Microbial Diversity: Its Exploration and Need of Conservation - Applied Environmental Biotechnology: Present Scenario and Future Trends - page 54

Table 4.1 Advantages and disadvantages of some molecular-based methods to study soil microbial diversity.
(Source: Fakruddin and Mannan 2013 and Kirk et al. 2004 )

Method Advantages Disadvantages

Mole % (G + C) Not influenced by PCR Requires large quantities of DNA

Includes all DNA extracted Dependent on lysing and extrac-

tion efficiency

Includes rare members of community Less sensitive resolution

Nucleic acid reassociation and
hybridization

Total DNA extracted Lack of sensitivity

Not influenced by PCR biases Sequences need to be in high copy

number for detection

Can study DNA or RNA Dependent on lysing and extrac-

tion efficiency

Can be studied in situ

DNA microarrays and DNA
hybridization

Same as nucleic acid hybridization Only detect the most abundant

species

Process thousands of genes simultaneously Need to culture organisms

If using genes or DNA, fragments specificity
increases

Only accurate in low diversity
systems

Single strand conformation
polymorphism (SSCP)

Same as DGGE/TGGE PCR biases

No GC clamp Some ssDNA can form more than

one stable conformation

No gradient

Denaturing and temperature gra-
dient gel electrophoresis (DGGE
and TGGE)

Large number of samples can be analyzed
simultaneously

PCR biases

Reliable, reproducible, and rapid Dependent on lysing and extrac-

tion efficiency
Way of sample handling can
influence community, i.e., the
community can change if stored
for too long before extraction
“One band-one species” is not
always true
Cannot compare bands between
gels
Only works well with short frag-
ments ( < 500 bp), thus limiting
phylogenetic characterization
Only detects dominant species

Restriction fragment length
polymorphism (RFLP)

Detect structural changes in microbial
community

PCR biases

Banding patterns often too
complex

Terminal restriction fragment
length polymorphism (T-RFLP)

Simpler banding patterns than RFLP Dependent on extraction and lys-

ing efficiency

Can be automated PCR biases

Can process large number of samples Type of Taq can increase

variability

Highly reproducible Choice of restriction enzymes will

influence community fingerprint

Ability to compare differences between
microbial communities