Culture independent methods

27 important questions on Culture independent methods

What are advantages of culture independent methods?

Cells can be identified without knowing their culture conditions, there is no culture bias and it presents an overview of the biodiversity

What are disavantages of culture independent methods?

It does not show activity, extraction bias, primer bias and unrepresentation of low-abundance microbes

What can fingerprinting methods be used for?

Determine the general community composition in a lot of different samples and monitoring it over a period of time
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What is Denaturing Gradient Gel Electrophoresis (DGGE)?

A technique used to separate short-to medium-length DNA fragments based on their melting characteristics

What is the migration based on in DGGE?

The GC content

What does it mean when there are more bands in DGGE?

The sample is more diverse

What does it mean when there are brighter bands in DGGE?

The organism is more abundant

What is a GC clamp?

When there is a G or C base present in the last 5 bases at the 3' end of the primer

What is the function of a GC clamp?

Help improve the specificity of primer binding

What is Terminal restriction fragment length polymorphism (TRFLP)?

A technique that utilizes specific PCR-fragment lengths of individual genotypes

What does the size of the fluorescent fragment correspond to in TRFLP?

The distance from the labelled primer to the nearest restriction site

What is dot-blot hybridization?

A technique where complementary single-stranded sequences of the probe hybridizes with single-stranded sequences of the samples

What is the advantage of dot-plot hybridization?

It does not involve running the sample on a gel matrix

What does each spot represent in a microarray?

A single gene

What is the disadvantage of a microarray?

It is expensive, labour intensive and not specific enough to differentiate between closely related species

What is illumina sequencing?

A sequencing technology that uses sequencing by synthesis to detect individual DNA bases as they are added to a growing strand

How are the DNA bases identified in illumina sequencing?

Fluorescently-labelled reversible terminator nucleotides are used and the fluorescence emitted from each added nucleotide is captured

What are advantages of Illumina sequencing?

It is high throughput and can be used to generate milions of sequences per sample

What are disadvantages of Illumina sequencing?

It only generates short reads, so no good phylogenetic analysis is possible

Fingerprint methods are not quantitive, what method is?

A quantitive PCR or real-time PCR

What is measured in real-time PCR?

The accumulation of amplication product

How is real-time detection of PCR products enabeled?

The inclusion of a fluoresent reporter molecule that yields increased fluorescence with an increasing amount of product DNA

What is the melting curve analysis?

A post-PCR method that detects differences in the melting temperature of DNA as it does from double to single stranded

What can the melting curve analysis be used for?

To determine the specificity of the amplicons

What happens in the melting curve analysis when the temperature increases?

The dsDNA denatures and the dye dissociates and thus the fluorescence decreases

What are primer dimers?

Complementary sequences at the 3' end of primers shorter than the target

What are non-specific amplicons?

When primers bind to sequences they shouldn't

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