The most rapid and reliable method to detect hGH-1 gene 6.7, 7.0, 7.6 Kb deletions in isolated growth hormone deficiency (IGHDIA), is polymerase chain reaction followed by the Smal restriction enzyme digestion. However, when analysing the Smal digested PCR products from normal subjects and 7.6Kb heterozygotes subjects, along with the expected fragments of about 1900bp, 762 bp, 711 bp and 448 bp a fragment of 1470 bp, derived from the coamplification of a third homologous sequence located downstream the gene is often visualized. Such a fragment is present in normal subjects as well as in heterozygotes for a 7.6Kb deletion and those heterozygotes for a 6.7 Kb deletion. To overcome this problem we have chosen a different reverse primer and more stringent PCR conditions, raising annealing and extensing temperature. We have studied four IGHDIA patients homozygous for a 6.7 Kb and 7.6 Kb deletion, eight heterozygous subjects for such deletions and three normal subjects. These changes avoid the primers interacting and extensing with the third homologous sequence located downstream the hGH-1 gene and prevent the appearance of this additional band which complicates the interpretation of the results.
|Number of pages||1|
|Journal||Endocrinology and Metabolism, Supplement|
|Publication status||Published - 1997|
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