Molecular Approaches
Once you get DNA out of an environmental sample and cut it with primers then there all kinds of things you can do with it. Such as:
(1) Cloning
– DNA fragments are incorporated into plasmids (each plasmid accepts only 1 fragment). Then you add a small amount of these fragment containing vectors (plasmids) to a very dense culture of bacteria (usually E. coli) so that is very unlikely that any single E. coli individual will take up more than 1 plasmid. In the vector you have genes encoding for antimicrobial resistance so after the culture grows you can select for E. coli that took up your plasmids. They do the work of amplifying the sequence many, many times. Your plate will contain single cell isolates – then you culture each single cell isolate into culture plates and then sequence these individual cultures. This was the only way to get mixed fragments separated from one another until high throughput sequencing came online.
(2) Fingerprinting approaches –> t-RFLPS & DGGE
-these are molecular fingerprinting techniques that give you information about the distribution of fragment lengths within a mixed DNA sample. DGGE separates the fragments using gel electrophoresis and you compare the # and intensity of bands between samples. t-RFLPS is conceptually similar but it sticks fluorescent tags onto the end of each fragment so that after they are clumped by size the fluorescence of similarly sized fragments can be quantified in a DNA sequencer.
(3) qPCR – add either reporter probes (designed for a specific gene) or a DNA binding dye that attached to double stranded DNA to your PCR mix. A qPCR machine can measure the fluorescence of these reporters or dyes during every PCR cycle
(4) high throughput sequencing
Functional Approaches
(1) Microbial biomass – chloroform fumigation
(2) Microbial biomass – substrate induced respiration
(3) Microbial biomass – Phospholipid fatty acids
(4) Fungal biomass – ergosterol or glomalin
(5) Microbial activity – enzyme profiles
(6) Microbial activity – C mineralization
(7) Microbial activity – N mineralization
(8) Microbial activity – trace gas (CH4 or N2O emissions)
(9) Microbial activity – denitrification potential, methanogenesis potential
