DNA Hybridisation

Question 1

What is the structure of a pentose sugar?

Answer 1

5 carbon ring – with an oxygen bridge between carbon 1 and carbon 4. C1 attached to nitrogenous base. C5 attached to phosphate group. C3 attached to hydroxyl group (OH)

Question 2

What are pyrimidines and purines?

Answer 2

Pyrimidines – nucleotide bases cytosine and thymine, which are single N-containing rings.
Purines- Adenine and guanine, which are double N-containing rings.

Question 3

How many hydrogen bonds do the base pairs form?

Answer 3

Cytosine-guanine = 3 H bonds, hence stronger

Thymine-adenine=2 H bonds

Question 4

What causes DNA to be denatured?

Answer 4

Heat, strong alkalis, urea, chemical denaturants- formamide

Question 5

What is the relationship between absorbance and strandedness, and hence hyperchromicity?

Answer 5

The separation of DNA strands increases absorbance of UV light (hence at the 290nm wavelength- uv light range). This works because single stranded DNA absorbs UV light to a greater extent than Double stranded DNA.

Question 6

What is Tm and what factors affect it?

Answer 6

Tm, which stands for melting temperature, is the temperature at which 50% of all strands are separated/molecules have melted. Stability, thus Tm, of DNA molecule are affected by many factors:

• GC Content
• PH of solution (alkali= denaturant)
• length of DNA molecule
• Number of mismatches (unmatched base pairs within a duplex)
• Salt concentration of solution

Question 7

How does GC content affect Tm? What is the equation?

Answer 7

GC base pairing have 3 hydrogen bonds, as opposed to 2, So have more hydrogen bonds and thus will form stronger base pairing. Thus, the more GC pairs you have, the more hydrogen bonds within the molecule, which gives it more stability and hence there is a higher Tm
%GC = (G + C)/ (G + C + A + T) X 100

Question 8

How does molecule length affect Tm?

Answer 8

The longer the length of the molecule, the more hydrogen bonds there are present within the duplex and hence the more stable the structure is – higher Tm. However, the temperature makes no difference to the stability of the structure at around >300bp.

Question 9

How does pH affect Tm?

Answer 9

Chemical denaturants (alkalis, urea, formamide) disrupt hydrogen bonds. Alkalis, such as NaOH dissociate into Na+ and OH-. OH ions disrupt hydrogen bond pairing, thus fewer hydrogen bonds = reducing the stability of the structure. High pH= destabilises duplex and hence lower Tm

Question 10

How do mismatches effect Tm?

Answer 10

A mismatch is a base pair combination that is unable to form hydrogen bonds. Mismatches reduce the number of hydrogen bonds within the duplex and also distort the structure and destabilise adjacent base pairing. These both combine to make the duplex less energetically favourable. Fewer H bonds = Lower Tm. Formation of a duplex that is imperfectly matched has a lower Tm than its corresponding perfectly matched pairing.

Question 11

How does salt concentration effect Tm?

Answer 11

Salt (Cations, e.g Na+) increase the stability of a duplex, at a given temperature, and hence increase Tm. Thus, high salt conditions (at a given temp) overcome the destabilising effect of mismatched base pairing within the duplex, thus reducing the specificity of base pairing. This means A duplex containing mismatches can still form and be stable at a given temp in high salt concentration. Whilst the same duplex would be unstable and dissociate at the same temperature in low salt conditions.

Question 12

What factors influence renaturation?

Answer 12

Renaturation is simply facilitated by altering the factor that led to the denaturation of the duplex, for example cooling or neutralisation. A duplex is in an equilibrium and the hydrogen bonds break and form spontaneously depending on the conditions- thus the position of this equilibrium is facilitated by the factors influencing denaturation. Thus depending on the energy and molecular composition of the system renaturation will occur and result in a change of free energy (as a result of cooling and neutralisation).

Question 13

What is the similarity and difference between renaturation and hybridisation?

Answer 13

• Both renaturation and hybridisation describe the same process – i.e. formation of a duplex. Both involve the formation of duplex structure of two DNA molecules that have been introduced to one another. The factors influencing renaturation are the same as the factors influencing hybridisation.
• The distinction between the two is dependent on the context. Renaturation is when a double stranded molecule is denatured and is reformed using the same strands from before and after the denaturation/renaturation, whereas Hybridisation involves the addition of a third molecule (after strands have separated) e.g. a primer, probe or other strand that is introduced prior to ‘renaturation.’

Question 14

What is stringency

Answer 14

The effect of manipulating conditions to form perfect matching duplexes. It involves limiting hybridisation between imperfectly matched sequences and hence increases specificity.

Question 15

Which conditions enable formation of perfectly matched base pairing?

Answer 15

Under High stringency conditions, only perfectly matched duplexes can form. Whereas in low stringency conditions, hybridisation is able to occur where mismatches are able to form.

Question 16

What determines high stringency? Why would we ever want low stringency conditions?

Answer 16

High stringency conditions are determined by a temperature near the Tm, and low salt concentration. Under these conditions, only perfectly matched duplexes form, and only complementary sequences are stable (high specificity). We can favour the formation of perfectly matched duplexes by hybridising at a temperature near the Tm of a perfectly matched duplex.
However, we may want low stringency conditions because the kinetics of hybridisation are faster. High salt conditions fall under low stringency conditions, they allow duplex to form containing mismatched base pairs, as they reduce the specificity and at a given temperature and are able to overcome the destabilising effect of mismatches.

Question 17

What techniques in molecular diagnostics make use of Hybridisation and complementarity? What do they rely on?

Answer 17

-Northern and southern blotting
-Microarrays
-Sequencing – Sanger and next generation
-Recombinant DNA techniques i.e. cloning
-PCR
These techniques rely on specificity of complementarity of base pairing, avoidance of mismatch pairing by using stringency and calculating Tm of duplex, manipulating conditions under which hybridisation is carried out.

Question 18

What can we use nucleic acid hybridisation techniques for?

Answer 18

Capture specific species of DNA
Amplify DNA segments
Quantify the number of molecules present containing the complementary sequence in the sample.

Question 19

What is a probe? What are its characteristics?

Answer 19

A probe is a short, labelled DNA molecule or oligonucleotide that is used to detect unique sequences and are part of a gene. They are designed to be complementary to a unique/specific segment of the target gene sequence.
Probes are single stranded (either ssDNA or RNA). They are 20-1000 bases in length. (usually 20-60 but can be larger). They are usually, not always, short synthetic molecules that are made chemically. They are labelled with fluorescent or luminescent molecules. They are unique to specific genes and, since they are complementary are, able to form perfectly matched duplex (under high stringency conditions) with their complement.

Question 20

What is a primer?

Answer 20

A primer is more typically a short single stranded polynucleotide, 16-30 bases in length. Usually unlabelled and are used in enzymatic reactions to ‘prime’ the reaction.

Question 21

What is northern blotting and what are the limitations to the technique?

Answer 21

Northern blotting is adapted from southern blotting and involves analysis of DNA or mRNA. It has many limitations.
-It is a gel technique, hence is messy and takes time
-Only detects one gene at a time and small numbers of samples.
Hence it is largely superseded by quantitative PCR and other techniques

Question 22

How is northern blotting carried out? What is the modern version?

Answer 22

Northern blotting separates the DNA or mRNA by gel electrophoresis. It is then transferred onto a nylon membrane through mass capillary flow of a buffer from a reservoir – carrying the nucleic acid with it. The RNA/DNA is captured and covalently bonded to the membrane. Labelled probes are added to this and hybridise to the mRNA transcript in the sample allowing us to detect the hybridisation.
The modern day technique uses electroblotting where the gel is sandwiched between electrodes (rather than mass capillary flow) and a voltage is applied so the negatively charged DNA/RNA is transferred to the nylon membrane electrophoretically.

Question 23

What are Microarrays?

Answer 23

Microarrays are an ordered assembly of thousands of nucleic acid oligonucleotides that are covalently fixed to a solid surface (such as a silicon or glass matrix), known as capture probes. Each probe has a specific location and hybridisation to that location identifies a corresponding target sequence/ SNP transcript/ gene and hence identifies the molecules present. Microarrays use thousands or millions of probes attached to a surface. The sample of interest is hybridised to the probes and the labels are visualised and measured.
This can simultaneously measure 50,000 different transcripts in a cell, tissue or organ or several different SNPs

Question 24

What can microarrays be used for?

Answer 24

Can simultaneously measure thousands of genes or transcripts.
Can be used to detect absence or presence of millions of individual SNPS (can detect 2.5 million SNPS simultaneously from the DNA from 1 person on 1 microarray) simply through hybridisation of genomic DNA to an array. Hence used in GWAS.
Can compare many different conditions simultaneously i.e. compare gene expression – gene expression profiling.

Question 25

What is the benefit of Microarrays over blotting techniques?

Answer 25

Microarrays can simultaneously measure the expression of different transcripts and compare multiple experimental conditions whereas northern blotting can only measure single gene targets