Genotyping of Transgenic Animals by Real-Time Quantitative PCR with TaqMan Probes

Abstract

Real-time quantitative PCR (qPCR) is a fast, sensitive, specific, and quantitative method for genotyping transgenic animals. Accurate quantitation of the number of transgenes helps to identify founders and to create and maintain pure lines of transgenic mice, thus reducing experimental variability. Here we describe an accurate method of genotyping using real-time quantitative PCR with primers and MGB TaqMan probes from Life Technologies. The first step in quantitating copy number is isolation of genomic DNA. To accurately compare the copies per genome (c/g) of a transgene in different mice, genomic DNA must be prepared by the same method for all the mice, with sample DNA and calibration standards dissolved in the same buffer. This chapter describes several “tried and true” methods, including an automatic system that isolates 16 samples at once in just 35–45 min, yielding DNA of excellent quality. Next, genomic DNA must be quantitated accurately so that similar amounts of DNA are added to each well. A fluorescent assay that is selective for dsDNA over RNA circumvents interference from RNA contamination and ensures more accurate DNA quantitation than A₂₆₀ measurements. It is also very important to use appropriate calibration standards for accurate quantitation of transgene copy number. The best calibrator is the DNA fragment used for microinjection, mixed with normal mouse DNA in such a way that the transgene is present in a range of concentrations spanning the expected copy number in the transgenic mice. This chapter provides guidelines and sample calculations for preparing calibration standards that will accurately reflect the number of transgenes in the mice being tested. Finally, guidelines for preparing primers and TaqMan probes and techniques to prepare and run a 384-well plate smoothly and without errors are presented.