Question

We will detect environmental microbes by microscopic, culture-based, and molecular approaches. ​State the advantages and disadvantages of each approach.

Answer 1

Method	Advantages	Disadvantages
Fingerprinting techniques DGGE/TGGE SSCP,T-RFLPs Ribosomal Intergenic Space Analysis (RISA/ARISA) Length Heterogeneity PCR (LH-PCR)	Quick profiling of spatialtemporal variability Simultaneous analysis of large number of samples Bands on DGGE/TGGE and SSCP gels can be excised, amplified and sequenced	Bias associated with PCR Only predominant species are detected No direct taxonomic identification Time consuming, requires postPCR analysis of samples Analysis of short sequences(s(<500 bp) DGGE, difficult comparison between gels T-RFLPs and ARDRA; difficult resolution of microbial profiles s(<500 bp) DGGE, difficult comparison between gels T-RFLPs and ARDRA; difficult resolution of microbial profiles
Fluorescent in situ hybridization (FISH) and catalyzed reporter deposition FISH (CARDeFISH)	Phylogenetic identification Visualization of non- cultivable microorganisms Highly sensitive and quantitative Detection of different microorganisms simultaneously by using multiple fluorescent dyes	Sequence information is required for probe design and specific detection Difficult to differentiate between live and dead cells Difficult accessibility to target gene
Cloning and Sequencing	Taxonomic and phylogenetic analysis	Time consuming and laborious Semi-quantitative Sequencing of a limited number of clones describe only the dominant members of the microbial communities
Highethroughput sequencing techniques (Roche 454 FLX, Illumina/Solexa Genome Analyzer, etc.)	Faster and less expensive than traditional Sanger sequencing Multiple samples can be combined in a run	High cost and time-consuming data analysis
Quantitative PCR (QePCR) or Real Time (RTePCR)	Highly sensitive and quantitative Fast and accurate gene quantification	RT-PCR; difficult to obtain enough and good quality RNA
DNAechip array/microarrays DNA/RNA	No bias associated with PCR Rapid evaluation with replication The intensity of the hybridization signal is proportional to the abundance of the target organisms	Very costly and highly trained personal is needed for data analysis
Biosensors	Fast detection	Very costly and highly trained personal is needed for data analysis