Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE).
Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.
Ljujic
Mila
M
Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O. Box 23, 11010 Belgrade, Serbia and Montenegro. qwert@eunet.yu <qwert@eunet.yu>
Nikolic
Aleksandra
A
Divac
Aleksandra
A
Djordjevic
Valentina
V
Radojkovic
Dragica
D
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005820:Genetic Testing; D014644:Genetic Variation; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D000515:alpha 1-Antitrypsin / Q000235:genetics*
D000515:alpha 1-Antitrypsin