The Correlation Research Between Lung Cancer And The Mutation Of Puma Gene

Objective:To explore the correlation between lung cancer and the mutation of puma gene.Methods:By explored the experiment conditions of polymerase chain reaction and selected the optimized amplification systems and reaction conditions of three high GC content Exons of puma,it make the Exon2 that GC content 82%be amplificationed succeefully.By using the optimized amplification systems and reaction conditions,the Exon2,Exon3 and Exon4 of puma gene in all 34 cases of lung cancer tissues,paracancer lung tissues and farcancer lung tissues,7 cases of non-cancer lung tissues were also amplificationed.The amplification productions were depurated and sequencing detected both mutation and small delete fragments.ResuLts:1.By explored the experiment conditions of polymerase chain reaction,the ultimate amplification systems and reaction conditions of three Exons of puma were established.(1)The optimized reaction systems of Exon2 were:dNTP200μmol/L, enhancer system MgSO₄ 3.0mmol/L,primers 500nmol/L,Taq DNA polymerase 0.1U/μl,templates more than 4000ng,1×PCR_x enhancer system buffer,enhancer solution 10μl,the total volum was 50μl。The cycling conditions were as follows:After 5 min at 95℃,reactions were cycled through 45s denaturation at 95℃,45s annealing at 64℃,and 45s extension at 72℃for 33;followed 72℃for 5 minutes.(2)The optimized reaction systems of Exon3 were:dNTP200μmol/L, enhancer system MgSO₄ 2.0mmol/L,primers 500nmol/L,Taq DNA polymerase 0.1U/μl,templates more than 4000ng,1×PCR_x enhancer system buffer,the total volum was 50μl。The cycling conditions were as follows:After 5 min at 95℃,reactions were cycled through 45s denaturation at 95℃,45s annealing at 64℃,and 45s extension at 72℃for 33;followed 72℃for 5 minutes.(3)The optimized reaction systems of Exon4 were:dNTP200μmol/L, common MgCl₂ 1.0mmol/L,primers 500nmol/L,Taq DNA polymerase 0.1U/μl,templates more than 4000ng,common 1×PCR buffer,the total volum was 50μl。The cycling conditions were as follows:After 5 min at 94℃,reactions were cycled through 45s denaturation at 94℃,45s annealing at 61℃,and 45s extension at 72℃for 33;followed 72℃for 5 minutes.2.By using the optimized amplification systems and reaction conditions, the Exon2,Exon3 and Exon4 of puma gene in all 34 cases of lung cancer tissues,paracancer lung tissues and farcancer lung tissues,7 cases of non-cancer lung tissues were also amplificationed.The amplification productions were depurated and sequencing detected both mutation and small delete fragments.The sequencing results contrast to standard DNA sequence by using Blast2,None of mutation and small delete fragments were found.Conclusions:1.The three Exons coded sequences of puma gene DNA sequence are stable and conservative,they are not easy influenced by all sorts of induction of mutation factors that come from surrounding and itself.2.Puma gene mutation might not be the important agent accompany the happen and development of lung cancer.3.In the process of lung cancer taking place and progressing,even if the expression of puma appears abnormal,the function protein quantity shows abnormal even absence,results in the apoptosis alteration both physiological and pathological, It shows that the series of changes are not induced by gene mutation,and it might induce by the changes or abnormal of regulation series and promoter region It might only take place in the level of transcriptional and/or translation of puma gene too.