taxonomically connected groups only differ from 3% to 5% (Tiedje et al. 1999).
However, similar base composition is not a confirmation of relationship, although it
suggests that the difference can be evidence of a missing relationship.
5.2 Nucleic Acid Hybridization and Reassociation
Nucleic acid hybridization is an important qualitative and quantitative tool in
molecular bacterial ecology (Clegg et al. 2000). Valuable spatial distribution information on microbial communities in natural environments is often provided by
hybridization methods, mostly using extracted DNA or RNA, or in situ. Probes
(oligonucleotide or polynucleotide) can be designed from well-known sequences,
ranging in specificity from domain to species, and can be tagged with markers at the
5
0 -end (Goris et al. 2007). After lysing the sample to release all nucleic acids, dot blot
hybridization with specific and universal oligonucleotide primers is used to quantify
rRNA sequences of interest relative to total rRNA. The relative abundance may
represent changes in the abundance in the population or changes in the activity and
hence the amount of rRNA content (Theron and Cloete 2000). Another approch is in
situ cellular level hybridization. The only limitation is the lack of sensitivity of
hybridization of nucleic acids, which requires the sequences to be present in high
copy number, such as those from dominant species; otherwise, the probability of
detection is low.
The kinetics of DNA reassociation estimates diversity by measuring the genetic
complexity of the microbial community (Torsvik et al. 1996). Total DNA is
extracted from environmental samples, purified, denatured, and allowed to reanneal.
The rate of reassociation depends on the similarity of sequences present, and as the
complexity of DNA sequences increases, the reassociation rate decreases (Theron
and Cloete 2000). Two parameters controlling the reassociation reaction are the
concentration of DNA product (Co) and time of incubation (t), usually described as
the half-association value, Cot1/2 (the time needed for half the DNA to reassociate).
The value of Cot1/2 can be used as a diversity index in special conditions, as it takes
into account both the amount and distribution of DNA reassociation (Torsvik et al.
1998). Thus, the similarity between communities of two different samples can be
studied by measuring the degree of similarity of DNA through hybridization kinetics
(Griffiths et al. 1999).
5.3 Quantitative PCR and RT-PCR
DNA amplification by PCR has been used in many studies to detect, characterize,
and identify pollutant-degrading microbial populations. PCR detection of genes
encoding for microbial monooxygenases and dioxygenases such as NAHA,
PHNAc, NIDA, and NARB are useful for the detection of pollutant-degrading
448
S. K. Shekhar et al.
However, similar base composition is not a confirmation of relationship, although it
suggests that the difference can be evidence of a missing relationship.
5.2 Nucleic Acid Hybridization and Reassociation
Nucleic acid hybridization is an important qualitative and quantitative tool in
molecular bacterial ecology (Clegg et al. 2000). Valuable spatial distribution information on microbial communities in natural environments is often provided by
hybridization methods, mostly using extracted DNA or RNA, or in situ. Probes
(oligonucleotide or polynucleotide) can be designed from well-known sequences,
ranging in specificity from domain to species, and can be tagged with markers at the
5
0 -end (Goris et al. 2007). After lysing the sample to release all nucleic acids, dot blot
hybridization with specific and universal oligonucleotide primers is used to quantify
rRNA sequences of interest relative to total rRNA. The relative abundance may
represent changes in the abundance in the population or changes in the activity and
hence the amount of rRNA content (Theron and Cloete 2000). Another approch is in
situ cellular level hybridization. The only limitation is the lack of sensitivity of
hybridization of nucleic acids, which requires the sequences to be present in high
copy number, such as those from dominant species; otherwise, the probability of
detection is low.
The kinetics of DNA reassociation estimates diversity by measuring the genetic
complexity of the microbial community (Torsvik et al. 1996). Total DNA is
extracted from environmental samples, purified, denatured, and allowed to reanneal.
The rate of reassociation depends on the similarity of sequences present, and as the
complexity of DNA sequences increases, the reassociation rate decreases (Theron
and Cloete 2000). Two parameters controlling the reassociation reaction are the
concentration of DNA product (Co) and time of incubation (t), usually described as
the half-association value, Cot1/2 (the time needed for half the DNA to reassociate).
The value of Cot1/2 can be used as a diversity index in special conditions, as it takes
into account both the amount and distribution of DNA reassociation (Torsvik et al.
1998). Thus, the similarity between communities of two different samples can be
studied by measuring the degree of similarity of DNA through hybridization kinetics
(Griffiths et al. 1999).
5.3 Quantitative PCR and RT-PCR
DNA amplification by PCR has been used in many studies to detect, characterize,
and identify pollutant-degrading microbial populations. PCR detection of genes
encoding for microbial monooxygenases and dioxygenases such as NAHA,
PHNAc, NIDA, and NARB are useful for the detection of pollutant-degrading
448
S. K. Shekhar et al.