Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution |
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Authors: | Takayuki Sakurai Akiko Kamiyoshi Satoshi Watanabe Masahiro Sato Takayuki Shindo |
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Institution: | (1) Department of Organ Regeneration, Graduate school of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto Nagano, 390-8621, Japan;(2) Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba Ibaraki, 305-0901, Japan;(3) Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 1-21-20 Korimoto, Kagoshima Kagoshima, 890-0065, Japan |
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Abstract: | We examined whether crude DNA extracts prepared from gene-engineered mouse tissues are suitable as a template for zygosity
determination by SYBR Green real-time genomic PCR. A crude DNA solution was prepared by brief incubation with lysis buffer
containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the β-galactosidase (β-gal) gene. Five serially
diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95°C, 60°C
and 72°C) reactions of real-time PCR to detect the β-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as
an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency
was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined
the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity,
wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg,
B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present
method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. Our method
will be particularly useful for experiments requiring rapid and accurate genotyping of gene-modified animals/fetuses. |
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Keywords: | Crude DNA solution Real-time PCR SYBR Green Zygosity Transgenic mice Gene-targeted mice |
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