5.5 Single-Strand Conformation Polymorphism (SSCP)
Single-strand conformation polymorphism (SSCP), which is also dependent on
electrophoretic separation based on differences in DNA sequences, allows differentiation of DNA molecules having the same length but different nucleotide sequences.
This technique was originally developed to detect known or novel polymorphisms or
point mutations in DNA (Peters et al. 2000). In this method, single-stranded DNA
separation on polyacrylamide gel was based on differences in mobility resulting
from their folded secondary structure (heteroduplex). As formation of folded secondary structure or heteroduplex and hence mobility is dependent on the DNA
sequences, this method reproduces genetic diversity in a microbial community. All
the limitations of DGGE are equally applicable for SSCP. Again, some singlestranded DNA can exist in more than one stable conformation, so that the same
DNA sequence can produce multiple bands on the gel (Tiedje et al. 1999). However,
it does not require a GC clamp or the construction of gradient gels and has been used
to study bacterial or fungal community diversity (Stach et al. 2001). SSCP has been
used to measure the succession of bacterial communities (Peters et al. 2000),
rhizosphere communities (Schmalenberger et al. 2001), bacterial population changes
in an anaerobic bioreactor (Zumstein et al. 2000), and arbuscular mycorrhizal fungi
(AMF) species in roots (Kjoller and Rosendahl 2000).
5.6 Restriction Fragment Length Polymorphism (RFLP)
Restriction fragment length polymorphism (RFLP), another method for reading
microbial range, depends on DNA polymorphisms. During previous years RFLP
packages have also been implemented to estimate diversity and network structure in
extraordinary microbial groups (Moyer et al. 1996). In this approach,
electrophoresed digests are blotted from agarose gels onto nitrocellulose or nylon
membranes and hybridized with suitable probes prepared from cloned DNA segments of associated organisms. RFLP can be very useful, especially in a mixture
with DNA–DNA hybridization and enzyme electrophoresis, for the differentiation of
carefully associated lines (Palleroni 1993); the technique also appears to be useful
for detection of intraspecies variants (Kauppinen et al. 1994). RFLPs can also
provide a easy and effective device for the identity of bacterial lines at and below
species level. This method is helpful for detecting structural modifications in microbial groups, although not for degree of diversity or detection of precise phylogenetic
agencies (Liu et al. 1997). Banding patterns in various communities are too complicated to analyze by RFLP because a single species may have four to six limit
fragments (Tiedje et al. 1999). However, one must be aware that a similar banding
pattern does not necessarily indicate a very close connection among the organisms
compared.
18 Molecular Technologies for Assessment of Bioremediation and. . .
451
Single-strand conformation polymorphism (SSCP), which is also dependent on
electrophoretic separation based on differences in DNA sequences, allows differentiation of DNA molecules having the same length but different nucleotide sequences.
This technique was originally developed to detect known or novel polymorphisms or
point mutations in DNA (Peters et al. 2000). In this method, single-stranded DNA
separation on polyacrylamide gel was based on differences in mobility resulting
from their folded secondary structure (heteroduplex). As formation of folded secondary structure or heteroduplex and hence mobility is dependent on the DNA
sequences, this method reproduces genetic diversity in a microbial community. All
the limitations of DGGE are equally applicable for SSCP. Again, some singlestranded DNA can exist in more than one stable conformation, so that the same
DNA sequence can produce multiple bands on the gel (Tiedje et al. 1999). However,
it does not require a GC clamp or the construction of gradient gels and has been used
to study bacterial or fungal community diversity (Stach et al. 2001). SSCP has been
used to measure the succession of bacterial communities (Peters et al. 2000),
rhizosphere communities (Schmalenberger et al. 2001), bacterial population changes
in an anaerobic bioreactor (Zumstein et al. 2000), and arbuscular mycorrhizal fungi
(AMF) species in roots (Kjoller and Rosendahl 2000).
5.6 Restriction Fragment Length Polymorphism (RFLP)
Restriction fragment length polymorphism (RFLP), another method for reading
microbial range, depends on DNA polymorphisms. During previous years RFLP
packages have also been implemented to estimate diversity and network structure in
extraordinary microbial groups (Moyer et al. 1996). In this approach,
electrophoresed digests are blotted from agarose gels onto nitrocellulose or nylon
membranes and hybridized with suitable probes prepared from cloned DNA segments of associated organisms. RFLP can be very useful, especially in a mixture
with DNA–DNA hybridization and enzyme electrophoresis, for the differentiation of
carefully associated lines (Palleroni 1993); the technique also appears to be useful
for detection of intraspecies variants (Kauppinen et al. 1994). RFLPs can also
provide a easy and effective device for the identity of bacterial lines at and below
species level. This method is helpful for detecting structural modifications in microbial groups, although not for degree of diversity or detection of precise phylogenetic
agencies (Liu et al. 1997). Banding patterns in various communities are too complicated to analyze by RFLP because a single species may have four to six limit
fragments (Tiedje et al. 1999). However, one must be aware that a similar banding
pattern does not necessarily indicate a very close connection among the organisms
compared.
18 Molecular Technologies for Assessment of Bioremediation and. . .
451