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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 methods2,8 failed to provide reproducible amplification of large (1.5 kb) DNA fragments, we developed a
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