Nucleic Acid Hybridisation

Question 1

Describe denaturation?

Answer 1

dsDNA is heated to partially denatured DNA to ssDNA. Can also denature DNA, but not RNA, using alkaline solutions.

Question 2

What is Tm?

Answer 2

Tm is the temperature at which 50% of the molecules remain double-stranded and 50% of the molecules are single-stranded = melting temperature.

Question 3

Describe hybridisation?

Answer 3

Denatured DNA cooled to or below Tm with other DNA strand and Watson-crick base paring allows annealing to form hybrid: G three bonds to C and A two bonds to T

Question 4

What can be used to create a hybrid?

Answer 4

A labelled, radiolabelled or
non-isotopically labelled,
probe, which can be DNA molecules,
RNA molecules or oligonucleotides.

Question 5

What is the DNA or RNA to be probed usually?

Answer 5

Immobilised on a solid support

Question 6

What does Tm depend on?

Answer 6

GC/AT composition of the probe region, higher GC content = higher Tm.
Salt (Na+) concentration: high salt stabilises even imperfect hybrids and low salt destabilises
Formamide: disrupts hydrogen bonds and so the presence if it lowers the Tm
The type of hybrid formed: DNA-DNA is low Tm, DNA-RNA is medium Tm and RNA-RNA is high Tm.

Question 7

What is homologous hybridisation?

Answer 7

When identical DNA sequences anneal; conditions are low salt and high temperature close to Tm.

Question 8

What is heterologous hybridization?

Answer 8

When similar but not identical sequences anneal, for example the same gene from different organisms.

Question 9

How can the extent if mismatching be altered during heterologous hybridisation?

Answer 9

By varying temperature, salt concentration and formamide concentration.

Question 10

What is the simple rule to calculate Tm?

Answer 10

= 2(A+T) + 4(G+C)

Question 11

What length oligonucleotides can use the simple rule to calculate Tm?

Answer 11

20-30 nt long

Question 12

What rule is more accurate for calculating Tm and when should it be used?

Don’t need to remember.

Answer 12

Tm (oC) = 81.5 + 16.6(log10 Na+) + 0.41 (%GC) – 0.72 (% formamide)- 600/length

Used for long DNA fragments > 60 nt. For oligos exclude the 600/length term

Question 13

What applications do nucleic acid hybridisation techniques have?

Answer 13

Identifying restriction fragments carrying a target gene, identifying mRNA size and concentration, screening gene libraries, analysis of complex restriction maps, identifying mutations and genes from gene libraries, analysing gene expression

Question 14

Describe the steps of nick translation?

Answer 14

1) With low level DNase 1 create random nicks in both strands
2) Add DNA pol 1, dNTPs alpha- 32 phosphate or other labelled nucleotide
3) Digestion by 5’ -3’ exonuclease activity and synthesis by polymerase activity occurs
4) Labelled DNA produced

Question 15

Describe how random hexamers can be used for primer extension to develop labelled probes?

Answer 15

1) Denature template DNA and anneal with random hexamer primers
2) Use a DNA pol without 5’ to 3’ exonuclease activity to anneal random hexamer primers
3) Add dNTPs including with one a32P labelled
4) Labelled probes produced.

Question 16

How are Riboprobes made?

Answer 16

1) Insert the cloned fragment into a vector containing specific phage promoters.
2) Linearise with restriction enzymes
3) Add T7 RNA polymerase, NTPs and labeled NTPs
4) Run off transcription to produce Riboprobes

Or PCR can also be used to add the phage promoters onto the probe fragment

1) Add T7 promoter sequence to the 5’ end of the PCR primer
2) Denature template DNA
3) Anneal primers by cooling
4) Carry out PCR

Question 17

What is end labelling?

Answer 17

The addition of a single or incorporation of multiple labels.

Question 18

How is end labelling carried out?

Answer 18

The gamma phosphate is transferred to DNA by polynucleotide kinase. Incorporates only a single label. Use alkaline phosphatase to produce all OH ends on DNA by removing the phosphate, use T4 polynucleotide kinase and the gamma-32 phosphate which is added onto the 5’ end.

Question 19

What is end labelling useful for?

Answer 19

Useful for tracking the ends of DNA molecules in some restriction experiment or for labelling oligonucleotides but not useful for longer pieces of DNA.

Question 20

What can end labelling do for oligonucleotides?

Answer 20

As they do not have 5’ phosphate when synthesised, it can add this and label it as hybridisation probes. One probe per oligo.

Question 21

What is end-labelling of oligonucleotides useful for?

Answer 21

Electrophoretic mobility shift and DNA-protein interaction studies.

Question 22

Give some example of radioactive labels and how they can be detected?

Answer 22

Usually 32P as alpha- dNTP or or gamma-NTP

Detection: autoradiography or phosphorimager

Question 23

Give some examples of non-radioactive labels and how they can be detected?

Answer 23

Biotin
Digoxigenin
Detection: Enzyme coupled to binding protein or antibody
Or Alkaline phosphatase and horseradish peroxidase are coloured

Question 24

How is digoxigenin incorporated?

Answer 24

As derivatized dUTP or UTP. Uses both primary and secondary or just primary anti-DIG antibody conjugated with alkaline phosphatase or horseradish peroxidase.

Question 25

How is biotin incorporated?

Answer 25

As biotinylated dUTP or UTP. Uses either furores neatly labelled street avidin or alkaline phosphatase/horseradish peroxidase conjugated to strept avidin.

Question 26

Describe a southern blot hybridisation?

Answer 26

1. Digest DNA with one or more restriction enzymes
2. Separate by size (agarose gel electrophoresis). Treat DNA on gel: fragment DNA by HCl, denature dsDNA with alkali and neutralise.
3. Blot onto nitrocellulose sheet
4. Fix ssDNA to filter by heat or UV to prehybridise.
5. Fix ssDNA with ss probe, labelled with 32P, biotin or digoxigenin.
6. Auto radiograph.

Question 27

How is northern blot analysis different to Southern blot?

Answer 27

RNA more sensitive to nucleases, ssRNA used, RNA is separated on a denaturation gel, containing formaldehyde or glyoxal (do not treat with alkali as it degrades RNA) and transferred to membrane and perform hybridisation using a labelled DNA or RNA probe in the same way as southern blots.

Question 28

What is a microarray experiment?

Answer 28

The analysis of expression of thousands of genes in a single reaction, involves hybridization of mRNA molecules to the DNA from the corresponding genes. The DNA immobilised on the microarray chip is normally oligonucleotides derived from each gene.

Question 29

How are microarrays analysed?

Answer 29

Each spot on the microarray contains a single sequence. mRNA samples isolated from target cells are labelled with fluorescent dyes to allow detection of the amount of mRNA binding to each template DNA. The microarray is scanned using a laser and the data on the intensity of the fluorescence of each spot is measured. The data generates a profile of gene expression in the cell.

Question 30

What are microarrays used for?

Answer 30

To compare differences in gene expression between cell populations, for example mRNA from cells treated in some way to see the effects of the treatment vs untreated or mRNA from cells from diseased tissue vs normal cells

Question 31

What application is in situ hybridisation used for?

Answer 31

Detection of mRNA temporal and spatial gene expression
Detecting infectious agents such as viruses,
-human papillomavirus (HPV); cervical cancer
-cytomegalovirus (CMV); range of symptoms
-Epstein Barr virus (EBV); Hodgkin’s lymphoma,
Burkitt’s lymphoma, nasopharyngeal carcinoma
Detection of gene expression levels for diagnostics and drug treatment decisions eg HER2
Detecting genetic defects and chromosome mapping