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      ANNEALING TEMPERATURE OF POLYMERASE CHAIN
       REACTION IS IMPORTANT FOR THE DETECTION OF
             HETEROZYGOUS GENE MUTATIONS
                      A. Takagi1, Y. Ikeda1, A. Yamamoto1,2
      1
          National Cardiovascular Center Research Institute, Suita, Osaka,
                2
                  Hyogo College of Medicine, Kobe, Hyogo, Japan
Objective: We reported that mild hypertriglyceridemia (type IV
hyperlipoproteinemia) results from superimposition of triglyceride
synthesis-stimulating factors such as a hyperinsulinemic state and/or a high
alcohol intake on a genetic state of a heterozygous mutation in the
lipoprotein lipase (LPL) gene. Identification of heterozygous LPL gene
mutations as an early diagnosis, therefore, is important for preventing the
development of hypertriglyceridemia and subsequent development of heart
disease by getting the patient to change to a more healthful lifestyle. We
aimed to develop and evaluate a PCR method for the amplification of
heterozygous LPL gene mutations. Methods: Subjects A and B with a
heterozygote of Y61X (T-438-A) of exon 3 in the LPL gene were studied.
PCR amplification of both alleles was performed at various annealing temp
of 45°C to 65°C. The 438-T normal allele (+) and the 438-A mutant allele
(–) were discriminated by digestion of PCR-products with EcoT22I, where
(+) and (–) mean that DNA is “digested” and “not digested”. Results: PCR-
RFLP assay at an annealing temp of 55°C revealed a heterozygote of the
438-T and –A alleles for the heterozygote of Y61X, but showed a
homozygote of the 438-A allele at an annealing temp of 65°C. This was due
to a SNP (Int 3-5’ a(21)c) in the primer annealing site of the 438-T normal
allele. Also, variation for the magnitude of amplification of normal and
mutant alleles at various annealing temp was observed in heterozygotes of
Int 3-3’ c(-6)t (LPLosaka). Conclusion: PCR amplification of DNA should
be done using a relatively low annealing temp for the detection of
heterozygous gene mutations, if nonspecific PCR-products are not detected
at its temp.

				
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posted:11/18/2011
language:English
pages:2