Culture-independent approaches

23 important questions on Culture-independent approaches

How do culture independent methods overcome the limitation that not all microorganisms can be cultured?

By studying nucleic acids directly extracted from environmental samples

What does 16S rRNA gene analysis give an overview of?

The overall miicrobial diversity, so who is there

What does functional gene analysis give an overview of?

The metabolic capabilities
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What do culture independent methods allow?

A snapshot of the biodiversity in the environment

What are disadvantages of culture independent methods?

They give no information about microbial activity and there is a DNA extraction and primer bias

Why is the 16S rRNA commonly used as a phylogenetic marker?

Because it is universally present in prokaryotes and very conserved and resistant to mutation

What is the function of the 16S rRNA?

Translating mRNA into proteins

What are general/universal primers?

Primers designed to bind to conserved regions and amplify variable regions

What can general/universal primers be used for?

To access the overall diversity in a sample

What are group specific primers?

Primers designed to amplify specific genes

What can group specific primers be used for?

To investigate the presence of microbes involved in a specific biogeochemical cycle

What is the difference between the ssRNA variable and conserved region?

The variable regions differ between microbes and conserved regions are similal between microbes

How can the product be visualized after a PCR?

By gel electrophoresis

What method can be used to differentiate between the cells that contain PCR product from the ones without the PCR product?

The blue-white screening

What does it mean when the colonies are white in the blue-white screening?

That the plasmid with PCR product is present in the E.coli

What does it mean when the colonies are blue in the blue-white screening?

That the plasmid isn't present in the E.coli

What three components are present in the selective agar for the blue-white screening?

An antibiotic, X-gal and IPTG

What is the function of the antibiotic in the selective media?

Selecting the cells that contain the plasmid

What is the function of the X-gal in the selective media?

Detecting the LacZ gene

What is the function of IPTG?

Inducing expression of the LacZ gene

How is the blue pigment in the blue-white screening caused?

If beta-galactosidase is present, it cleaves X-gal, releasing a product that forms an blue pigment

What happens if the LacZ gene is disrupted?

No functional beta-galactosidase is produced and X-gal remains uncleaved

What is a rarefaction curve?

A plot of the number of species as a function of the number of samples

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