DNA Hybridisation

Question 1

What are the different components of DNA?

Answer 1

Nucleotides make up DNA and RNA- A, G, C and T
Pentose sugar:
5 carbons that form a cyclical structure with oxygen bridge
Carbons are numbered 1-5
Nitrogenous base joined to carbon-1
Phosphate group joined to carbon 5
Hydroxyl group carbon 3

Question 2

What are the different types of bonds in a nucleotide chain of DNA?

Answer 2

Sugar phosphates- linked by phosphodiester bonds
Base stacking- hydrophobic interactions lead to an arrangement of bases set above each other internalised to the structure and excludes water
Van der Waals forces- individually small but contributes to the stability
3’ to 5’ linkage between the pentose ring

Question 3

What does it mean when DNA is denatured?

Answer 3

Conversion of a double stranded molecule → of single stranded molecules
Disruption of Hydrogen bonds within the double helix
Occurs when DNA in solution is heated
Can also be induced by strong alkali or urea

Question 4

How can denaturation be measured?

Answer 4

Denaturation can be measured optically by absorbance at 260nm

Question 5

What is hyperchromicity?

Answer 5

Hyperchromicity- Increased absorption of light at 260nm on denaturation

Question 6

What is meant by Tm?

Answer 6

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

Question 7

What are the different factors affecting Tm?

Answer 7

Tm depends largely on hydrogen bonds:
GC content 
Length of DNA molecule
Salt concentration i.e. the molecular environment
pH (alkali is a denaturant)
Mismatches (unmatched base pairs)

Question 8

Why does GC content affect Tm and how can we calculate it?

Answer 8

Higher GC content= more hydrogen bonds= higher Tm
%GC =[ (G+C)/(G+C+A+T) ] x 100

where G is the number of guanine nucleotides, C is the number of cytosine nucleotides etc

Three h-bonds in G:C vs two in A:T pair

Question 9

How does molecule length affect Tm?

Answer 9

The longer the contiguous duplex, the higher Tm
More hydrogen bonds within the molecule means greater stability
However little further contribution beyond 300 bp

Question 10

How does salt concentration affect Tm?

Answer 10

Salt stabilises DNA duplexes
High [Na+] = High Tm
Increasing the salt concentration stabilises the structure increases the Tm and thus overcomes the destabilising effect of mismatched base pairing
High salt reduces the specificity of base pairing at a given temperature

Question 11

How does pH affect Tm?

Answer 11

Chemical denaturants disrupt hydrogen bonds
Alkali, formamide, urea

NaOH —->

Question 12

How do mismatched affect Tm?

Answer 12

A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
Reduces Number of Hydrogen bonds, Fewer = lower Tm
Shorter contiguous stretches of double stranded sequence = lower Tm
Mismatches also distorts the structure and destabilises adjacent base pairing

Question 13

What is renaturation and how can it occur?

Answer 13

Denaturation is reversible- we call this Renaturation
Reversal of denaturation:
Formation of structure favours energy minimisation driven by change in free energy ΔG
Facilitated by:
Slow Cooling
Neutralisation

Question 14

What is stringency in terms of DNA hybridisation?

Answer 14

Perfect matches have a higher Tm
Are thermodynamically favoured over Mismatches
We can use this property to form a complementary molecule with no mis-matches
Stringency:
Manipulating conditions- limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity

Question 15

What are complementarity and hybridisation useful in?

Answer 15

Complementarity and hybridisation are at the heart of many nucleic acid-based techniques:
Northern blotting
Southern blotting
Microarrays
Dideoxy and Next-Gen Sequencing
PCR
Cloning

Question 16

How can nucleic acid hybridisation be used?

Answer 16

Nucleic acid hybridisation techniques:
Identifies the presence of NA containing a specific sequence of bases
Allows the absolute or relative quantification of these sequences in a mixture
Hybridisation uses the ability of NA to form specific duplexes
Uses the complementarity and hybridisation of labelled nucleic acids
These molecules are referred to as “probes”

Question 17

What is a probe?

Answer 17

An ssDNA (or RNA) molecule
Typically 20-1000 bases in length
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
In some techniques thousands or millions of probes are used simultaneously

Question 18

What are the examples of nucleic acid blotting techniques? (What is northern and southern blotting)

Answer 18

Northern blotting an adaptation of Southern blotting:
Analysis of mRNA or DNA
Limited technique only detects one gene at a time and small number of samples
The gel based techniques are time consuming and messy
Largely superseded by quantitative PCR or other techniques

Question 19

Recall the steps of Northern and Southern blotting?

Answer 19

Extract RNA or DNA
Gel electrophoresis
Transfer to nylon membrane
Add labelled probe
Probe hybridises to mRNA transcript in sample
Detect hybridisation
Mass flow trough gel by capillary action passively carries the nucleic acid
Potential applied across the gel electrophoretically transfers the -ively charged nucleic acid Nylon or nitrocellulose membrane capture nucleic acid and hybridises it with a label probe.
More typically today the gel is sandwiched by two electrodes and a voltage is applied across the two electrodes. The negatively charged DNA migrates towards the positive electrode and is transferred to the membrane electrophoretically. Again the DNA or RNA is captured by and covalently bonded to the membrane and then hybridised with the label probe.

Question 20

What are microarrays?

Answer 20

An ordered assembly of thousands nucleic acid probes
Probes are fixed to a solid surface, then sample of interest is hybridised to the probes
Simultaneously measuring 50,000 different transcripts in a cell, tissue or organ
Probes are fixed to a solid surface such as glass or silicone

Question 21

What are microarrays used for?

Answer 21

Microarrays can also assess millions of SNPs
DNA from 1 person on 1 microarray
Detects 2.5 million SNPs simultaneously
Result- homozygous or heterozygous for each SNP e.g. rs1333049: CC, GG or CG
Used in Genome Wide Association studies