Symbiotic Fungi: Principles and Practice (Soil Biology)

10 Assessing the Mycorrhizal Diversity of Soils and Identification of Fungi 181
ends need to be trimmed off. In addition, it might be necessary to repeat the
sequencing, sequence with another primer, or clone the PCR products if they appear
to be mixed, rather then having occasional point errors or single nucleotide polymorphisms
(SNPs). DNA analysis software such as used for phylogenetics, as well
as BLAST-n (Altschul et al. 1997) can deal with the ambiguity codes for nucleotides
(see Table 10.2, Cornish-Bowden 1985).
10.6.6 Cloning
If no good sequence can be obtained, e.g., because the specimen or culture is impure
or the ribosomal DNA is heterogeneous, cloning is necessary. It is simplest to use a
T/A or U/A cloning kit (Mead et al. 1991) where the PCR products, which should
have A overhangs due to the peculiarity of Taq polymerase, are ligated into a
special cloning vector for the bacterium E. coli. In that case, one can then directly
add a little pipette tip full of E. coli single colony material into a PCR mix such as
above (barely touch individual colony on plate!), but using primers anchored in the
vector, such as recommended by the cloning kit manufacturer. The sequencing of
the colony PCR product also has the advantage of getting a clean sequence from the
start, as the otherwise usually ‘‘messy’’ start and the region where dye blobs would
Table 10.2 Meaning of letters (IUPAC codes) and symbols in nucleotide sequences
Code Meaning Complement Ribonucleotide type
A A (adenine) T purine
C C (cytosine) G pyrimidine
G G (guanine) C purine
T T (thymine) A pyrimidine
U U (uracil) A pyrimidine (in RNA only)
I I (inosine) N, X purine (RNA; can cost-efficiently
replace N in primers, Candrian
et al 1991)
M AorC K
R A or G Y purine
W AorT W
S CorG S
Y C or T R pyrimidine
K GorT M
V AorCorG B
H AorCorT D
D AorGorT H
B CorGorT V
N, X G or A or T or C N, X
(in alignment) Indel (insertion/deletion)
* (in alignment) Identical