*3.2.6 Temperature gradient gel electrophoresis*

*Human Microbiome*

[37], for example, DGGE and qPCR.

*3.2.4 Genetic fingerprinting of gut microbiota*

*3.2.5 Denaturing gradient gel electrophoresis*

new strains without the availability of a compatible probe.

in the stool or gut mucosa samples. In this technique, fluorescent probes or dye molecules are used that intercalate between the double strand of DNA molecules or 16 s RNA amplicons. These probes send a strong signal, and its intensity is directly proportional to the amount of DNA sample present. Sometimes sequence-specific oligonucleotide probes are linked with molecular markers or complementary DNA sequence [34]. The primers designing is a crucial step in the RT-PCR technique; therefore, primers must be specific for all bacterial phyla or taxa or species present in a sample [35]. Real-time PCR has been used to investigate the state of the ecological environment in normal and obese persons [36]. Quantitative PCR technique is also used solely or in combination with other gel and non-gel-based techniques. This combination of protocols is used to understand the functional microbial diversity of gut microbiota in the patient of age and effect of antibiotics on gut microbes

Real-time PCR-based methods are suitable for the prediction of accurate phylogenetic analysis. The appropriate primers provide great help to know the composition of a microbial community and microbial load. The protocol is simple to complex, and all chemicals and consumables are easily available in laboratories. But, this is also suffering due to PCR biases, which percolate at each step of the protocol. Quantitative PCR cannot be used to detect new bacterial strains in the gut

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

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

microbiota without prior information of primers or probe.

**10**

of humans [39].

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 appropriate hybridization processes.

## *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* and *Actinobacteria*.

### *3.2.8 Probe hybridization-based methods*

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 the microbial ecosystem.

#### *3.2.8.1 Fluorescence in situ hybridization*

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 FISH. In the studies of microbial communities, the 16S rRNA gene amplicons are prepared and denatured in a solution. After that, both fluorescent probe and DNA strands are also added in the hybridization solution. In order to allow maximum hybridization process, some cross-linking agents like aldehyde or any precipitating agent (methanol) are also added and incubated in the reaction mixture and kept at 65–75°C for 12 h [45]. After ensuring that the hybridization process is completed, the intensity of fluorescence is measured by using suitable laser available fitted in the flow cytometry instrument. The combination of FISH and flow cytometry is a sort of high-throughput method used in the genome comparison of two different species in the gut sample [46]. The FISH technique is efficiently applied to compare two types of microbial communities such as breast- and formula-fed newborns, and two different species *Bifidobacterium* and *Atopobium* are identified [47]. The merits of this method are that it is semiquantitative and rapid. Due to the availability of diverse probes for specific phyla or species, FISH can be widely used in microbiome studies. But the technique completely failed to identify de novo identification of a bacterial strain. Some researchers have used FISH to estimate the time of sample stability and change in their species compositions with the passage of time and storage conditions.

## *3.2.8.2 DNA microarrays*

DNA microarray technology or DNA chip method is widely applied to learn more about the microbial ecosystem, particularly in gut microbiota. The component of the DNA microarray is a small chip containing a large number of microscopic spots on a solid surface which are used to immobilize fluorescent probes. DNA spots hold pico-level DNA, which is sufficient for hybridization process of a small part of a gene or its regulatory element with cDNA already immobilized on a DNA chip under suitable reaction environments. The microarray protocol includes the following: firstly, the 16S rRNA amplicon or extracted DNA from the samples is processed to make them fluorescent. Secondly, oligonucleotide probes are spotted and immobilized on the surface of the microarray chip [48]. Finally, hybridization is allowed between 16S rRNA amplicons and fluorescent probes. The fluorescence intensity after complete hybridization is quantified by using a laser. The microarray can identify the expression of hundreds of genes in a single experiment. The effect of *C. difficile* infection and its successful cure by fecal microbiota transplantation (FMT) is studied by microarray [49]. This method is quite fast and rapid and offers a high-throughput method for phylogenetic analysis of gut microbiota. It requires a very small amount of DNA for accurate analysis. The most noticed demerit of a microarray experiment is the possibility of cross hybridization, i.e., binding of multiple oligonucleotide probes to a single DNA fragment. In the absence of the probe, a microarray cannot identify a new bacterial species.
