could therefore be discarded from further consideration, yielding an ADO rate of 7.9% (7/89). Further analysis revealed that 3 of 7 remaining apparent ADO samples generated a signal that was outside the range of all corresponding homozygotes, thereby enabling a further reduction of the ADO rate to 4.7% (4/86). Thus, narrowing the range of signal characteristics acceptable for diagnosis increases the accuracy of the assay without seriously reducing its efficiency. We recently tested a cell lysis protocol that reduces sample handling and may further improve diagnostic accuracy.
Conclusions: We have developed an assay for CF alleles utilizing an improved form of asymmetric PCR that generates linear signals. Analysis of signal kinetics increases diagnostic accuracy, because most samples with ADO do not fit the criteria of homozygous samples. The assay can be carried out in an affordable real-time PCR machine. These are important steps toward introduction of clinically convenient real-time PCR assays for preimplantation genetic diagnosis.