PHÁT HIỆN SỰ BẮT CẶP KHÔNG ĐẶC HIỆU CỦA MỒI 27F, 1527R KHI KHUẾCH ĐẠI GEN 16S rRNA TỪ DNA ĐA HỆ GEN VI KHUẨN TRONG MẪU PHÂN TRẺ EM

EFFECT OF ANNEALING TEMPERATURE ON NON-SPECIFIC AMPLIFICATION OF PRIMERS 27F, 1527R FROM METAGENOMIC DNA OF BACTERIA IN CHILDREN FECES Primers 27F and 1527R are universal primers capable of amplifying nearly complete 16S rRNA gene of various bacteria in an environment for diversity analysis. In this study, the primers were used for amplification of multigene 16S rRNA of bacteria extracted from stool samples of a healthy child and a dirrheal child with unknown-pathogenic agent by three PCR programs: specific, tough-up and tough-down. The tough-up amplified DNA fragments of about 250 bp with high content from metagenomic DNA samples extracted by two methods from stool sample of diarrheal child. Sequences of the DNA fragments were 100% identical with each other, both contained 247 nucleotides. The DNA was the highest similarity (99.13%) with 22 genes encoding carbohydrate binding proteins (CBP) from B. longum. The investigating the binding sites of the two primers on the homologous genes showed that primers 27F, 1527R has a sequence of six nucleotides at the 3' terminal similarity to a sequence in the CBP gene, especially the 5/6 nucleotides at 3’ terminal of both primers were purines. Primer 27F was able to bind well but primer 1257R may not bind well to the 16S rRNA gene of B. longum. Thus, the low primer anealling temperature can help the primers binding well to the sequences which contains 6 nucleotides identical to the sequence at the 3' terminal of the primers.