Email updates

Keep up to date with the latest news and content from BMC Genetics and BioMed Central.

Open Access Highly Accessed Methodology article

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

Richard R Bennett1*, Hal E Schneider1, Elicia Estrella1, Stephanie Burgess1, Andrew S Cheng2, Caitlin Barrett1, Va Lip2, Poh San Lai3, Yiping Shen2, Bai-Lin Wu2, Basil T Darras4, Alan H Beggs15 and Louis M Kunkel167

Author Affiliations

1 Program in Genomics and Division of Genetics, and The Manton Center for Orphan Disease Research, Children's Hospital Boston, Boston, Massachusetts, USA

2 Department of Laboratory Medicine, Children's Hospital Boston, Boston, Massachusetts, USA, and Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

3 Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

4 Department of Neurology, Children's Hospital Boston, Boston, Massachusetts, USA

5 Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

6 Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

7 Howard Hughes Medical Institute, Children's Hospital Boston, Boston, Massachusetts, USA, and Harvard Medical School, Boston, Massachusetts, USA

For all author emails, please log on.

BMC Genetics 2009, 10:66  doi:10.1186/1471-2156-10-66

Published: 18 October 2009



One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.

These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.

The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput.


An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper.


This automated process allows laboratories to discover DNA variations in a short time and at low cost.