Sequencing of the Coding Region

In a first step it was necessary to characterize the sequence of the human -adrenergic receptor. Firstly, sequencing the complete structure gene of the receptor from both, salt resistant and salt sensitive subjects was performed. Secondly, the revealed sequences were compared with published gene sequences[77, 78]. Based on the results obtained and the mutations discovered screening methods for rapid screening of these mutations had to be developed. Furthermore, the sequencing results were used as references for the scanning methods described later on (see chapter 4).

• Sequencing Strategy
• PCR of the Coding Fragment
• Subcloning the Insert in a pUC18 plasmid
  • Preparation of the Vector
  • Dephosphorylation of the Linearized Vector
  • Preparation of the Insert
  • Ligation of Insert into the Vector
• Transformation of Bacteria
  • Preparation of Competent Hosts
  • Transformation of Competent DH5 Cells
  • Isolation of the Plasmid after Alkaline Lysis
• Sequencing
  • Alkali Denaturation of DNA Template
  • Sequencing Protocol
  • Denaturing Polyacrylamide Gels
• Results
  • Mutations of the -Adrenergic Receptor Gene