DNA Hybridisation: DNA complementarity, hybridisation & its application

Question 1

How many nucleotides makeup DNA?

Answer 1

4

Question 2

What are the 3 components of a nucleotide?

Answer 2

A nitrogenous base, a ribose sugar and a phosphate group:

Question 3

Describe the structure of the individual components of a nucleotide

Answer 3

• The nitrogenous base comprises of either a single or double ring containing nitrogen ( shown in blue) and carbon (grey), the polar or charged groups result in Watson and crick base pairing
• This ring structure is attached to a 5 carbon pentose sugar (ribose). Ribose has a cyclical structure formed by an oxygen bridge between carbon 1 and 4 . The nitrogenous base is attached via carbon 1 of the sugar, it has a hydroxyl group attached at the position 3 of the ring.
• Also attached to the carbon 5 of the ribose sugar is one or more a phosphate group, in the slide a nucleotide monophosphate containing cytosine is shown

Question 4

Name the four nucleotides that make up DNA

Answer 4

Cytosine, Guanine, Thymine and Adenine.

Question 5

Describe the ring structure of Cytosine, Guanine, Thymine and Adenine.

What are purines and what are pyrmidines?

Answer 5

These fall into two molecular structures consisting of either a single or double nitrogen containing ring either a Pyrimidine or Purine, the easiest way to remember these pyrimidine contains a Y and include Cytosine and Thymine, Purines don’t and thus include Guanine and Adenine

Question 6

How is RNA different to DNA?

Answer 6

In RNA Uracil substitutes Thymine and base pairs with Adenine in RNA to form duplex structure. And uracil is of course a pyrimidine like thymine

Question 7

How do nucleotides join together?

Answer 7

Hydrogen bonding

Question 8

What groups do the hydrogen bonds form between?

What do the bases pair with and how many bonds do they form?

Describe the bonding in RNA or dsRNA

Answer 8

• This involves amine and carboxyl groups or amide and nitrogen’s within the ring structure
• In each pair there is a single purine and pyrimidine
• The consequence of this is that, Cytosine – Guanine pairing forms 3 bonds and is stronger than Thymine-Adenine forming just 2 bonds
• In dsRNA or RNA secondary structure Cytosine – Guanine pairing also occurs along with Uracil-Adenine pairing, but the rule still applies and GC pairing is stronger than AU pairing.

Question 9

What is the most common formation of DNA.

Answer 9

B-DNA

Question 10

What is the backbone of the DNA molecule formed from and what does it connect?

Answer 10

• The backbone of the molecule is formed from a phospho-diester linkage
• This connects the 3 and 5 prime carbons of the deoxyribose sugar of DNA

Question 11

What determines the stability of the DNA molecule?

Answer 11

• The stability of the structure is determined by the free energy of the molecule and energy minimisation, just as we discussed in protein structure at the beginning of semester 1.
• This makes double stranded DNA a dynamic molecule that is influenced by its molecular environment.

Question 12

What other bonding is involved in DNA?

Answer 12

This structure derives its stability from hydrogen bonding, and the internal arrangement of the bases; gaining additional stability by base stacking which is a form of hydrophobic interactions excluding water from the internal structure alongside van der waals forces

Question 13

What is the sugar phosphates joined by?

Answer 13

linked by phosphodiester bonds

Question 14

What is base-stacking?

Answer 14

hydrophobic interactions ->arrangement of bases set above each other internalised to the structure & excludes water

Question 15

What is the purpose of Van Der Waals forces

Answer 15

individually small but contributes to the stability

Question 16

Why is DNA said to be anti-parallel?

Answer 16

they have an opposite orientation

Question 17

Where are the bases of DNA found?

Why has DNA got an overall negative charge and what process is this utilised by?

Answer 17

• The bases are on the inside, forming stacked bases and the negatively charged phosphates (the dominant red and yellow colour in the model) are external giving DNA an overall negative charge and is the characteristic we use in gel electrophoresis

Question 18

What happens to the double stranded DNA structure when it is denatured?

Answer 18

• Conversion of a double stranded molecule → of single stranded molecules

Question 19

What does denaturing DNA interupt?

Answer 19

• Disruption of Hydrogen bonds within the double helix

Question 20

What condition to we change to denature DNA?

Answer 20

1. Occurs when DNA in solution is heated
2. Can also be induced by strong alkali or urea. This is achieved in solution by either heat or chemical denaturants such as formamide, urea or alkali

Question 21

How can we measure the denaturation of DNA?

Why does it work?

Answer 21

We can measure the denaturation of a DNA duplex using optical density measured at a wavelength of 260 nm

• This works because single stranded DNA absorbs UV light to a greater extent than double stranded DNA, this property is termed hyperchromicity

Question 22

What increases as temperature increases?

Answer 22

the optical density increases

Question 23

What does denaturation depend on?

Answer 23

• The denaturation of a DNA duplex depends upon the stability of the structure determined by its sequence of bases

Question 24

What is the term for the temperature at which 50% of the molecules have melted

Answer 24

Tm , this shown by the arrow on the graph

Question 25

What 5 factors does tm depend on?

Answer 25

• GC content of a molecule
• The Length of DNA molecule
• The Salt concentration of the solution ie the molecular environment
• pH (alkali is a denaturant)
• Number of Mismatches (unmatched base pairs within a duplex)

Question 26

What does more GC content mean?

Answer 26

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

Question 27

The longer the DNA molecule the more… and therefore?

Answer 27

hydrogen bonds the duplex will have and the more stable the structure is.

• However there is a diminishing return on this, and as you can see from the graph, a length beyond about 300 base pairs contributes little or no more to the stability of the structure
• Longer the contiguous duplex, the higher Tm
• More Hydrogen bonds within the molecule greater stability
• However little further contribution beyond 300 bp

Question 28

When we talk about salt solution what is the main ion we refer to?

Answer 28

Na+

Question 29

What effect on DNA does increasing the salt concentration have?

What does this mean?

Answer 29

Thus, increasing the salt concentration stabilises the structure and increases the Tm, thus it overcomes the destabilising effect of mismatched base pairs reducing specificity of base pairing in high salt environments.

Thus, adjusting the salt concentration is a common means of manipulating duplex formation and specificity of this process.

• A duplex containing mismatches may form and be stable at a given temperature in the presence of high salt concentration (lower part of the slide , whilst the same duplex would be unstable and dissociate at the same temperature in low salt (upper part of the slide)
• This is simply because of the stabilising effect of salt and its effect on changing the melting temperature of the duplex
• 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

Question 30

How does akali solutions such as NaOH and OH- ions impact on the stability of DNA? (3)

Give some example of alkali solutions

Answer 30

• Thus, fewer hydrogen bonds result in lower stability of the structure - OH- disrupts H bonding, so lower Tm
• So high pH (alkalinity) destabilises DNA duplexes
• Chemical denaturants disrupt hydrogen bonds
• Alkali, formamide, urea

Question 31

Define mis-match

Answer 31

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

Question 32

What does a mis-match cause? (3)

Answer 32

• Reduces Number of Hydrogen bonds, Fewer = lower Tm
• Shorter contiguous stretches of double stranded sequence = lower Tm

These combine to make the formation of a duplex less energetically favourable reducing the change in free energy on duplex formation

Question 33

When does renaturation occur?

What are the conditions?

What does the formation of the structure favour?

What is it facilitated by?

Answer 33

Therefore depending upon the energy and molecular composition of the system renaturation will occur and result in a change in the free energy for example as a result of
• Cooling
• Or neutralisation
• Formation of structure favours energy minimisation driven by change in free energy DG
• Facilitated by:
Slow Cooling
Neutralisation

Question 34

What is hybridisation?

Answer 34

The concept of hybridisation is similar to renaturation simply that it involves two DNA molecules that have been introduced to each other for example a short synthetic DNA (or primer) and genomic DNA.

Question 35

What factors influence hybridisation?

Answer 35

The factors influencing hybridisation are the same as renaturation and both are simply examples of duplex formation

Question 36

Stringency

Answer 36

Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity

Question 37

Under high stringency conditions how are complementary sequences made stable?

Why would we want low stringency?

Answer 37

Manipulating conditions: Limiting hybridisation between imperfectly matched sequences increases specificity
• Under High stringency: only
• complementary sequences are stable determined by a
• Temperature near Tm
• or
• Low salt concentration

So why would you ever think about employing conditions of low stringency, well the simple answer is the kinetics of hybridisation are much faster under low stringency conditions

Question 38

What is the primary aim of most hybridisation techniques?

Give some examples

Answer 38

The primary aim of most hybridisation techniques is to indicate the presence or absence of specific sequences of bases within a mixed population of nucleic acids as is represented on the slide

The different techniques may allow the determination of relative, or absolute abundances of those sequences within a sample

• For example by labelling a particular short synthetic oligonucleotide we sometimes call a probe or a primer and hybridising this to a mixed population of DNA in a sample we can use these to
• capture specific species of DNA,
• amplify segments of DNA
• or use them to quantify the number of molecules present containing the complementary sequence in the sample
• Identifies the presence of NA containing a specific sequence of bases
• Allows the absolute or relative quantitation of these sequences in a mixture

Question 39

What is a Probe?

How long are they?

What are they labelled with?

Answer 39

• Are always single stranded molecules but can be made from either DNA or RNA depending on the technique
• They are usually between 20 and 1000 bases in length but are generally, but not always, relative short synthetic molecules that are made chemically
• A probe is typically labelled with a fluorescent, or luminescent molecule
• In some techniques thousands or even millions of capture probes are used simultaneously such as in a microarray
A 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 40

What is northern blotting?

Does it have any limitations?

Answer 40

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

• It has a number of limitations and as a consequence is largely superseded by other techniques such as qPCR, digital PCR, microarrays or even RNASeq (a form of next generation sequencing)
• Southern blotting is the original technique which applies to DNA and was developed by Ed Southern

Question 41

Describe the process of nothern/ southern blotting

Answer 41

• The original technique of Southern or Northern blotting uses DNA or RNA respectively that is separated by gel electrophoresis
• which is then transferred by mass capillary flow of a buffer from a reservoir to a nylon membrane carrying the nucleic acid with it
• It is captured by and covalently bonded to the membrane and then hybridised with a labelled probe

Question 42

What are mircoarrays?

What do they form?

What are the practical details?

Answer 42

• 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

• Microarrays are one such technique and use probes attached to a specific location on a surface and hybridisation of molecules to that location identifies the molecules present you will learn more about these in subsequent lectures
• But they essentially form an ordered assembly or array of thousands or millions nucleic acid probes
• The Probes are covalently fixed to a solid surface such as a silicon or glass matrix, then a sample of interest is hybridised to the probes and the label visualised and measured as in the lower right image
Thus this approach can Simultaneously measure 50,000 different transcripts in a Cell, Tissue or Organ or several million single nucleotide polymorphisms (SNPs)

Question 43

How can a microarray be used for gene expression profiling, for example, a comparison of drug-treated cells and untreated cells

Answer 43

• For this RNA is extracted
• Labelled usually with a fluorescent molecule
• Hybridised to the array and the amount and location of the label measured
• We can then compare these measurements and determine changes in specific genes, identify signatures that relate to specific diseases or conditions
• This tells us how much of each and everyone of the transcripts in the human genome are being expressed and is an alternative to RNASeq, a next generation sequencing alternative that you will also learn about

Question 44

How can microarrays assess millions of SNPs

Answer 44

• 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 (GWAS)
• Similarly we can use microarrays to assess the presence or absence of millions of individual SNPs simply through hybridisation of genomic DNA to an array.
• This type of approach is still more common and cost effective than next generation sequencing and is how many Genome wide association studies have been done