Sequencing and expression of Elastase II gene in Polymorphoneuclear leukocytes in healthy individuals for determination of single nucleotide polymorphism (SNP) by RT-PCR
Abstract
Background: ELA2 gene responsible for coding human neutrophil elastase, a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins. The NE gene consist of 5 exons and 4 introns.
Methods: Peripheral blood obtained from healthy individuals. Total RNA was isolated using RNA standard techniques from fresh separated cells by polymorphoprep. RNA was analyzed by employing PCR amplification of reveres transcribed using a total of ten specific primers. We amplified five exon of ELA2 gene separately and sequenced each exon. Mutational analysis was performed by directed capillary sequencing method.
Findings: We have found new single nucleotide polymorphism (SNP) in exon II codon 44. It was a silent mutation G to A substitution but no changes in amino acid sequences were seen. The codon sequence was GCG that has changed to GCA.
Conclusion:
The purpose of SNP detection is mainly recognition of susceptible people to certain diseases and providing suitable drugs based on each person genetic information. SNP information in Iranian patients can be used for analysis of drug response to human diseases.
Methods: Peripheral blood obtained from healthy individuals. Total RNA was isolated using RNA standard techniques from fresh separated cells by polymorphoprep. RNA was analyzed by employing PCR amplification of reveres transcribed using a total of ten specific primers. We amplified five exon of ELA2 gene separately and sequenced each exon. Mutational analysis was performed by directed capillary sequencing method.
Findings: We have found new single nucleotide polymorphism (SNP) in exon II codon 44. It was a silent mutation G to A substitution but no changes in amino acid sequences were seen. The codon sequence was GCG that has changed to GCA.
Conclusion:
The purpose of SNP detection is mainly recognition of susceptible people to certain diseases and providing suitable drugs based on each person genetic information. SNP information in Iranian patients can be used for analysis of drug response to human diseases.
Keywords
Elastase II gene (ELA2), neutrophil elastase (NE), single nucleotide polymorphism (SNP), capillary sequencing,
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