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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