Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a beta-thalassemia fetal mutation in maternal plasma

Abstract

Objective. Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C → T), in maternal plasma DNA.

Methods. The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C → T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples.

Results. Our results demonstrated that this approach to detect a low abundance IVS2 654(C → T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C → T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA.

Conclusions. PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.

Keywords:

• Prenatal diagnosis
• ligase detection reaction
• capillary electrophoresis
• maternal plasma

Acknowledgements

The authors acknowledge Bing Deng (Diagnostic Centre of Molecular Biology, the Affiliated Children's Hospital, Chongqing Medical University) for kindly providing DNA samples. The authors would like to thank Zhong Chen (Cytogenetics Laboratory, Department of Pediatrics, University of Utah School of Medicine) for technical assistance and for revising the paper. This work was supported by the National Natural Science Foundation of China (No. 30672249) and by the National Key Technology R&D Program (No. 2006BA105A05).