Primers' design of cloning aldehyde-alcohol dehydrogenase fragment for bacteria B49

In order to clone the aldehyde-alcohol dehydrogenase gene and study its important role in the ethanol fermentation bio-hydrogen,multiple alignment analyses were conducted on twelve aldehyde-alcohol dehydrogenase protein sequences of different bacteria using clustalw software,and six blocks were obtained.Then degenerate primers were designed according the six blocks,and nine degenerate primers of the aldehyde-alcohol dehydrogenase gene were acquired,named ADHJ1,ADHJ2,ADHJ3,ADHJ4,ADHJ5,ADHJ6,ADHJ7,ADHJ8 and ADHJ9.The high-biohydrogen bacteria B49 genome DNA as the template and nine degenerate primers as primers were used to degenerate PCR,two of them achieved anticipative results.Their PCR products were transformed into E.coli DH5α through being linked with pMD18-T vector and sequenced after filtration.Similarity alignment showed that the products were the most similar to the aldehyde-alcohol dehydrogenase gene of Bacillus cereus,and the identities was 69%.The results indicate that these degenerate primers designed by the clustalw software can be used to obtain the specific gene fragment.Although the degeneracy of ADHJ5 primers are 1048576 and 98304 respectively,the length of ADHJ5 primers are 30 bp and 26 bp,the PCR result is very good.The results show that these degenerate primers designed by this software can be used to obtain specific PCR products.