**4. Discussions**

RAPD and ISSR methods were generally used for genetic diversity and populations study; those methods also could be used to analyze the breeding relationship. For species identification and genetic resource/diversity analysis, RAPD and microsatellitsa method were recommended (Liu & Cordes, 2004). The RAPD techniques has been used for discrimination of populations of species of the genus Barbus, grouper, Nile perch and wreck fish, salmonids, among others (Partis & Wells, 1996; Callejas & Ochando, 2001; Asensio et al., 2002; Jin, Cho, Seong, Park, Kong & Hong, 2006). Genetic analysis with RAPD markers is relatively easy, fast, and efficient. RAPD analysis, however, may not be practical for identifying interbreed species (Martinez, Elvevoll & Haug, 1997). SSRs are inherited in a codominant fashion. This allows one to discriminate between homo- and heterozygous state, and increases the efficiency of genetic mapping and population genetic studies. ISSR markers have recently been used successfully for genetic analysis in hatchery and wild *Paralichthys olivaceus* strains. It indicates that molecular marker systems contribute greater levels of capability for the detection of polymorphism, and provide a better solution for the assessment of genetic variations (Shikano, 2005; Liu, Chen, Li & Li, 2006).

Fig. 8. UPGMA consensus dendrogram of dissimilarity among individuals analysed using the primer ISSR 15.

According to our results, RAPD and ISSR method can be effectively discriminate giant grouper from different sources, such as cultivate and wild, even if fish are from different cultivate farms. But cobia and red coral trout were less discriminate ability. The study found that ISSR and RAPD methods were positively high correlations. Giant grouper species have highly genetic diversity. A comparison of RAPD and ISSR patterns in 14 giant grouper samples, ISSR primers have higher polymorphism and fewer bands than those of RAPD primers. It could provide simple and convenient method to discriminate genetic variation of giant grouper samples. In this study, ISSR method could distinguish genetic variation within specie and different populations. Some reports also have suggested that ISSR may reveal a much higher numbers of polymorphic fragments per primer than those of RAPD (Esselman, et al., 1999). Among these markers, microsatellite DNAs have revolutionized the use of molecular genetic markers in the applications mentioned before, and the markers are destined to dominate this type of studies in the coming years (Asensio, 2007). It also has been revealed as important tools in studies regarding the genetic structure of populations, phylogeographic relations and phylogenetic reconstruction in fish (Antunes, et al., 2010).

### **5. Conclusion**

92 Gel Electrophoresis – Advanced Techniques

For screening ISSR primers, the ISSR primer15 (ISSR15: 5'-(AG)8TG -3') was better distinguished than other primers. The result was shown in Fig 7. The primer ISSR 15 was generated 10 to 16 bands and in size from 200 to 2000 bp. For dendrogram analysis, three groups were identified. Group I, the 71412, 71413, 71422, and 82011 were clustered in the group. Group II, including 81621, 91321, 71411, and 91712 samples; group III including 71423, 80622, 91322, 71421, and 81623 samples. All the nodes of the dendrograms ranged

Fig. 7. ISSR profiles of the 14 red coral trout obtained using the primer ISSR 15. Lanes M: Bio-100 bp DNA Ladder. Lanes: 4, 5, 6, 8, 9, 10, and 12 (71411, 71412, 71413, 82011, 91321, 91711, and 91712) red coral trout fish wild population; Lanes: 1, 2, 3, 7, 11, 13, and 14 (71421, 71422, 71423, 81621, 80622, 81623, and 91322) red coral trout fish cultivate population.

RAPD and ISSR methods were generally used for genetic diversity and populations study; those methods also could be used to analyze the breeding relationship. For species identification and genetic resource/diversity analysis, RAPD and microsatellitsa method were recommended (Liu & Cordes, 2004). The RAPD techniques has been used for discrimination of populations of species of the genus Barbus, grouper, Nile perch and wreck fish, salmonids, among others (Partis & Wells, 1996; Callejas & Ochando, 2001; Asensio et al., 2002; Jin, Cho, Seong, Park, Kong & Hong, 2006). Genetic analysis with RAPD markers is relatively easy, fast, and efficient. RAPD analysis, however, may not be practical for identifying interbreed species (Martinez, Elvevoll & Haug, 1997). SSRs are inherited in a codominant fashion. This allows one to discriminate between homo- and heterozygous state, and increases the efficiency of genetic mapping and population genetic studies. ISSR markers have recently been used successfully for genetic analysis in hatchery and wild *Paralichthys olivaceus* strains. It indicates that molecular marker systems contribute greater levels of capability for the detection of polymorphism, and provide a better solution for the

assessment of genetic variations (Shikano, 2005; Liu, Chen, Li & Li, 2006).

**3.4 ISSR methods of red coral trout**

**4. Discussions** 

from 90 to 100%. The result was shown in Fig 8.

We developed DNA molecular marker techniques which could be used to generate information for fish genetic diversity, species identification, trace genetic variation between different individuals in aquaculture, authenticate fish, fishery products and provide good reference resources for species sources and relationships.

### **6. Acknowledgment**

This research project was supported by the Council of Agriculture (COA) (97AS-4.1.2-AI-I2).

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**6** 

*Brazil* 

**Gel Electrophoresis for Investigating Enzymes** 

Enzymes are vital biological catalysts with essential action in the metabolism of all living beings. Moreover, enzymes have a very significant role in various industrial sectors, including baking, brewery, detergents, textile, pharmaceutical, animal feed, cellulose pulp biobleaching, biofuels and others (Polizeli et al., 2005; 2009; 2011). Industrial enzymes are especially produced from microorganisms, such as bacteria, yeasts and filamentous fungi. Here, we describe some basic knowledge about microbial enzymes with potential application in industry, their properties and some biochemical methods for the detection of amylase, pectinase, proteases, and xylanase activities on polyacrylamide gel electrophoresis. These methods are more qualitative procedures than quantitative, once they may be used to confirm the electrophoretic homogeneity of purified enzymes through chromatographic process. In addition, we described the principle of each method approached in this chapter,

Enzymes are optimum biological catalysts present in all living beings, and they, under adequate conditions, catalyze in their active sites the natural substrates from the metabolic route reactions. Quite often, the metabolic enzymes act in a sequence, and the product

Some properties of the enzymes make them excellent competitors against traditional chemical catalysts, due to their great catalytic efficacy (kcat), considering that the main

enzyme1 Substrate1 Product1 Enzyme1 (1)

enzyme1 Substrate 2( Product 1) Product 2 Enzyme2 (2)

generated in a reaction becomes the substrate for the following phase, as diagramed:

which grants a better understanding of each procedure.

**1.1 Overall considerations about industrial enzymes** 

**1. Introduction** 

Maria de Lourdes T. M. Polizeli1\*, Simone C. Peixoto-Nogueira1, Tony M. da Silva1, Alexandre Maller2 and Hamilton Cabral3

**with Biotechnological Application** 

*Sciences and Letters of Ribeirão Preto, São Paulo University*

*School of Medicine of Ribeirão Preto, São Paulo, São Paulo University* 

*School of Pharmaceutical Science of Ribeirão Preto, São Paulo University* 

*1Biology Department, Faculty of Philosophy* 

*2Biochemistry and Immunology Department* 

*3Science Pharmaceutical Department* 

