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Molecular Biology > Hybridization > Flashcards

Hybridization Flashcards

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1
Q

Renaturation of DNA

A
  • reannealing of ssDNA to dsDNA

- also called hybridization

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2
Q

Hyperchromic effect

A

increase in absorbance of DNA when it is denatured

The bases become unstacked and can thus absorb more light.

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3
Q

Hypochromic effect

A

decrease in absorbance of DNA when it hybridizes

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4
Q

Tm (melting temperature)

A

temperature where the absorbance of UV light is 50% between the maximum and minimum, i.e. where 50% of the DNA is denatured

= 4 x (number of G/C) + 2 x (number of A/T)

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5
Q

Hybridisation Technique

A
  1. heat and separate DNA
  2. attach ssDNA to filter
  3. add ssDNA second sample to filter bound DNA
  4. hybridisation occurs if the two strand have any related sequences

Result: more closely related sequences = more hybrid molecules and high proportion of labelled DNA bound to filter

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6
Q

Factors affecting Tm

A

Increases:

  1. increasing G-C content
  2. increasing Na+ content
  3. increasing hybrid length
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7
Q

Probe Sequence

A

use to search for identical or similar sequences in the sample

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8
Q

Stringency of Hybridisation

A

High stringency: temperature of hybridisation closer to Tm for perfect match
- find exact sequence
Low stringency: temperature of hybridisation much lower than Tm for related sequences

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9
Q

Homologous vs Heterologous Hybridisation

A

Homo: probe and target contain identical sequences (high stringency)
Hetero: probe and target contain similar but non identical sequences (low stringency)

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10
Q

Nucleic Acid Labelling

A
  • labelling DNA can be generated by end labelling of random priming
  • can be isotopic or nonisotopic labelling (direct or indirect)
    END labelling: polynucleotide kinase (forward or exchange reactions) with Klenow (fill in reaction)

*check this mechanism**

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11
Q

Random Priming

A
  • 6 base long fragments
  • random sequence
  • denature and anneal in presence of mixture of random nucleotides
  • Klenow subunits of DNA polymerase elongates sequence
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12
Q

Blotting

A

transfer of nucleic acid/protein to a solid membrane support

buffer movement upwards takes sample with it to botom surface of membrane (stained)

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13
Q

Southern Blotting

A
  • DNA sample, ssDNA/RNA probe
  • DNA separated by size on agarose gel + transferred on nitrocellulose, nylon membrane
  • radio labelled DNA fragment (probe) incubated with membrane
  • hybridised strands detected via film exposure
  • used to detect specific DNA sequence within DNA samples
  • good sensitivity and efficiency
  • used for things like fingerprinting or genome mapping
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14
Q

Northern Blotting

A
  • RNA analysis, ssDNA/RNA probe
  • RNA separated by size on agarose gel and transferred onto membrane
  • radiolabelled fragment (probe) incubated with membrane hybridized bands visualised
  • used to identify what is transcribed and the level of transcription
  • RNA needs to be stretched and relaxed
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15
Q

Colony Hybridization

A
  • colony (phage) screening

- if a bacterial colony or plaque is potentially carrying a fragment of interest, it can be screened via hybridization

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16
Q

DNA Microarrays

A
  • thousands of genes hybridised simultaneously
  • Probe: DNA complementary to gene of interest is generated and positioned in microscopic quantities on solid surfaces at specific positions
  • Target: DNA generated by sample RNA is fluorescently tagged and allowed to hybridize to DNA chip. 2 sample target populations analysed simultaneously

Monitor fluorescence with laser excitation. Different target DNA samples have different fluorophores

17
Q

In Situ Hybridisation

A
  • RNA/DNA fixed and probed in situ, ie. in position it occupies in living tissue
  • cut into sections, hybridized with probe, examine with microscope
  • used to examine levels of expression/transcription
18
Q

FISH

A
  • chromosome painting
  • fluoresence in situ hybridisation
  • chromosome specific probes
    laboratory technique for detecting and locating a specific DNA sequence on a chromosome. The technique relies on exposing chromosomes to a small DNA sequence called a probe that has a fluorescent molecule attached to it.
  • gene identification on a chromosome
  • can be used to visualise abnormalities

Molecular Biology (11 decks)

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