Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution.
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 beta-galactosidase (beta-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95 degrees C, 60 degrees C and 72 degrees C) reactions of real-time PCR to detect the beta-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.
Sakurai
Takayuki
T
Department of Organ Regeneration, Graduate school of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan. tsakurai@sch.md.shinshu-u.ac.jp
Kamiyoshi
Akiko
A
Watanabe
Satoshi
S
Sato
Masahiro
M
Shindo
Takayuki
T
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification*; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes; D006579:Heterozygote; D006720:Homozygote; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic / Q000235:genetics*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011506:Proteins / Q000235:genetics; D011804:Quinolines; D022661:RNA, Untranslated; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D001616:beta-Galactosidase / Q000235:genetics
D052160:Benzothiazoles; D017931:DNA Primers; D004274:DNA, Recombinant; D003959:Diamines; D005456:Fluorescent Dyes; C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009930:Organic Chemicals; D011506:Proteins; D011804:Quinolines; D022661:RNA, Untranslated; C545506:Ramp2 protein, mouse; C545507:Ramp3 protein, mouse; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; C098022:SYBR Green I; D001616:beta-Galactosidase