Numerous innovative and high-throughput techniques have been established to identify human disease genes. However, DNA sequencing of candidate genes still remains as a major limitation in the identification of causative mutations. Much of this limitation is due to the time and labor needed for the polymerase chain reaction (PCR) optimization and reaction setup. Toward this end, we have established a simplified protocol that utilizes a single PCR amplification condition. PCR purification is accomplished via enzymatic digestion and all products can be sequenced using universal primers. This combination of a single amplification condition, single-step purification, and sequencing setup using universal primers all contribute to a simple and high-throughput mutation screen.
