Gene Cluster Degenerate primers 

Organisms with the homologues of dnd gene cluster


Fig. 1. dndABCDE gene cluster in Streptomyces lividans 1326 encoding DNA degradation (Dnd) system.


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PCR assay with degenerate primers targeting dndC gene
1. degenerate primers targeting dndC gene
Fig. 2. Sequence alignment of dndC in eight representative bacteria (Forward primer KRf:118-135, Reversed primer KRr:403-422, referring to Streptomyces lividans dndC gene sequence location)
2. PCR primers and PCR reaction conditions
Degenerate Primers used in this work were KRf 5’-CCN TGG RTN RTY GGN TWY AGY GG-3’ (Tm 64.2), and KRr 5’-ATY TTY ADN CKN TCD GTR CAC C-3’ (Tm 57.8), here B = C, G or T; D = A, T or G; H = A, C or T; K = G or T; M = A or C; N = A, T, C or G; R = A or G; S = C or G; V = A, C or G; W = A or T; Y = T or C.

PCR reactions (30 µl) containing 25-50 ng template DNA, 1 pM of each primer (VH Bio Ltd), 0.2 mM dNTPs (BioLINE), 1 unit GoTaq polymerase in 1×PCR buffer (1.5 mM MgCl2 at pH 8.5) (Promega Corporation) were performed using the Gene Amp PCR System 9700 (Applied Biosystems).

PCR cycling conditions were as follows: initial denaturation at 95°C for 300s, followed by 35 cycles of denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 20s, finally hold at 4 °C for 15min.

PCR products were purified from 1.5% agarose (BioLINE) gels using YORBIO PCR DNA Gel Extraction Kit (Yorkshire Bioscience) and subsequently inserted into pCR®4-TOPO® vector (InvitrogenTM) for sequencing.
Fig. 3a (left) Dnd phenotype determination of total DNA from serven isolates.
Lane 1, E. coli B7A;
lane 2, Klebsiella pneumoniae 100;
lane 3, Klebsiella pneumoniae 104;
lane 4, Klebsiella pneumoniae 105;
lane 5, Pseudomonas fluorescens PfO-1;
lane 6, Salmonella enterica serovar Livingstone 663;
lane 7, Salmonella enterica serovar Cerro 87.
Fig. 3b (right) Survey of the dndC homolog in bacterial strains using degenerate primers KRf and KRr. The size of expected band is 327bp.
Lane 1, E. coli B7A;
lane 2, Klebsiella pneumoniae 100;
lane 3, Klebsiella pneumoniae 104;
lane 4, Klebsiella pneumoniae 105;
lane 5, Pseudomonas fluorescens PfO-1;
lane 6, Salmonella enterica serovar Livingstone 663;
lane 7, Salmonella enterica serovar Cerro 87.

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