Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2 Zdrave Street, 1431 Sofia, Bulgaria.
Mol Biotechnol. 2010 Jun;45(2):150-4. doi: 10.1007/s12033-010-9260-y.
We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient's genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat's number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.
我们报告了一个统一的快速甜菜碱 - PCR 方案，用于扩增亨廷顿病（HD）的（CAG）n 区和脆性 X 综合征（FXS）的（CGG）n 区，然后在自动测序仪上进行电泳分离，以精确确定三核苷酸的数量。高浓度的甜菜碱（2.5 M 甜菜碱）允许精确扩增 CAG 和 CGG 重复序列。对 10 名 HD 受影响的患者和来自 HD 家族的 10 名健康个体进行了重新评估。对于 FXS，该方案可以精确扩增正常个体和大约 100 个重复的 CGG 区的重复序列。对 10 名无关的 FXS 前突变携带者和 24 名智力低下的非 FXS 受影响的男孩进行了重新检查。结果与以前的结果完全一致。该方案是一种快速筛选测试的不错选择。在 24 小时内，我们可以获得有关患者遗传状况的初步信息。正常个体、CGG 前突变携带者高达 100 个重复，以及携带多达 50 个 CAG 重复的 HD 患者，都可以轻松明确。这占了我们实验室进行遗传分析的疑似 HD 和 FXS 患者的相对较大比例（约 90％）。重复次数的计算对于正确解释结果、筛选测试和遗传咨询更准确。
