L25: something

Question 1

What is an overview of the steps in DeLongs paper

Answer 1

1. environmental sample collection
2. DNA extraction
3. DNA denaturation
4. Serial dilution and blotting; quantitative slot blot

Question 2

What is an overview of the steps in DeLongs paper, about probes

Answer 2

5. hybridisation of probes vs primers
6. detection of labelled probes like dot blot of soil DNA hybridised with radioactive probes

Question 3

illustrate nucleic acid hybridaisation

Answer 3

Often denature DNA prior to application to membrane DNA is immobilized on membrane via baking in an Oven (often under vacuum) prior to prehybridization
draw it

Question 4

What are hybridisation probes

Answer 4

– single-stranded DNA (~20 nt) homologous to target genes
–produced via a DNA synthesizer
– homologous to parts of the 16S rRNA gene
– end-labeled with 32 P (radioactive)
– DeLong used taxon-specific probes and control probes:
–bacterial-, archaeal-, and eucaryal-specific probes
–universal probe to set a 100% value (bacteria + archaea + eucarya) to allow determination of the group-specific % values
–negative control probe is DNA that is not homologous to any gene (non-specific binding ‘background noise’ to be subtracted from total, to normalize all probes for background)
– probes can be made to be genus-specific, etc, or homologous to functional genes