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Genomics - SGUL (Sem 3) > DNA Hybridisation > Flashcards

Flashcards in DNA Hybridisation Deck (24)
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1
Q

As a recap, describe the structure of a nucleotide.

A

The structure of a nucleotide includes a nitrogenous base, comprising of either a single or double ring containing nitrogen and carbon. The polar/charged groups on these are important for base pairing.

This ring structure is attached to a 5 carbon or pentose sugar (ribose) with at least one hydroxyl group attached to the 3’ position. Attached to the 5’ carbon of the ribose sugar is a phosphate group.

2
Q

As a recap, describe the 4 nucleotides.

A

The 4 nucleotides fall into two molecular structures consisting of either a single or double nitrogen-containing ring (so either a pyrimidine (1) or a purine (2)).

An important difference in the structures resides in the charged or polar groups, providing the specificity of base pairing.

IN RNA, Uracil substitutes Thyamine and base pairs with Adenine in RNA to form a duplex structure.

3
Q

What is Watson and Crick bonding?

A

Watson and Crick’s bonding is the pairing of nucleotide bases within DNA, and it is largely determined by hydrogen bonding between oppositely charged groups.

4
Q

What is the consequence (bonding-wise) of the difference in structure in pyrimidines and purines?

A

An important difference between the pairs is the number of hydrogen bonds formed (GC has 3, while TA has 2).
As a consequence, the GC pairing is stronger than the TA pairing (and the UA pairing).

5
Q

Give examples of three bondings in a DNA double helix that give it its stability.

A
  • sugar phosphates: linked by phosphodiester bonds
  • base stacking: hydrophobic interactions (the arrangement of bases as they are set above each other is internalised to the structure and excludes water)
  • Van der Waals forces: individually small but they contribute to the stability
6
Q

What are some ways in which DNA can get denatured?

A
  • by the addition of chemicals (such as strong alkali or urea)
  • by addition of heat
  • basically, by disruption of the hydrogen bonds within the double helix
7
Q

How can we measure denaturation?

A

Denaturation can be measured optically by absorbance at 260 nm.

Single-stranded DNA absorbs UV light to a greater extent than double-stranded DNA; this property is termed hyperchromicity.

The denaturation of a specific DNA depends upon the stability of the specific structure.

8
Q

What is hyperchromicity?

A

Hyperchromicity is the increased absorption of light at 260 nm on denaturation.

9
Q

What is the Tm?

A

The point at which 50% of all strands separate is called the melting temperature or Tm.

This characteristic is specific to an individual double helical structure, and we can use this knowledge to control the formation of the duplex.

10
Q

What is the correlation between Tm and GC content?

A

The higher the GC content, the more hydrogen bonds, the higher the Tm.

%GC = (G+C)/(G+C+A+T) x 100

11
Q

What is the correlation between Tm and molecule length?

A

The longer the continuous duplex, the more hydrogen bonds within the molecule giving it greater stability, the higher the Tm.

However, there is a diminishing return on this, and a length beyond about 300 base pairs contributes little or no more to the stability.

12
Q

What is the correlation between Tm and salt concentration [Na+]?

A

Salt stabilises DNA duplexes.
Increasing the salt concentration stabilises the structure, increases the Tm, and overcomes the destabilising effect of mismatched base pairs, reducing the specificity of base pairing.

13
Q

Explain the mechanism behind salt stabilising DNA.

A

DNA is a structure that is a balance between the repulsion of the charged backbones, and the attraction between the bases.

Adding salt reduces the charge repulsion, and shifts the balance towards attraction - increasing stability.

14
Q

How does pH affect Tm?

A

Chemical denaturants can disrupt hydrogen bonds.

With fewer hydrogen bonds, the stability of the structure is lowered. Thus, a high pH destabilises DNA duplexes.

15
Q

What is a mismatch?

A

A mismatch is defined as a base pair combination that is unable to form hydrogen bonds.

16
Q

What are some effects of mismatches on the DNA duplex?

A
  • they reduce the number of hydrogen bonds formed within the duplex
  • they distort the duplex and destabilise the adjacent base pairs.
  • they make the formation of a duplex less energetically favourable, reducing the change in free energy on duplex formation
17
Q

Denaturation is reversible: renaturation.

Explain this phenomenon, and what it is facilitated by.

A

Hydrogen bonds form and break spontaneously depending on the conditions. Thus, the breaking of hydrogen bonds is reversible. This is facilitated by the reversal of the factors influencing denaturation.

Depending upon the energy of the system and the corresponding molecules, renaturation will occur as a result of a change in free energy upon:

  • slow cooling
  • neutralisation
18
Q

What is the difference between renaturation and hybridisation?

A

Renaturation is the joining of two strands of DNA to form a duplex molecule that was originally a duplex molecule in the first place.

Hybridisation is the joining of two new strands of DNA to make a new duplex molecule.

19
Q

What is stringency?

A

Stringency is the concept of manipulating the conditions to select duplexes with a perfect match only.

20
Q

Complementarity and hybridisation are at the heart of many nucleic acids based techniques. List some.

A
  • northern blotting
  • southern blotting
  • microarrays
  • dideoxy and Next Gen sequencing
  • PCR
  • cloning

These all rely upon the specificity of complementary base pairing and the avoidance of mismatches using Tm and manipulating the conditions under which hybridisation is carried out.

21
Q

What is a probe?

A

It is a ssDNA (or RNA) molecule. Probes that are used to detect nucleic acids are designed to be complementary to a specific region of a target gene sequence which is unique to that gene.

It is usually between 20 and 1000 bases in length, depending on the technique that it is used for. It is typically labelled with a fluorescent, luminescent or radioactive marker.

22
Q

Describe Northern blotting.

A

Northern blotting is a technique adapted from Southern blotting for analysing the genes that are expressed by a cell, tissue or organ.

It has a number of limitations, and, as a consequence, is largely superseded by other techniques, such as qPCR or microarrays/
Southern blotting is the original technique, which applies to DNA, and was developed by Ed Southern.

23
Q

Describe the technique of Northern/Southern blotting.

A

The technique of Southern or Northern blotting uses DNA or RNA respectively that is separated by gel electrophoresis. That is then transferred by mass capillary flow to a nylon membrane.

It is covalently bound to the membrane and then hybridised with a labelled probe. The probe is then visualised by some means.

24
Q

Describe microarrays.

A

Microarrays are an ordered assembly of thousands of nucleic acid probes. The probes are fixed to a solid surface, then a sample of interest is hybridised to the probe.
It simultaneously measures 50,000 different transcripts in a cell, tissue or organ.