Genotype identification of Math1/LacZ knockout mice based on real-time PCR with SYBR Green I dye.
Knockout mice are widely used in all fields of biomedical research. Determining the genotype of every newborn mouse is a tedious task, usually performed by Southern blot hybridization or Polymerase Chain Reaction (PCR). We describe here a quick and simple genotype identification assay based on real-time PCR and SYBR Green I dye, without using fluorescent primers. The discrimination between the wild type and targeted alleles is based on a PCR design that leads to a different melting temperature for each product. The identification of the genotype is obvious immediately after amplification, and no post-PCR manipulations are needed, reducing cost and time. Therefore, while the real-time PCR amplification increases the sensitivity, the fact that the reactions tubes are never opened after amplification, reduces the risk of contamination and eliminates errors, which are common during the repeated handling of dozens of samples from the same mouse line. The protocol we provide was tested on Math1 knockout mice, but is general, and may be utilized for any knockout line and real-time thermocycler, without any further modification, accessories or special reagents.
Krizhanovsky
Valery
V
Department of Cell and Animal Biology, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Golenser
Esther
E
Ben-Arie
Nissim
N
D000483:Alleles; D000818:Animals; D000831:Animals, Newborn; D051792:Basic Helix-Loop-Helix Proteins; D052160:Benzothiazoles; D001921:Brain / Q000166:cytology / Q000254:growth & development; D004247:DNA / Q000032:analysis / Q000235:genetics; D003959:Diamines; D017930:Genes, Reporter / Q000235:genetics; D005838:Genotype; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics* / Q000254:growth & development; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D009421:Nervous System Malformations / Q000235:genetics*; D009930:Organic Chemicals; D011804:Quinolines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D014157:Transcription Factors / Q000172:deficiency* / Q000235:genetics
D051792:Basic Helix-Loop-Helix Proteins; D052160:Benzothiazoles; D004247:DNA; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D014157:Transcription Factors; C500943:Atoh1 protein, mouse; C098022:SYBR Green I