RAPID PRENATAL-DIAGNOSIS OF SICKLE-CELL-ANEMIA BY A NEW METHOD OF DNA ANALYSIS
RAPID PRENATAL-DIAGNOSIS OF SICKLE-CELL-ANEMIA BY A NEW METHOD OF DNA ANALYSIS
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DOI：
10.1056/nejm198703123161103
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发表时间：
1987-03-12
影响因子：
158.5
通讯作者：
ERLICH, HA
中科院分区：
文献类型：
--
作者：
EMBURY, SH;SCHARF, SJ;ERLICH, HA
我们已经使用了一种新的DNA分析方法，用于快速产前诊断镰状细胞性贫血的两个胎儿在这种疾病的风险。这种检测镰状基因的方法是对标准限制性内切酶技术的改进，只需要少量的DNA。第一步是对特定的β进行20万倍的酶扩增。-珠蛋白DNA序列可能携带镰状突变。这为分析提供了足够数量的DNA。接下来，一个短的放射性标记的合成DNA序列同源于正常。将a -珠蛋白基因序列与扩增的靶序列杂交。然后用两个限制性内切酶依次消化杂交的“双酶体”。β的存在。A或。beta。s -珠蛋白基因序列的扩增目标DNA从患者决定是否。a -杂交探针退火完美或与单核苷酸不匹配。这种差异影响了DNA的限制性酶切和由此产生的放射标记的消化产物的大小，这可以通过电泳和放射自显影来区分。这种方法足够灵敏和快速，可以在获得胎儿DNA的同一天进行镰状细胞性贫血的产前诊断。也可应用于血红蛋白C病的诊断。
We have used a new method of DNA analysis for the rapid prenatal diagnosis of sickle cell anemia in two fetuses at risk for this disease. This method of detecting the sickle gene is a modification of standard restriction-enzyme techniques and requires only a small amount of DNA. The first step involves a 200,000-fold enzymatic amplification of the specific .beta.-globin DNA sequences that may carry the sickle mutation. This provides a sufficient quantity of DNA for the analysis. Next, a short radiolabeled synthetic DNA sequence homologous to normal .beta.A-globin gene sequences is hybridized to the amplified target sequences. The hybrid "duplexes" are then digested sequentially with two restriction endonucleases. The presence of .beta.A- or .beta.S-globin gene sequences in the amplified target DNA from the patient determines whether the .beta.A-hybridization probe anneals perfectly or with a single nucleotide mismatch. This difference affects the restriction-enzyme digestion of the DNA and the size of the resulting radiolabeled digestion products, which can be distinguished by electrophoresis followed by autoradiography. This method is sufficiently sensitive and rapid that the prenatal diagnosis of sickle cell anemia can be made on the same day that the fetal DNA is made available. It can also be applied to the diagnosis of hemoglobin C disease.