Mone C M, Nigro V, Rotondi M, Del Buono A, Mazziotti G, Riondino M, Sinisi A M, Ghizzoni L, Phillips J A, Bellastella A, Carella C
Institute of Endocrinology, Second University of Naples, Italy.
J Pediatr Endocrinol Metab. 1998 Jul-Aug;11(4):563-8. doi: 10.1515/jpem.1998.11.4.563.
hGH-1 gene deletions are detected by simultaneous PCR amplification along the two homologous DNA sequences flanking the hGH-1 gene on both sides and are differentiated by SmaI restriction enzyme digestion. We have observed that among the SmaI digested PCR products from normal homozygous subjects, from those heterozygous for the 7.6 kb deletion and from those heterozygous for a 6.7 kb deletion, along with the expected fragments there is an unexpected 1470 bp fragment. This fragment arises from the co-amplification of a third homologous sequence located downstream from the hGH-1 gene and it confuses differentiation between normal homozygous and heterozygous for 7.6 kb subjects from the 6.7 kb heterozygous subjects. To overcome this problem we have improved PCR conditions using a different reverse primer. These changes avoid the interaction of the primers with the third homologous sequence located downstream from the hGH-1 gene and prevent the appearance of this additional band that complicates the interpretation of the results. We conclude that the new reverse primer sequence avoids the amplification of the downstream hGH-1 gene sequence and the production of the 1474 bp band after SmaI endonuclease enzyme digestion and makes it possible to differentiate homozygous normal subjects and those who are heterozygous for a 7.6 kb deletion from those who are heterozygous for a 6.7 kb deletion.
通过对hGH - 1基因两侧的两个同源DNA序列同时进行PCR扩增来检测hGH - 1基因缺失，并通过SmaI限制性内切酶消化进行区分。我们观察到，在来自正常纯合子受试者、7.6 kb缺失杂合子受试者以及6.7 kb缺失杂合子受试者的SmaI消化后的PCR产物中，除了预期的片段外，还有一个意外的1470 bp片段。这个片段来自hGH - 1基因下游第三个同源序列的共扩增，它混淆了正常纯合子和7.6 kb缺失杂合子受试者与6.7 kb缺失杂合子受试者之间的区分。为了克服这个问题，我们使用了不同的反向引物改进了PCR条件。这些改变避免了引物与hGH - 1基因下游第三个同源序列的相互作用，并防止了这个使结果解释复杂化的额外条带的出现。我们得出结论，新的反向引物序列避免了hGH - 1基因下游序列的扩增以及SmaI内切酶消化后1474 bp条带的产生，使得区分纯合正常受试者、7.6 kb缺失杂合子受试者和6.7 kb缺失杂合子受试者成为可能。
