The ISME Journal

TABLE 2

FROM:

Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities

Martin Mühling, John Woolven-Allen, J Colin Murrell and Ian Joint

BACK TO ARTICLE

Table 2. Nested PCR approach with DGGE primers

Figure and tables index
Primers used for group-specific PCR Primers used for re-PCR for DGGE a AT (semi-) nested PCR (°C) Denaturing gradient used for DGGE (%) b
Alf28f/Alf684r341f-GC/518r5640–60
Beta359f/Beta682r518f-GC/Beta682r6040–55
Gamma395f/Gamma871r518f-GC/785r5640–60
CFB555f/CFB968rCFB555f-GC/907r6440–60
CYA361f/CYA785r518f-GC/CYA785r5640–55
Plancto352f/Plancto920r518f-GC/907r6040–60
Firm350f/Firm814r518f-GC/785r5640–60
9bfm/1512uRc 341f-GC/518r5640–60

 Abbreviations: AT, annealing temperature; DGGE, denaturing gradient gel electrophoresis.

 PCR products produced with the group-specific primers were reamplified using a set of bacterial or universal DGGE primers.

a Nucleotide sequences of the primers, which have been published previously, and the GC clamp are: 341f, CCTACGGGAGGCAGCAG (Muyzer et al., 1993); 518f, CCAGCAGCCGCGGTAAT (Muyzer et al., 1993); 518r, ATTACCGCGGCTGCTGG (Muyzer et al., 1993); 785r, CTACCAGGGTATCTAATCC (Lee et al., 1993); 907r, CCGTCAATTCMTTTGAGTTT (Muyzer et al., 1998); GC clamp, CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG (Muyzer et al., 1993); Please note, the sequence of the bacterial primer 785r (E. coli position 785–803) matches 81.2% of all 16S rRNA gene sequences in the ROSE database, but only 1% of the Cyanobacteria/chloroplast 16S rRNA gene sequences.

b 100% denaturant contains 7 M urea and 40% (v/v) formamide.

c The approach taken for DGGE analysis of the domain bacteria involved reamplification also.

Figure and tables index
BACK TO ARTICLE