*2.4.2 Gene cloning*

It depends upon the phenolic content or its composition that are present in mutants that can altered incapable cloning of the mutated gene and cloning of the wild type of the gene [normal]. If a plant lacks in a particular phenolic compound as which is the results of a mutation, the wild type of the gene will indicate as the wildtype allele that plays an important role in the compound for biosynthesis process. The sequence of the protein encoded by the gene and this sequence of the gene can

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*Phenolic Compounds*

at 94 °C.

*DOI: http://dx.doi.org/10.5772/intechopen.96740*

and length of the primer.

*2.4.3 Insertional mutagenesis*

lethal [10, 11].

*2.4.4 Map-based cloning*

*2.4.5 Candidate-gene approach*

*2.4.6 Quantitative trait locus mapping*

mapped to the sector of chromosome [10, 11].

be used to deduce the amino acid present in the compound. The polymerase chain

a.Denaturation: To separate the two DNA strands of the template performed

b.Primer annealing: It was performed at 45-70 °C depending on the GC-content

c.Extension: It was performed at specific temperature recommended by the manufacturer of the enzyme Particularly at 72 °C, during which the polymerase synthesizes the DNA delineated by the primers. This procedure is repeated 20-40 times, and in each cycle every template strand is being replicated. PCR is the preferably used technique in plant, animal and microbial biology also as medicine. PCR is also considered for cloning of genes and cDNA's, and taken

On the basis of known sequence, which is also referred as tag in the gene of

1.Tagging: A spontaneous or chemically induced mutant, which was been identified with no information accessible about the mutant gene.

2.Random Tagging: It is based on Principle of insertion of any gene. All the genes managing the trait of interest were rarely covered, until the mutation is not

It involves identification of the molecular marker(s) which are associated with the mutation. The mutant plants will show mutation when closely linked with predominant marker allele from the mutant parent, whereas wild-type parent marker allele will show wild-type plant Once the accurate mapping is achieved, the gene sequence will be obtained on the availability of genome sequence [10, 11].

This approach is possible once there is establishment of protein databases and large DNA then the candidate gene is defined as defective gene that can originate the mutant phenotype. Once the candidate gene is identified the sequence databases were searched to identify DNA or protein sequences from the candidate gene [10–12].

This can show the position of the identified candidate gene. QTL; the abbreviation for the quantitative trait loci), that can be defined as a genetic locus described by two molecular markers on a genetic map affecting a quantitative trait which is identified in 2 parental lines which differs from each other and are identified in F2 population. The F2 population are evaluated to separate the genetic and environmental effects on the trait in several locations and for years. Later the QTL was

reaction was revolutionized the clone genes and it involves three steps:

for genotyping using molecular markers [10, 11].

interest. There are two methods done for cloning purposes.

## *Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.96740*

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

1.It is widely distributed in all plants or in a specific plant.

3.Phenolic component exists as polymers.

**2.4 Biosynthesis of phenolic compounds**

*2.4.1 Protein isolation and purification*

the level of observation [8, 10, 11].

*2.4.2 Gene cloning*

follows: [10].

Ribéreau-Gayon (1972) classified the phenols into three origins which is as

2.It is less widely distributed to known in confined number of compounds.

The biosynthesis of PC to exhibit the origin of the various families which precursors. The review of accepted pathways, newly illuminated steps in the biosyn-

The conventional methodology for the isolation of proteins includes the techniques for the separation of biochemical, where the protein is isolated from the other proteins based on its special chemical and physical properties. This involves molecular mass i.e. size, shape, net charge and hydrophobicity. The isolation procedure starts with the mixture of a cell extract is recognize in the course of the enzyme activity. This mostly involves crushing of the tissue in an extraction buffer and the contents of the cell [proteins] become accessible. To avoid proteolytic degradation of the enzyme, add protease inhibitors i.e. phenylmethyl sulphonylfluoride (PMSF), in the mixture of the extraction buffer. The first step is a centrifugation where the enzyme is precipitated or otherwise ends up in the supernatant which is bound to the cell wall or the cell membrane, Soluble proteins are usually separated from one another by their solubility in high-salt solutions with supported variation. Add a salt, followed by centrifuge to remove the precipitated proteins that will be fruitful strategy for proteins to separate from each other. During saturation, ammonium sulfate is usually used to precipitate proteins, as most of the proteins in the solution of this salt. The quantity of ammonium sulfate must be mixed in order to avoid subset of the proteins in the extract which can be determined by enzyme activity assays on the various fractions. Likewise, other salts, trifluoroacetic acid, protamine sulfate, polyethylene glycol, apolar solvents etc. are often used for the proteins precipitation. The next step used for further purifying the enzyme is chromatography such as High-performance liquid chromatography (HPLC), Hydrophobic interaction chromatography (HIC), Ion exchange chromatography(IEC) Bio-affinity chromatography (BAC), fast protein liquid chromatography (FPLC) and Gel filtration or size exclusion chromatography (GFC). When a fraction is obtained is the only protein during this procedure then purification is completed to homogeneity, or any contaminants are remained below

It depends upon the phenolic content or its composition that are present in mutants that can altered incapable cloning of the mutated gene and cloning of the wild type of the gene [normal]. If a plant lacks in a particular phenolic compound as which is the results of a mutation, the wild type of the gene will indicate as the wildtype allele that plays an important role in the compound for biosynthesis process. The sequence of the protein encoded by the gene and this sequence of the gene can

thesis for isolation of protein, gene cloning, and protein characterization.

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be used to deduce the amino acid present in the compound. The polymerase chain reaction was revolutionized the clone genes and it involves three steps:

