Comparison with the NCBI database using BLAST
revealed that all 37 nirK and all 31 nosZ clones amplified
with primers F1aCu:R3Cu-GC and nosZF:
nosZ1622R-GC, respectively, showed homology with
known nirK and nosZ sequences. No heteroduplex
molecules were observed. The resolution of nirK and
nosZ amplicons was satisfactory. For the 24 nirK bands
that were excised, five bands contained two sequences
and the bands PK6, UK4, BK3 and BK4 held three
sequences. For nosZ, five of the 22 bands were composed
of two different sequences and three sequences
were found in bands BZ5 and HZ9. Sequences appearing
in the same band were in most cases closely related
and were therefore difficult to separate. However, a few
bands contained distantly related nirK and nosZ clones,
indicating co-migration of DNA. This is not unusual
[45], and to resolve these sequences either cloning or
running the fragment on new DGGE can be employed.
The cloning approach is often used for sequencing