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Development of a direct DNA sequencing method for detecting heterozygous mutations of the human lipoprotein lipase
1Department of Etiology and Pathophysiology, National Cardiovascular Center Research Institute, Osaka, Japan.
Objective:
The purpose of this study was to develop an improved method of direct DNA sequencing, which makes it possible to identify heterozygous mutations of the lipoprotein lipase (LPL) gene in order to understand the underlying genetic disorder of type IV hyperlipoproteinemia.
Methods And Results:
The direct sequencing method was improved by devising primers for amplifying the LPL gene and for sequencing DNA amplified by the polymerase chain reaction (PCR)T since the reported base sequences of the introns flanking exons of the LPL gene were limited to 40 bases. Improvement was achieved by attaching nine additional bases to both the PCR amplification primer and sequencing primer, and by optimizing the Tm value of the sequencing primers by adjusting the sequence of the nine extra bases. Use of the sequencing primers having suitable Tm values (48 degrees C-58 degrees C) made it possible to reduce nonspecific bands on the sequence ladder pattern and to identify heterozygous mutation sites in LPL gene exons 5 and 6 as model cases.
Conclusion:
Our improved direct sequencing method is useful for identifying heterozygous mutation sites in human LPL gene exons and splicing consensus regions.