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Letters to the Editor |
I. Dept. of Obstet. and Gynaecol., Semmelweis Univ. Med. School, Budapest, Hungary
a Address correspondence and reprint requests to this author at: I. Dept. of Obst. and Gynaecol., Semmelweis Univ., Baross u. 27, Budapest, H-1088, Hungary.
To the Editor:
Recently Muglia et al. (1) reported in your journal a nonisotopic detection of (CAG)n repeats in the Huntington disease (HD) gene. We have read this article with great interest, as we use a similar method for the detection of the above (2)(3).
There are many difficulties in the PCR conditions of examining CAG repeats in the HD gene and in the detection of PCR products on nondenaturating polyacrylamide gel by silver staining. As yet there is no standardized protocol for the detection of CAG repeats in the HD gene. Muglia et al. (1) reported factors that affect the utility of the PCR product from the HD region by using the known HD-1 and HD-3 primers (4). They used a relatively high MgCl2 concentration (2.0 mmol/L) without dimethyl sulfoxide in 35 cycles. When they applied >35 PCR cycles, they obtained high background of nonspecific bands. We do not see these bands in our method with the lowest possible MgCl2 concentration (0.625 mmol/L), dimethyl sulfoxide (120 g/L) (3) instead of formamide, and 36 cycles.
Figure 1
demonstrates four patients' results obtained by use of 8%
nondenaturating polyacrylamide gel stained by the silver staining
method (5) applied by Muglia et al.
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We found that the amount of mispriming and other nonspecific products mainly depends on the origin of Taq polymerase and other conditions, such as the number of PCR cycles and the use of enhancer components. The latter were investigated by Muglia et al., but the origin of Taq polymerase was not indicated in the article. We found that AmpliTaq (Perkin-Elmer, Norwalk, CT) and Taq polymerase from Promega (Madison, WI) gave sharper bands and less bacground than some others.
References
Ist. di Med. Sperimentale e Biotecnol., CNR 87050 Mangone, (Cosenza) Italy
To the Editor:
Tóth et al. report a method of detection of (CAG)n repeats causing Huntington disease. They suggest some technical variations to our procedure. The major differences are the use of a lower MgCl2 concentration (0.625 mmol/L) and the use of dimethyl sulfoxide (DMSO) (12%) instead of formamide. Before deciding to use formamide, we tried performing PCR with DMSO as reported in the published article. However, we achieved the optimum results by using formamide (35 mmol/L). This difference in results may be attributed to the fact that we did not attempt to decrease the MgCl2 concentration while using DMSO. It is possible that the use of DMSO together with a lower concentration of MgCl2 yields similar results to those obtained when using formamide and a MgCl2 concentration of 2 mmol/L.
With respect to the Taq polymerase, we used Taq polymerase from Promega with the incubation buffer specified in our article.
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