**1. Introduction**

50 Gel Electrophoresis – Advanced Techniques

Zuppini, A., Gerotto, C. & Baldan, B. (2010). Programmed cell death and adaptation: two

Zurek, R. & Bucka, H. (2004). Horizontal distribution of phytoplankton and zooplankton

*Physiology*, Vol. 51, pp. 884-895, ISSN 0032-0781

*Hydrobiological Studies*, Vol. 33, pp.69-81, ISSN: 1730-413X

different types of abiotic stress response in a unicellular chlorophyte. *Plant and Cell* 

from the littoral towards open waters under wind stress. *Oceanological and* 

Molecular tools in environmental microbiology have been applied extensively in last decades because of the limitations in culture-dependent methods (Amann *et al.*, 1995; Muyzer *et al.*, 1996; Head *et al.*, 1998). Despite isolation techniques are provided detailed knowledge about the single species in terms of morphology, biochemistry, and also genetic (Bitton, 2005), they have important drawbacks. The first one is to find the selective media favoring the desired microbial group. Additionally, isolated species cannot reflect their behaviors in the natural environment. Until today, 19.000 microbial species have been isolated (DSMZ, 2011; http://www.dsmz.de), however it is accepted that this number is only a small portion of real diversity (Amann *et al.*, 1995). Besides, using the molecular tools in natural and engineering systems, we can find the answer to the questions such as 'which species do exist?', 'which species are active?', 'how many microorganisms are there?', which species do utilize the specific compounds?'.

Microbial ecology studies need identification of species based on a comprehensive classification system that perfectly reflects the evolutionary relations between the microorganisms (Pace, 1996). Zuckerkandl and Pauling (1965) indicated that nucleic acids could document evolutionary history. Due to the pioneering studies, nucleic acids, especially 16S rRNA, are the ultimate biomarkers and hereditary molecules probably because of their essential role in protein synthesis, making them one of the earliest evolutionary functions in all cellular life-forms (Olsen *et al.*, 1986; Pace *et al.*, 1986; Woese, 1987; Stahl *et al.*, 1988). In particular, 16S rRNA and 16S rDNA have been used in phylogenetic analysis and accepted as ideal evolutionary chronometer.

Genetic fingerprinting techniques are one of the most applied molecular tools based on 16S rRNA in microbial ecology studies. These techniques such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), amplified ribosomal DNA restriction (ARDRA) or restriction fragment length polymorphism (RFLP), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP), have been developed for estimation of diversity in ecosystems, screening clone libraries, following the diversity changes with respect to time

Gel Electrophoresis Based Genetic Fingerprinting Techniques on Environmental Ecology 53

changes (Woese, 1987, Amann *et al.*, 1995), their variable regions allow phylogenetic

As a result, to design general or specific primers and probes for 16S rDNAs and 16S rRNAs provides study options about identification and evolution of microorganisms because this molecule is fairly large (≈1500 nucleotides) including sufficient sequence information. Also the abundance is high within most cells (103 to 105 copies) and they can be detected easily (Amann *et al.*, 1995). While even secondary structure of 16S rRNA molecule is highly conserved, many variable regions randomly change during evolution. This differential variation explains the relationship between microorganisms evolutionarily. Data obtained from this analysis are adequate to compare statistically significant phylogenetic relations (Olsen *et al.*, 1986). Therefore 16S rRNA and its encoding gene have been widely used to investigate community diversity. The rapidly growing 16S rDNA sequence data bank, accessible (http://www.ebi.ac.uk/) provides the opportunity to get information about 16S

rDNA sequences of the determined cultured and uncultured species (Dahllöf, 2002).

libraries and sequences retrieved from banding patterns (Dahllöf, 2002).

**3. Fingerprinting techniques and their application areas** 

In spite of the advantages of using 16S rRNA molecule for phylogenetic analysis, the main limitations are that the heterogeneity between multiple copies of this molecule in one species interferes pattern analysis, confuses the explanation of diversity obtained from clone

Fingerprinting techniques provide a separation in microbial community according to their genetic pattern or profile (Muyzer, 1998). A variety of fingerprinting techniques such as *denaturing/temperature gradient gel electrophoresis, amplified ribosomal DNA restriction analysis, terminal restriction fragment length polymorphism, and single strand confirmation polymorphism* has been developed to assess diversity and dynamics in the ecosystem (Hofman-Bang, 2003). The first fingerprinting technique was used in 1980's, which based on the electrophoretic separation in high-resolution polyacrylamide gels of 5S rRNA and tRNA

determination on different taxonomic level (Amann *et al.*, 1995; Head *et al.*, 1998).

Fig. 1. The rRNA phylogenetic tree of life (Madigan *et al.*, 2009).

and location and also identification of species (Hofman-Bang, 2003). This approach comes into prominence because of fast, less labor-intensive features (Muyzer and Smalla, 1998).

These methods have been used to characterize the microbial diversity in different environments such as activated sludge (Liu *et al.*, 1997; Curtis and Craine, 1998), anaerobic reactors (Leclerc *et al.*, 2004), sediments (Muyzer and De Wall, 1993, Cetecioglu *et al.*, 2009), lake water (Ovreas *et al.*, 1997), hot springs (Santegoeds *et al.*, 1996), biofilm (Santegoeds *et al.*, 1998). The method can be used for as both qualitative and semi-quantitative approaches on biodiversity estimations.

In this chapter, these genetic fingerprinting techniques based on gel electrophoresis are discussed. Also exemplarily applications are presented.
