**3.2 Amplified Ribosomal DNA Restriction Analysis (ARDRA)**

Recognition site of restriction enzymes are changed for different microbial species. The principle of amplified ribosomal DNA restriction analysis (ARDRA), also called as restriction fragment length polymorphism (RFLP), is based on this knowledge. The combination of PCR and restriction can, for example, be used for enhanced amplification of minor DNA templates (Green and Minz, 2005).

In the first step of this technique, ribosomal DNA is amplified by PCR to avoid undesired and/or dominant DNA templates. Then, the 16S rDNA PCR products are digested into specific DNA fragments by restriction enzymes. At the final step, the fragments are loaded to high-resolution gel for electrophoresis. The schematic representation of the principle of ARDRA is given in Figure 4. The main advantage of this technique is to provide rapid comparison of rRNA genes (Moyer *et al.*, 1994).

DGGE/TGGE is used for several purposes in microbial ecology. The first and the most common application is to reveal and to compare community complex of the microbial diversity within different environments. Curtis and Craine (1998) used this technique to show the bacterial complexity of different activated sludge samples. Connaughton *et al.* (2006) used PCR-DGGE method to find out bacterial and archaeal community structure in a high-rate anaerobic reactor operated at 18 C. This technique was used to reveal the microbial community in a lab-scale thermophilic trickling biofilter producing hydrogen (Ahn *et al.*, 2005). Another biofilm study showed the bacterial diversity in a river by 16S rDNA PCR-DGGE method (Lyautey *et al.*, 2005). In another study, the authors showed that the different bacterial and archaeal profiles within the highly polluted anoxic marine sediments in the different locations from the Marmara Sea (Cetecioglu *et al.*, 2009). Ye *et al.* (2011) showed the temporal variability of cyanobacteria in the water and sediment of a

Furthermore the scientists use these techniques, mostly DGGE, to analyse the community changes over time. Santagoeds *et al.* (1998) used PCR-DGGE method to monitor the changes in sulphate reducing bacteria in biofilm. Ferris and Ward (1997) also performed similar approach to reveal seasonal changes in bacterial community from hot spring microbial mat. Kolukirik *et al.* (2011) used 16S rDNA PCR-DGGE technique to represent the local and seasonal bacterial and archaeal shifts in hydrocarbon polluted anoxic marine

These fingerprinting techniques are widely used to monitor simple communities instead of complex environments. It is one of the detection methods to analyse the cultivation/ isolation approaches and to determine the enrichment cultures (Santagoeds *et al.*, 1996;

Also DGGE/TGGE are commonly chosen for comparison of the efficiency of the DNA extraction protocols (Heuer and Smalla, 1997; Lieasack *et al.*, 1997) and the screening of the clone libraries (Heuer and Smalla, 1997; Lieasack *et al.*, 1997, Kolukirik *et al.*, 2011) because rapid and reliable results are caused to perform less time (Kowalchuk *et* 

Recognition site of restriction enzymes are changed for different microbial species. The principle of amplified ribosomal DNA restriction analysis (ARDRA), also called as restriction fragment length polymorphism (RFLP), is based on this knowledge. The combination of PCR and restriction can, for example, be used for enhanced amplification of

In the first step of this technique, ribosomal DNA is amplified by PCR to avoid undesired and/or dominant DNA templates. Then, the 16S rDNA PCR products are digested into specific DNA fragments by restriction enzymes. At the final step, the fragments are loaded to high-resolution gel for electrophoresis. The schematic representation of the principle of ARDRA is given in Figure 4. The main advantage of this technique is to provide rapid

Ward *et al.*, 1996; Teske *et al.*, 1996; Muyzer, 1997; Bucholz-Cleven *et al.*, 1997).

**3.2 Amplified Ribosomal DNA Restriction Analysis (ARDRA)** 

minor DNA templates (Green and Minz, 2005).

comparison of rRNA genes (Moyer *et al.*, 1994).

**3.1.2 Application area** 

lake.

sediments.

*al.*, 1997).

Fig. 4. Steps of ARDRA (a: Genomic DNA extraction, b: PCR reaction for specific region, c: restriction digestion, d: gel electrophoresis) (Dijkshoorn *et al.*, 2007).

The application areas of this technique are also similar to DGGE. It is varied from detection isolates or clones to determination of whole community in an environment. For these different purposes, different gel types can be used. While agarose gel is sufficient to detect isolates or clones, polyacrylamide gels are necessary for better resolution in the community analysis (Martinez-Murcia *et al.*, 1995).

In the literature, there are different studies performed by ARDRA. Lagace *et al.* (2004) identified the bacterial community of maple trees. A wide variety of the organisms were detected from different groups. Barbeiro and Fani used this technique to investigate more

Gel Electrophoresis Based Genetic Fingerprinting Techniques on Environmental Ecology 59

Single Strand Conformation Polymorphism (SSCP) is also a fingerprinting technique to separate same-length DNA fragments according to their differences in mobility caused by the secondary structure. The principle of this technique is represented in Figure 6. None of denaturant is used in this method to detect the mobility of the secondary structure of DNA fragments. Each band on SSCP gel corresponds to a distinct microbial sequence, indicating the presence of a microbial strain or species retrieved from the sample (Leclerc *et al.*, 2001; Lee *et al.*, 1996). The main limitation of SSCP, which is similar to DGGE/TGGE, is that one single strand DNA sequence can form more than one stable conformation and this fragment can be represented by multiple bands (Tiedje *et al.*, 1999). The advantage of this technique compared to other fingerprinting methods is that it does not require GC-clamp and gradient

SSCP is mostly performed to determine the microbial community profile in different environments such as bioreactor and natural ecosystems. Firstly Lee *et al.* (1996) applied this method to obtain genetic profile of microbial communities. Then Schwieger and Tebbe (1998) used SSCP to determine the community profile including up to 10 bacterial strains. In another study, this method was combined with colony PCR to determine population levels of single and multiple species within plant and environmental samples (Kong *et al.*, 2005). Schmalenberger *et al.* (2008) investigated bacterial communities in an acidic fen by SSCP following by sequencing analysis. In this study, each representative

Fig. 5. Steps of T-RFLP (Kaksonen, 2011).

gel. SSCP is easier and more straightforward.

**3.4 Single Strand Conformation Polymorphism (SSCP)** 

specific bacterial group, Acinetobacteria, within 3 sewage treatment plants (1998). In 1995, Vaneechoutte and his colleagues performed similar study for Acinetobacter strains. They showed that this technique is less prone to contamination problems for detection. In another study, ARDRA was used to screen bacterial and archaeal clone libraries to detect the microbial community within an anaerobic reactor to treat fodder beta silage (Klocke *et al.*, 2007). Also there are some studies to investigate the microbial community in soil (Smith *et al.*, 1997; Viti and Giovannetti, 2005).

## **3.3 Terminal Restriction Length Polymorphism (T-RFLP)**

Terminal Restriction Fragment Length Polymorphism (T-RFLP) is another fingerprinting technique to obtain profiles of microbial communities. The principle of this method is to separate the genes according to position of their restriction site closest to a labelled end of an amplified gene (Figure 5). The main difference from ARDRA is that the restriction enzymes using in T-RFLP only detect terminal restriction fragments (T-RF). Also this method is used qualitative and quantitative analysis like DGGE (Liu *et al.*, 1997).

The method is carried out in a series of steps including PCR, restriction enzyme digestion, gel electrophoresis and recognition of labelled fragments. Like most other fingerprinting techniques, PCR amplification of a target gene is the first step of T-RFLP.

After DNA extraction, target gene amplification is carried out using one or both the primers having their 5' end labelled with a fluorescent molecule. Then amplicons are digested by restriction enzymes. Following the restriction reaction, the digested DNA fragments are separated using either capillary or polyacrylamide gel electrophoresis in a DNA sequencer with a fluorescence detector so that only the fluorescently labelled terminal restriction fragments (TRFs) are visualized. At the final step, electropherom is obtained as a result of T-RFLP profiling. Using this graph, electropherom, only target restricted DNA fragments are detected and also satisfactorily quantified by automated electrophoresis systems. Quantification analysis gives an opportunity to make various statistical methods, such as similarity indices, hierarchical clustering algorithms, ordination methods, and selforganizing maps (Liu *et al.*, 1997).

In the literature, T-RFLP was carried out for different purposes like other fingerprinting techniques. In 1997, while Liu *et al.* used this technique to characterize microbial diversity in different environments such as activated sludge, enriched sludge from lab-scale bioreactor, aquifer sand, termite, Moeseneder and his colleagues (1999) optimized T-RFLP to determine marine bacterioplankton communities and to compare this technique to DGGE. In 2000, Horz and his colleagues reported major sub-groups of ammonia oxidizing bacteria by using amoA functional gene. Methane-oxidizing bacteria from landfill site cover soil were detected by T-RFLP combined with RNA dot-blot hybridization (Stralis-Pavese *et al.*, 2006). Also in the same study, RFLP method is used to screen clone libraries. Lueders and Friedrich tried to determine PCR amplification bias by T-RFLP in 2003. Blackwood and his colleagues used T-RFLP for quantitative comparison of microbial communities from different environments such as soil and bioreactors (2003). Additionally this technique was used to screen clone libraries (Moeseneder *et al.*, 2001). Liu *et al.* (2011) performed T-RFLP to determine the microbial shift during bioremediation of petroleum hydrocarbon contaminated soil.

Fig. 5. Steps of T-RFLP (Kaksonen, 2011).

58 Gel Electrophoresis – Advanced Techniques

specific bacterial group, Acinetobacteria, within 3 sewage treatment plants (1998). In 1995, Vaneechoutte and his colleagues performed similar study for Acinetobacter strains. They showed that this technique is less prone to contamination problems for detection. In another study, ARDRA was used to screen bacterial and archaeal clone libraries to detect the microbial community within an anaerobic reactor to treat fodder beta silage (Klocke *et al.*, 2007). Also there are some studies to investigate the microbial community in soil (Smith *et* 

Terminal Restriction Fragment Length Polymorphism (T-RFLP) is another fingerprinting technique to obtain profiles of microbial communities. The principle of this method is to separate the genes according to position of their restriction site closest to a labelled end of an amplified gene (Figure 5). The main difference from ARDRA is that the restriction enzymes using in T-RFLP only detect terminal restriction fragments (T-RF). Also this method is used

The method is carried out in a series of steps including PCR, restriction enzyme digestion, gel electrophoresis and recognition of labelled fragments. Like most other fingerprinting

After DNA extraction, target gene amplification is carried out using one or both the primers having their 5' end labelled with a fluorescent molecule. Then amplicons are digested by restriction enzymes. Following the restriction reaction, the digested DNA fragments are separated using either capillary or polyacrylamide gel electrophoresis in a DNA sequencer with a fluorescence detector so that only the fluorescently labelled terminal restriction fragments (TRFs) are visualized. At the final step, electropherom is obtained as a result of T-RFLP profiling. Using this graph, electropherom, only target restricted DNA fragments are detected and also satisfactorily quantified by automated electrophoresis systems. Quantification analysis gives an opportunity to make various statistical methods, such as similarity indices, hierarchical clustering algorithms, ordination methods, and self-

In the literature, T-RFLP was carried out for different purposes like other fingerprinting techniques. In 1997, while Liu *et al.* used this technique to characterize microbial diversity in different environments such as activated sludge, enriched sludge from lab-scale bioreactor, aquifer sand, termite, Moeseneder and his colleagues (1999) optimized T-RFLP to determine marine bacterioplankton communities and to compare this technique to DGGE. In 2000, Horz and his colleagues reported major sub-groups of ammonia oxidizing bacteria by using amoA functional gene. Methane-oxidizing bacteria from landfill site cover soil were detected by T-RFLP combined with RNA dot-blot hybridization (Stralis-Pavese *et al.*, 2006). Also in the same study, RFLP method is used to screen clone libraries. Lueders and Friedrich tried to determine PCR amplification bias by T-RFLP in 2003. Blackwood and his colleagues used T-RFLP for quantitative comparison of microbial communities from different environments such as soil and bioreactors (2003). Additionally this technique was used to screen clone libraries (Moeseneder *et al.*, 2001). Liu *et al.* (2011) performed T-RFLP to determine the microbial shift during bioremediation of petroleum hydrocarbon

*al.*, 1997; Viti and Giovannetti, 2005).

organizing maps (Liu *et al.*, 1997).

contaminated soil.

**3.3 Terminal Restriction Length Polymorphism (T-RFLP)** 

qualitative and quantitative analysis like DGGE (Liu *et al.*, 1997).

techniques, PCR amplification of a target gene is the first step of T-RFLP.

## **3.4 Single Strand Conformation Polymorphism (SSCP)**

Single Strand Conformation Polymorphism (SSCP) is also a fingerprinting technique to separate same-length DNA fragments according to their differences in mobility caused by the secondary structure. The principle of this technique is represented in Figure 6. None of denaturant is used in this method to detect the mobility of the secondary structure of DNA fragments. Each band on SSCP gel corresponds to a distinct microbial sequence, indicating the presence of a microbial strain or species retrieved from the sample (Leclerc *et al.*, 2001; Lee *et al.*, 1996). The main limitation of SSCP, which is similar to DGGE/TGGE, is that one single strand DNA sequence can form more than one stable conformation and this fragment can be represented by multiple bands (Tiedje *et al.*, 1999). The advantage of this technique compared to other fingerprinting methods is that it does not require GC-clamp and gradient gel. SSCP is easier and more straightforward.

SSCP is mostly performed to determine the microbial community profile in different environments such as bioreactor and natural ecosystems. Firstly Lee *et al.* (1996) applied this method to obtain genetic profile of microbial communities. Then Schwieger and Tebbe (1998) used SSCP to determine the community profile including up to 10 bacterial strains. In another study, this method was combined with colony PCR to determine population levels of single and multiple species within plant and environmental samples (Kong *et al.*, 2005). Schmalenberger *et al.* (2008) investigated bacterial communities in an acidic fen by SSCP following by sequencing analysis. In this study, each representative

Gel Electrophoresis Based Genetic Fingerprinting Techniques on Environmental Ecology 61

DGGE is easier and more effective and also less equipment is necessary

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band was cut, then cloned and sequenced to identify species. Also SSCP was carried out to determine the bacterial profile in an aerobic continuous stirred tank reactor (CSTR) treating textile wastewater (Khelifi *et al.*, 2009). Also this technique was applied for determination of *Clostiridum* sp. based on difference their [Fe-Fe]-hydrogenase gene (Quemeneur *et al.*, 2010).

Fig. 6. Steps of SSCP (a: denaturation of ds DNA, b: electrophoresis) (Gasser *et al.*, 2007).

#### **4. Conclusion**

The principles of all fingerprinting techniques are similar. DGGE/TGGE, ARDRA, T-RFLP and (SSCP) have been developed to screen clone libraries, to estimate the level of diversity in environmental samples, to follow changes in community structure, to compare diversity and community characteristics in various samples and simply to identify differences between communities. While some of the scientists have showed that sensibilities and resolution of all these techniques are similar, DGGE is still more common application compared to other mentioned techniques. The main reasons of it are that the application of DGGE is easier and more effective and also less equipment is necessary for it.

#### **5. References**

60 Gel Electrophoresis – Advanced Techniques

band was cut, then cloned and sequenced to identify species. Also SSCP was carried out to determine the bacterial profile in an aerobic continuous stirred tank reactor (CSTR) treating textile wastewater (Khelifi *et al.*, 2009). Also this technique was applied for determination of *Clostiridum* sp. based on difference their [Fe-Fe]-hydrogenase gene

Fig. 6. Steps of SSCP (a: denaturation of ds DNA, b: electrophoresis) (Gasser *et al.*, 2007).

The principles of all fingerprinting techniques are similar. DGGE/TGGE, ARDRA, T-RFLP and (SSCP) have been developed to screen clone libraries, to estimate the level of diversity in environmental samples, to follow changes in community structure, to compare diversity and community characteristics in various samples and simply to identify differences between communities. While some of the scientists have showed that sensibilities and resolution of all these techniques are similar, DGGE is still more common application compared to other mentioned techniques. The main reasons of it are that the application of

(Quemeneur *et al.*, 2010).

**4. Conclusion** 


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**4** 

*Badacsony Hungary* 

**Gel Electrophoresis of Grapevine** 

Gizella Jahnke, János Májer and János Remete *University of Pannonia Centre of Agricultural Sciences* 

*Research Institute for Viticulture and Enology* 

**(***Vitis vinifera* **L.) Isozymes – A Review** 

Several articles were written from the beginning of the fifties about the presence of plant enzymes in multiple forms. The major discussion was questioning whether these forms are artifacts that rose during the purification or not. To show that these forms are not artifacts in 1952 Jermyn divided his original peroxidase juice into two parts by acidic precipitation. The precipitate contained the A and B, while the supernatant the C and D points. Two components were found in the purified peroxidase solution; one migrated to the anode, the

The first major step for the starting up of isozyme analysis was the development of starch gel electrophoresis by Smithies (1955). The second major step was the demonstration of the direct visualization of isozymes in the stach gel by specific histochemical stains by Hunter

The term isozyme was formed by Market and Moller (1959), using this word for different

Proteins - as the primary products of structural genes - are very alluring for the direct genetic studies. Variation in the DNA coding sequences frequently (but not all the cases) causes variation in the primary conformation of the proteins. In un-natural environments the detection of this variation is very difficult, because in such conditions the base of the separation is only the size of the protein (molecular weight). In natural environments the change of a single amino acid can detectably modify the migration. The extraction from a single tissue can contain a lot of proteins, which - in the case of non-specific (e.g. Comassie blue) staining - can result in a complex pattern, that makes it difficult to identify the homolog (allelic) and non-homolog enzymes. This problem can be solved by the application of enzyme-specific staining after the electrophoresis (Shields et al. in Tanksley and Orton,

The analysis of isozymes and their functions is the subject of functional genomics. The study of the gene expression in the level of RNA and proteins can give answers to a lot of open

**1. Introduction** 

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