Hybridization

Question 1

Renaturation of DNA

Answer 1

• reannealing of ssDNA to dsDNA

- also called hybridization

Question 2

Hyperchromic effect

Answer 2

increase in absorbance of DNA when it is denatured

The bases become unstacked and can thus absorb more light.

Question 3

Hypochromic effect

Answer 3

decrease in absorbance of DNA when it hybridizes

Question 4

Tm (melting temperature)

Answer 4

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)

Question 5

Hybridisation Technique

Answer 5

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

Question 6

Factors affecting Tm

Answer 6

Increases:

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

Question 7

Probe Sequence

Answer 7

use to search for identical or similar sequences in the sample

Question 8

Stringency of Hybridisation

Answer 8

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

Question 9

Homologous vs Heterologous Hybridisation

Answer 9

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

Question 10

Nucleic Acid Labelling

Answer 10

• 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**

Question 11

Random Priming

Answer 11

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

Question 12

Blotting

Answer 12

transfer of nucleic acid/protein to a solid membrane support

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

Question 13

Southern Blotting

Answer 13

• 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

Question 14

Northern Blotting

Answer 14

• 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

Question 15

Colony Hybridization

Answer 15

• colony (phage) screening

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

Question 16

DNA Microarrays

Answer 16

• 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

Question 17

In Situ Hybridisation

Answer 17

• 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

Question 18

FISH

Answer 18

• 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