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Easily Amplify Genomic DNA with Long-Distance PCR

Ex5aFOR

Ex8REV

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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
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