Preparation of Copy Standards for PCR Genotyping Sensitivity and Southern Blot Copy Number Determination

PCR screens must be designed to detect transgene DNA at the single copy level or 0.1 copy level. Southern Blots analysis of transgenic mice need copy standards to estimate copy number. Copy standards are prepared by mixing non-transgenic tail DNA with a known amount of transgene DNA is to produce transgene copy standards.

For PCR, these standards can be used to determine the sensitivy of the PCR assay. You must obtain single copy sensitivity in your PCR prior to transgene submission. When you test DNA from potentially transgenic founder mice, run your single copy PCR test sample to ensure that your PCR assay is sensitive enough to avoid false negatives.

Southern Blots are commonly used to determine transgene copy number and the number of integration sites in transgenic founder mice.

View a pdf file illustrating Southern Blot analysis of transgenic founders.

 

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 per 1 ug genomic DNA

transgenic mice will be hemizygous for the transgene, not homozygous

1.83pg transgene should be added to 2 ug of genomic DNA

Thus, to prepare a 1 copy standard: add  1.83 pg of transgene DNA to 2 microgram tail DNA
0.1 copy          0.183 pg
10 copy            18.3 pg
50 copy            91.5  pg
100 copy           183  pg

For use as a transgene PCR standard, use 200 ng of the spiked tail DNA as a substrate in a 25 ul PCR reaction as described: 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.

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