Quantifiction of transgenes, homo / heterozygisty
testing
This method is follows in principle the following paper:
Transgenic Res. 2004 Apr;13(2):191-4. Links
Simple method of zygosity identification in transgenic mice by
real-time quantitative PCR.
Shitara H, Sato A, Hayashi J, Mizushima N, Yonekawa H, Taya C.
Laboratory for Transgenic Technology, The Tokyo Metropolitan Institute of Medical
Science, Tokyo 113-8613, Japan. shitara@rinshoken.or.jp
To determine zygosity in transgenic (Tg) mice, a new technology, real-time
quantitative PCR, has recently been introduced in transgenic research to overcome
several drawbacks (time-consuming, specialized techniques and/or ambiguity in the
results) of previously established methods, for example, Southern blot hybridization,
dot blot hybridization, fluorescence in situ hybridization (FISH), etc. However, the
previous real-time quantitative PCR method still possesses several drawbacks, for
example, it needs two sets of primers/probes and the complicated setting up of
appropriate conditions, both of which are expensive and remain time-consuming. We
therefore developed an improved real-time quantitative PCR system for determination
of zygosity, which is easy, rapid and less expensive, because the technique needs only
two experimental processes: estimation of DNA concentration and CYBR Green
PCR. We found that homozygous, hemizygous and non-Tg animals could easily be
distinguished among F1 littermates in crosses of hemizygous EGFP- and DsRed2-Tg
mice. Our improved method will be applicable to any Tg mouse strains, when a
primer set is matched to the corresponding transgene.
We have found that the following modification is cost efficient but increases the signal intensity:
-using Sito9 from Invitrogen instead of SYBRgreen.
Principle
The method works without probes. Any primer pair that is specific for the gene
of interest can be used. The idea is, that the homozygous sample will have
exactly double as much of the gene of interest as the heterozygous sample. In
a PCR reaction double the amount equals logarithmically to 1cycle difference.
Hence the homozygous sample should have a crossing point one cycle earlier
compared to the heterozygous sample.The concept also relies on the fact that
absolutely equal amounts of DNA are brought into the PCR reaction.
Therefore the DNA concentration has to be measured carefully and it has to
be pipetted accurately. As one cycle difference is difficult to determine the
samples have to be run in at least triplicates, ideally 5x or more. The average
of the triplicates (or more) will show the one cycle difference.
Protocol:
-Your sample material should be purified DNA (not crude extracts because of
PCR inhibitors)
-measure the DNA on the Nanodrop. Measure the same sample 3 times!
Form the average of the 3 measurements and dilute to 20ng/ul.
-measure your dilution again and confirm that it is 20ng/ul. Otherwise dilute
again and measure again to confirm final concentration of 20ng/ul.
-set up a Mastermix of your primers, Sito9 and any TAQ, dNTPs, MgCl2 and
buffer (we recommend to use 2x Sensi-dt from Bioline)
-dilute Sito9 (5mM) dye 1:200 (in 10ul DMSO+90ul water) and use 1ul of this
per reaction
1x Sensi-dt
5ul 2x Mastermix
1ul primer for
1ul primer rev
1ul Sito9
1x Immolase
1ul 10xbuffer
1ul dNTPs (2mM)
0.5ul MgCl2 (50mM)
1ul Sito9
0.15ul Taq (immolase 5units/ul)
1ul primer for
1ul primer rev
2.5ul water
- aliquot the Mastermix into all wells
- prepare a no-template-control (NTC) adding 2ul of water
- repeat each sample to be tested 5 times
- repeat each control (if you have) also 5 times
5x 2ul of 20ng/ul
homozygous control
1
2
3
4
5
6
7
A
B
C
D
E
F
G
H
8
9
10
11
12
5x 2ul of 20ng/ul
heterozygous control
5x 2ul of 20ng/ul
sample1
5x 2ul of 20ng/ul
sample 2
5x 2ul of 20ng/ul
sample 4
5x 2ul of 20ng/ul
sample 3
- Run for 40 cycles with annealing temp of your primers in Fam-detection channel
with quantification at 72C (sensi and immolase only need 7 minutes initial denaturing)