*3.2.4 Genetic fingerprinting of gut microbiota*

There are many culture-independent methods which mainly rely on gel-based separation and hybridization of 16sRNA sequences with the probe, for example, T-RFLP , DGGE, TGGE, and a combination of FISH and flow cytometry [38]. These methods are also known as fingerprinting methods have been used to investigate microbial diversity. In the last two decades, fingerprinting methods have offered more information related to the composition of gut microflora. This group of techniques does not provide information about the phylogenetic compositions of the gut ecosystem. But the disturbance in the composition of gut microbiome, which is also known as "gut dysbioses," caused by various environmental perturbations, including foreign bacterial species and antibiotics, could be investigated in the case of humans [39].

### *3.2.5 Denaturing gradient gel electrophoresis*

It is the most widely used method built on the separation of 16S rRNA gene amplicons on polyacrylamide gel electrophoresis from the complex mixture of DNA fragments that have the same length but different nucleotides sequence [40]. The electrophoretic separation of DNA fragments is influenced by the gel gradient generally produced by denaturant agents, for example, urea and/or formamide. Actually, when the current passes through the electrophoresis gel, 16S rRNA gene amplicons/DNA fragments get separated at various positions on gel according to their molecular weight in linear order, and it continues till their complete denaturation. Consequently, a heterogeneous mixture of DNA sequences is separated in the form of bands on the gel due to their compositions and denatured gradient present in the gel. DGGE is a semiquantitative technique and practiced in the comparison of two different types of microbial communities, i.e., from a healthy or diseased person. The technique is fast and can be used for the separation of multiple samples in single experiments [41]. The main disadvantage of DGGE is that the final results are influenced by PCR-originated bias and not suitable for direct identification of new strains without the availability of a compatible probe.

**11**

*Genomic Techniques Used to Investigate the Human Gut Microbiota*

It is well known that the DNA sequence influenced the value of the melting temperature (Tm) of a fragment. The high GC content is mainly responsible for high Tm, while the high AT content, for lesser Tm. That can be attributed to the fact that base pairing between G and C contains three hydrogen bonds, while A and T form two hydrogen bonds. Therefore, GC base pairing is more stable than AT in a DNA fragment. In the case of TGGE, denaturant agents are replaced with a temperature gradient. The final results of TGGE protocol mainly depend on amplicon stability and melting behavior, which are determined by GC content. Therefore, when current is passed through the slab gel, intact DNA strands get separated under the influence of temperature gradient inside the gel, but simultaneously, their movements are halted. Consequently, a banding pattern is produced under the influence of the temperature gradient; it is also known as fingerprinting or TGGE [42]. The technique of TGGE is fast and semiquantitative, but like DGGE, its results are also influenced by PCR predispositions. TGGE is not suitable for direct identification of microbes and phylogenetic analysis in absence of sequence-based suitable probes or

*DOI: http://dx.doi.org/10.5772/intechopen.91808*

appropriate hybridization processes.

and *Actinobacteria*.

the microbial ecosystem.

*3.2.8 Probe hybridization-based methods*

*3.2.8.1 Fluorescence in situ hybridization*

*3.2.7 Terminal restriction fragment length polymorphism assay*

RFLP is a classical molecular biological technique used for genetic fingerprinting in the case of animals and plant samples. Its variant T-RFLP is applied to compare the microbial communities and the microbial diversities of gut microbiota. In the process of T-RFLP technique, 16sRNA gene amplicons are isolated from different stool samples and then amplified by PCR. Next, 16sRNA gene amplicons are cut by using different types of restriction enzymes that produced restriction fragments of varying lengths following the isolation of the electrophoresis gel. So that due to different length/M. wt, restriction fragments move to different distances on gel, thus producing a banding pattern. Being fluorescent, each terminal fragment can be identified, whereby each band represents an individual species in the gut community. T-RFLP is used in the comparison of two ecological communities [43]; it is a fast and cheap technique, but not suitable for direct phylogenetic analysis of bacterial strains. Moreover, incompatibility between primer and target genomic DNA influences the T-RFLP results [44]; therefore, it can underrepresent the crucial species, for example, *Lactobacillus*

Probe hybridization techniques are mainly used for species identification and their quantification in particular samples. These methods depend on the complementarity between specific oligonucleotide probes and specific target DNA sequences in the bacterial genome. Two major techniques, namely, FISH and DNA microarrays, are included in this class of probe hybridization-based methods which are mainly used in phylogenetic identification and quantification of species living in

Basically, FISH is a cytogenetic technique that is applied to pinpoint a specific DNA sequence on the chromosomal landscape by using a suitable fluorescent probe. But, it is also widely used in gut microbiome studies, also known as bacterial

*3.2.6 Temperature gradient gel electrophoresis*
