Preparation of Copy Standards for PCR Genotyping

Preparation of Copy Standards for PCR GenotypingPCR screens must be designed to detect transgene DNA at the single copy level. To demonstrate this level of sensitivity, non-transgenic tail DNA is spiked with a known amount of transgene DNA is to produce transgene copy standards. These standards should be re-run at the time tail DNA samples from potentially transgenic animals are tested.

Calculation of Copy Number Standards

Assumption: the Haploid content of a mammalian genome is 3 X 10⁹ bp
Assumption: you have 2 micrograms of tail DNA available

Since the transgenic founder mice are hemizygous:

 mass of transgene DNA     =     N bp transgene DNA
1 microgram genomic DNA       3 X 10⁹ bp genomic DNA

Example: for a 5,480 bp transgene insert or plasmid

 mass of transgene DNA      =     5,480 bp cloned DNA          or
1 micrograms genomic DNA       3 X 10⁹ bp genomic DNA

mass of transgene DNA = (5,480 bp cloned DNA) X (1 µg genomic DNA)         or
                                                              3 X 10⁹ bp genomic DNA

mass of transgene DNA = 1.83 picograms

Thus, to prepare a 1 copy standard: add  1.83 pg of transgene DNA to 2 microgram tail DNA
              10 copy            18.3 pg
              50 copy            91.5 pg
             100 copy         183 pg

For use as a PCR standard, use the single copy spiked DNA as a substrate to test the PCR assay you devised for genotyping transgenic mice .

For use in Southern blot analysis, digest the tail DNA as you would for Southern analysis, and add the transgene insert DNA (not the entire plasmid) just before you load your gel. Remember to reserve one lane for genomic DNA only with no spike. For an example of copy standards in Southern blots, refer to Camper SA. 1987. Research applications of transgenic mice. Biotechniques 5, 638-650. Click here for more review articles.