( Using TaqPlus Long DNA...)Easily Amplify Genomic DNA with Long-Distance PCR

Using TaqPlus Long DNA polymerase to genotype mice

Evgeny Loukianov Tanya Loukianova Muthu Periasamy
Cardiovascular Research Center, University of Cincinnati, Cincinnati, OH

We developed a simple, reliable procedure to genotype mice using long-distance PCR, whereby genomic DNA is isolated from mouse ear clips, and large DNA fragments are amplified using TaqPlus Long DNA polymerase.*^,,

PCR is the method of choice for screening transgenic mice¹ because it is fast, allows a large number of samples to be screened at one time, is less laborious than Southern blotting, and does not require purified DNA or the use of radioactive materials. Genomic DNA for PCR screening of mice is generally prepared from tail biopsies² or blood samples³; however, the routine procedure of clipping the ear or toe to mark the mice⁴ provides enough tissue to extract DNA for PCR amplification. The ear clip procedure is advantageous because not only are mice identified and genotyped simultaneously, they tolerate ear clipping better than cutting of the tail or toes.

In many cases, it is beneficial to amplify large DNA fragments to verify the structure of a modified genetic locus. For example, when using targeting vectors with long arms,⁵ conventional PCR is not suitable for mouse genotyping.⁶ Hence, we developed a procedure to amplify large DNA fragments with TaqPlus Long DNA polymerase using genomic DNA from mouse ear clips.

Genomic DNA Prepared from Mouse Ear Clips

The integrity of the DNA template is crucial for reproducible long-distance PCR.⁷ Because DNA prepared from ear clips by standard methods^2,8 failed to provide reproducible amplification of large (1.5 kb) DNA fragments, we developed a simple procedure to quickly isolate high-integrity genomic DNA.

Three-week-old mice were marked by ear clipping.⁴ The ear clips were transferred into 1.5-l microcentrifuge tubes containing 150 l of digestion solution (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 0.1% SDS, and 1 mg/ml proteinase K). The tubes were incubated overnight at 55C on a rotating platform. Genomic DNA was precipitated by adding 50 l of 10 M ammonium acetate and 600 l of ice-cold ethanol. After centrifuging at 15,000 x g for 10 minutes, the pellets were washed with cold 70% ethanol, air dried, and dissolved in 20 l of TE buffer (5 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA). The yield was 0.5 to 2.5 g of genomic DNA per sample.

PCR Amplification with TaqPlus Long DNA Polymerase

Figure 1

To evaluate whether genomic DNA isolated from ear clips is suitable for amplifying large DNA fragments with TaqPlus Long DNA polymerase, we genotyped the smooth muscle myosin heavy chain (SMHC) gene (Figure 1).

Two microliters of DNA were added to 16 l of PCR mixture [2 l of 10X TaqPlus Long low-salt PCR buffer (Stratagene), 2 l of dNTPs (2.0 mM each), 11.8 l H₂O, and 0.2 l (1 U) of TaqPlus Long DNA polymerase (Stratagene)]. To amplify DNA fragments larger than 4 kb, 2 l of 10X TaqPlus Long PCR high-salt buffer (Stratagene) were used instead of the 10X low-salt buffer. Reactions were initiated using a hot-start protocol.

The first PCR cycle included two steps: For the first step, the tubes were incubated at 85C for 20 minutes [after 2 minutes at 85C, 2 l of a mixture of the appropriate upstream and downstream primers (to a final concentration of 0.25 M each) were added to each tube]. In the second step, the tubes were incubated at 94C for 2 minutes.

After the first cycle, the mixtures were cycled 45X at 94C for 30 seconds, 55C for 30 seconds, and 72C for 2, 4, or 6 minutes to amplify 2, 4, or 6 kb DNA fragments, respectively (1 minute per ~1 kb of template) (see Table 1). Following amplification, 5-l aliquots of each reaction mixture were analyzed by electrophoresis in 0.5X TBE 0.9% agarose gels, and the amplification products were visualized by staining with ethidium bromide.

Table 1
Conditions Used to Amplify Mouse SMHC Gene Fragments with Specific Primers

The Amplified Fragment

Specific Primers

Stratagene 10X TaqPlus Long
PCR Buffer Used

Time at 72C
(Extension Step) (Minutes)

Size of the Fragment (kb)

Upstream

Downstream

#1

Ex5aFOR

Ex5bREV

low salt

2

2.1

#2

Ex5bFOR

Ex6REV

low salt

2

1.9

#3

Ex5aFOR

In5aREV

low salt

2

2.1

#4

In5bFOR

Ex6REV

low salt

2

1.9

#5

Ex5aFOR

In5bREV

low salt

2

2.4

#6

Ex5aFOR

Ex6REV

low salt

4

4.0

#7

Ex5bFOR

Ex8REV

low salt

4

3.9

#8

Ex5bFOR

Ex7REV

low salt

4

3.2

#9

In5bEx6

Ex7REV

low salt

2

1.4

#10

In5bEx6

Ex8REV

low salt

2

2.1

#11

In5bFOR

Ex7REV

low salt

4

3.2

#12

In5bFOR

Ex8REV

low salt

4

3.9

#13

Ex5aFOR

Ex7REV

high salt

6

5.3

#14

Ex5aFOR

Ex8REV

high salt

6

6.0

Fourteen different pairs of primers were used to amplify the specific overlapping fragments of the mouse SMHC gene to examine the reproducibility of the procedure with different primers (Figure 1, Table ). DNA from SMHC genomic clone lMHC17 (20 pg per reaction) was used in parallel as a template for control amplifications (Figure 2, Panel A). This phage carries 19 kb of genomic DNA containing a portion of the mouse MHC gene (Figure 1).

In Figure 2, Panel B, all 14 specific overlapping fragments, ranging from 1.4 to 6 kb, were successfully amplified from the DNA template of mouse ear clips. Primers Ex5aFOR and Ex6REV were used to verify that the different DNA samples were amplified reproducibly. In Figure 2, Panel C, a specific 4-kb DNA fragment was amplified from all 14 samples.

Because the DNA concentration in ear clip preparations may vary, we checked the reliability of the procedure using different amounts of template DNA. In Figure 2, Panel D, a specific 4-kb DNA fragment was amplified (with primers Ex5aFOR and Ex6REV) using from 12.5 to 200 ng of template genomic DNA without significant difference in the intensity of the amplified band.

These results suggested that the procedure works within a wide range of DNA concentrations and can be employed with as little as 12.5 ng of template DNA, so DNA prepared from ear clips can be used directly for PCR amplification.

Conclusions

This procedure allows fast genotyping of a large number of mice with long-distance PCR using TaqPlus Long DNA polymerase. Usually, the results are ready the day after mice ear clipping. Additionally, by amplifying several specific overlapping fragments and analyzing these fragments, a more detailed characterization of the region of interest can be realized. This procedure will have wide application in transgenic mice technology and may be useful for detailed analysis of chromosomal loci with extensive modifications, either introduced by homologous recombination or naturally occurring (i.e., deletions and insertions).

REFERENCES

1. Abbott, C., et al. (1988) Trends Genet. 4: 325.

2. Hanley, T. and Merlie, J.P. (1991) Biotechniques 10: 56.

3. Winberg, G. (1991) PCR Methods Appl. 1: 72-74.

4. Hogan, B., Constantini, F., and Lacy, E. (1986) Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

5. Hasty, P. and Bradley, A. (1995) In: Joyner, A.L. ed., Gene Targeting, A Practical Approach. Oxford University Press, New York. pp. 1-31.

6. Kim, H.-S. and Smithies, O. (1988) Nucleic Acids Res. 16: 8887-8903.

7. Cheng, S., et al. (1995) PCR Methods Appl. 4: 294-298.

8. Nadon, N.L. and Draeger, K. (1996) Transgenic Res. 5: 209-211.

* U.S. Patent No. 5,556,772 and patents pending.