Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution
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- 作者：Takayuki Sakurai ; Akiko Kamiyoshi ; Satoshi Watanabe ; Masahiro Sato and Takayuki Shindo
- 关键词：Crude DNA solution ; Real ; time PCR ; SYBR Green ; Zygosity ; Transgenic mice ; Gene ; targeted mice
- 刊名：Transgenic Research
- 出版时间：February, 2008
- 出版年：2008
- 期刊代码：118_09628819
- 类别：bio
- 卷：17
- 期：1
- 页码：149-155
- 数据来源：sp
摘要
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掳C, 60掳C and 72掳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.