Table 2.

16S rRNA-based molecular approaches for studying microbial ecology in the chicken gut (64–67).

Approach	Sample capacity	Applications	Challenges and confines	Advantage
SEQUENCING ANALYSIS TARGETED AMPLICONS
16S rDNA sequencing	Limited w/ Sanger sequencing. Non-limiting w/ next-gen sequencing	16S rRNA gene sequence, wide range identification of genus/ species/ strain, as database rich	Bias in DNA extraction and Primers, PCR amplification and numbers of clones, costly, laborious	Each clone represents single molecule of rDNA, Allows precise identification of a relatively small number of OTUs
Real-time PCR (RT-PCR)	Limited	Specific gene expression in targeted groups, high in sensitivity	Bias in DNA extraction and RT-PCR, costly
PROFILING APPROACHES
Fingerprinting DGGEa, TGGEb, TTGEc, T-RFLPd, and SSCPe	Good	Amplify common 16S rDNA sequences, diversity profiles within the targeted group, rapid, comparative	Bias in DNA extraction, primers, inter and intra laboratory reproducibility remains a major challenge. Provides relatively coarse taxonomic resolution, data usually is qualitative or semi-quantitative	Amplicons may be used from sequencing
GENE QUANTIFICATION
FISH6	Limited	Enumeration of the bacterial population	Laborious at the species level	Sensitivity has been improved using fluorescent probes
DNA MICROARRAY TECHNOLOGY
Diversity arrays	High	Diversity profiles, different gene expression levels	Laborious in development, costly
DNA microarrays	High	Transcriptional fingerprint, comparative	Bias in nucleic acids extraction and their labeling, costly

^aDGGE, denaturing gradient gel electrophoresis;

^bTGGE, temperature gradient gel electrophoresis;

^cTTGE, temporal temperature gradient gel electrophoresis;

^dT-RFLP, terminal restriction fragment length polymorphism;

^eSSCP, single strand conformation polymorphism; ^f FISH, fluorescence in situ hybridization.