RESUMO
The highly polymorphic human alpha-1 antitrypsin (AAT) gene codes for the most abundant circulating plasma serine protease inhibitor. Previously, genetic variants of the AAT gene were reported from different regions of the world. In the present study, the AAT gene was characterized in an Indian sample. The AAT gene was isolated and cloned from a liver biopsy sample through RT-PCR and the full-length gene was sequenced. Nucleotide sequence comparison with the human genome and the AAT sequences available in the GenBank (NCBI) demonstrated four unique variations--(i) an A to G variation at position 286 (Thr96Ala), (ii) an A to G variation at position 839 (Asp280Gly), (iii) a T to C variation at position 1182 that did not result in any change in the protein sequence (TTT to TTC both code for Phe) and (iv) an A to C variation at position 1200 (Glu400Asp) that resulted in replacement by an amino acid of similar nature. Other variations found were T to C at position 710 (Val273Ala) and T to C position 863 (Val288Glu), which were also reported earlier. In conclusion, this study reports the entire 1257 bp nucleotide sequence of protein coding region of the human AAT gene from an Indian sample. This preliminary finding is significant, as it reports for the first time the AAT gene sequence in the Indian sample.
Assuntos
Alelos , Sequência de Aminoácidos , Sequência de Bases , Biópsia , Códon , Variação Genética , Genoma Humano , Humanos , Índia , Fígado/metabolismo , Dados de Sequência Molecular , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores de Serina Proteinase/genética , alfa 1-Antitripsina/genéticaRESUMO
In the present protocol for extraction of RNA, hexadecyltrimethylammoniumbromide (CTAB) and insoluble polyvinylpyrrolidone were used followed by LiCl precipitation, CsCl ultracentrifugation and finally poly (A)+ mRNA was isolated with the help of oligo(dT)-cellulose columns. The isolated poly (A)+ mRNA was found to be suitable for cDNA-AFLP and suppression subtractive hybridization applications. It is a modified and consolidated protocol based on previously described methods for isolated steps and works better for medicinal and aromatic plants. High yield of poly (A)+ mRNA coupled with its amenability for downstream reactions like RT-PCR, northern blotting and cDNA synthesis for library construction is a key feature of the present protocol.