29
variation means, single-point mutations, or smaller changes in nucleotides, on the
basis of differences in electrophoretic mobility. Mutated DNA bands have different
mobility rates in comparison to wild-type DNA (Hayashi 1991). SSCP is a pivotal
technique for evaluating microbe ecology and diversity in the soil system. The dissimilar electrophoretic mobilities and conformational differences are separated
through nondenaturing polyacrylamide gel electrophoresis (Widjojoatmodjo et al.
1995).
SSCP is comparatively simpler in approach than DGGE or TGGE as it does not
depend on denaturing gradient gels, GC clamp primers, or a specific apparatus.
However, the technique is most suitable for 150- and 400-bp DNA fragments
(Muyzer 1999). Yet, the increased frequency of DNA strands reannealing after
denaturation in cases of higher DNA concentration is a limiting factor in deciphering high-diversity communities (Selvakumar et al. 1997). Again the problem lies
with the presence of more than one band from the double-stranded DNA-based PCR
product after electrophoresis, as similarly confirmation of complementary singlestranded products of double-stranded DNA may lead to the detection of less than
three products from a single organism (Lee et al. 1996). The poor detection limit of
the technique can be minimised using a fragment (~400 bp) of the microbial 16S
rRNA gene (V4 and V5 sequence regions), which is PCR amplified with universal
primers with the 5′-end of a primer phosphorylated. However, despite certain limitations, the technique has been effectively used for microbial community analysis in
various habitats. SSCP was successfully applied to differentiate between interspacer
regions of 16S–23S rRNA of bacterial strains (Scheinert et al. 1996). Further SSCP
has been employed to distinguish the soil microorganisms Pseudomonas fluorescens, Bacillus subtilis, and Sinorhizobium meliloti (Schwieger and Tebbe 1998).
SSCP-based exploration of the Medicago sativa and Chenopodium album rhizospheres revealed that bacterial communities are shaped according to plant species
despite both plants growing in the same soil because rhizospheric bacterial communities varied for each plant.
The technique further revealed the diversity and activity of bacterial biofilm
communities growing on hexachlorocyclohexane (HCH)-contaminated soil polluted by the pesticide-producing factories residues and waste material in Egypt
(Gebreil and Abraham 2016). Moreover, the effectiveness of tomato-linked rhizospheric bacteria applied in single and consortium mode for control of tomato stem
rot by Sclerotinia (Abdeljalil et al. 2016) was assessed using SSCP. Similarly, under
arsenic (As)-polluted soils, 16S rDNA-based capillary electrophoresis single-strand
conformation polymorphism confirms the altered soil microorganism community in
response to concentration gradients of As (Quemeneur et al. 2016).
3.6 Single-Strand Conformation Polymorphism (SSCP)
variation means, single-point mutations, or smaller changes in nucleotides, on the
basis of differences in electrophoretic mobility. Mutated DNA bands have different
mobility rates in comparison to wild-type DNA (Hayashi 1991). SSCP is a pivotal
technique for evaluating microbe ecology and diversity in the soil system. The dissimilar electrophoretic mobilities and conformational differences are separated
through nondenaturing polyacrylamide gel electrophoresis (Widjojoatmodjo et al.
1995).
SSCP is comparatively simpler in approach than DGGE or TGGE as it does not
depend on denaturing gradient gels, GC clamp primers, or a specific apparatus.
However, the technique is most suitable for 150- and 400-bp DNA fragments
(Muyzer 1999). Yet, the increased frequency of DNA strands reannealing after
denaturation in cases of higher DNA concentration is a limiting factor in deciphering high-diversity communities (Selvakumar et al. 1997). Again the problem lies
with the presence of more than one band from the double-stranded DNA-based PCR
product after electrophoresis, as similarly confirmation of complementary singlestranded products of double-stranded DNA may lead to the detection of less than
three products from a single organism (Lee et al. 1996). The poor detection limit of
the technique can be minimised using a fragment (~400 bp) of the microbial 16S
rRNA gene (V4 and V5 sequence regions), which is PCR amplified with universal
primers with the 5′-end of a primer phosphorylated. However, despite certain limitations, the technique has been effectively used for microbial community analysis in
various habitats. SSCP was successfully applied to differentiate between interspacer
regions of 16S–23S rRNA of bacterial strains (Scheinert et al. 1996). Further SSCP
has been employed to distinguish the soil microorganisms Pseudomonas fluorescens, Bacillus subtilis, and Sinorhizobium meliloti (Schwieger and Tebbe 1998).
SSCP-based exploration of the Medicago sativa and Chenopodium album rhizospheres revealed that bacterial communities are shaped according to plant species
despite both plants growing in the same soil because rhizospheric bacterial communities varied for each plant.
The technique further revealed the diversity and activity of bacterial biofilm
communities growing on hexachlorocyclohexane (HCH)-contaminated soil polluted by the pesticide-producing factories residues and waste material in Egypt
(Gebreil and Abraham 2016). Moreover, the effectiveness of tomato-linked rhizospheric bacteria applied in single and consortium mode for control of tomato stem
rot by Sclerotinia (Abdeljalil et al. 2016) was assessed using SSCP. Similarly, under
arsenic (As)-polluted soils, 16S rDNA-based capillary electrophoresis single-strand
conformation polymorphism confirms the altered soil microorganism community in
response to concentration gradients of As (Quemeneur et al. 2016).
3.6 Single-Strand Conformation Polymorphism (SSCP)