DNA complimentarity: hybridisation and it's application

Question 1

What consist of nucleotides? Describe a nucleotides structure.

Answer 1

DNA and RNA.

A nucleotide consists of a phosphate group, a pentose sugar and a nitrogenous base.

Question 2

What is a nitrogenous base?

Answer 2

A ring composed of both carbon and nitrogen.

Question 3

What is a pentose sugar and what is attached to the 1st, 3rd and 5th carbons?

Answer 3

5 carbon atoms in a cyclical structure, including an oxygen bridge.

The nitrogenous base is joined to carbon 1. A hydroxyl group is joined to carbon 3. The phosphate group is joined to carbon 5.

Question 4

What is a polynucleotide? Give an example.

Answer 4

A biopolymer with 13 or more nucleotide monomers with covalent bonds between them in a chain. An example is DNA.

Question 5

How many types of nucleotide in DNA?

Answer 5

4.

Question 6

Describe a DNA nucleotide via the nucleotides 3 components.

Answer 6

The nitrogenous base: Either a single or double ring. Contains N and C.

The pentose sugar: Attached to the nitrogenous base ring at the 1st C. It is a ribose sugar. It has a -OH (hydroxyl) group attached to it at the 3rd position.

The phosphate group: Attached at the 5th carbon of the ribose.

Question 7

What are the 4 types of nitrogenous bases?

Answer 7

Adenine, Thymine, Cytosine, Guanine.

Question 8

Which of the 4 nitrogenous bases are pyrimidine and purine?

Answer 8

Cytosine and Thymine (and uracil) are pyrimidine, so have a single N containing ring.

Adenine and Guanine are purine, so have a double N containing ring.

Question 9

What on the nitrogenous groups are important for base pairing and why?

Answer 9

The polar (charged) groups on them, which provide specificity for the base pairing.

Question 10

What is different about the RNA molecule in terms of the nitrogenous base?

Answer 10

Instead of Thymine, RNA has Uracil. The Uracil therefore binds to Adenine.

Question 11

With what force do nitrogenous bases bind and how?

Answer 11

Hydrogen bonds that occur between oppositely charged groups (amine and carboxyl groups).

Question 12

Can there ever be 2 purines in a base pair?

Answer 12

No - each pair has a single purine and pyrimidine nitrogenous base.

Question 13

How many hydrogen bonds form between A and T and C and G? What is the consequence of this?

Answer 13

A to T has 2 bonds, C to G has 3 bonds. This means that the C to G pair is stronger than the A to T.

Question 14

What is dsRNA/ RNA secondary structure?

Answer 14

Double stranded RNA molecules (usually they are single stranded only)

Question 15

What occurs with Uracil-Adenine pairing and how does it compare to AT and CG bond pair strengths?

Answer 15

Cytosine-Guanine pairing which is still the strongest of them all

CHECK

Question 16

What is a phosphodiester bond and where in the DNA molecule are they?

Answer 16

2 bonds connecting to the negative phosphate group, which is part of every nucleotide.

It connects the 5’ C of one nucleotides pentose sugar and the 3’ C’s -OH of the adjacent nucleotides pentose sugar

Question 17

What are the interactions that occur in a DNA molecule that ensure stability of the molecule and thus influences it’s behaviour and shape?

Answer 17

• Hydrophobic interactions between bases that have stacked excludes water, giving a hydrophobic internal structure to the DNA helix
• Van der Waals forces; small
• H bonds found between bases (2 between A and T and 3 between C and G)
• Covalent bonds in the sugar phosphate backbone (the phosphodiester bonds)

Question 18

How are the 2 strands of DNA orientated?

Answer 18

Antiparallel

Question 19

What are the negatively charged phosphate groups used for by DNA?

Answer 19

To interact with proteins + it gives DNA it’s overall charge

Question 20

What is DNA denaturation and how does it occur?

Answer 20

Denaturation is the breaking of the H bonds between the DNA’s bases, causing the 2 strands to separate

This occurs when chemicals (e.g. NaOH, alkali, urea) or heat (when DNA is in solution) is applied to the DNA

Question 21

What happens after a DNA denatures?

Answer 21

It forms a randomly structured coil

Question 22

How is denaturation of DNA measured?

Answer 22

The DNA’s absorbance of 260nm UV rays is measured

Question 23

What is hyperchromicity? So what happens to the absorption of 260nm rays as denaturation occurs?

Answer 23

Single stranded DNA can absorb more UV light than double stranded DNA. So, as DNA denatures, absorption increases resulting in a tendency towards hyperchromicity

Question 24

What is the melting temperature of a molecule, Tm? How is it related to absorbance?

What does it indicate?

Why is it important?

Answer 24

The temperature at which 50% of the DNA molecules have denatured, measured based on relative absorbance of the DNA molecule at different temperatures.

The Tm indicates the stability of the DNA duplex - the higher it is, the higher the stability.

It is important as every molecule has it’s own specific Tm, + the Tm can be used to help design probes that allow for hybridisation (forming a duplex using 2 different molecules’ single strands)

Question 25

What does Tm depend on?

Answer 25

Mainly hydrogen bonds.

Since G-C bonds have the most hydrogen bonds, GC content is very important.

Other important factors include the length of DNA, the salt concentration (i.e. the env. in which the molecule resides), the pH and mismatches

Question 26

How does Tm relate to GC content?

Answer 26

The higher the GC content, the more H bonds, the higher the Tm

Question 27

How does Tm relate to molecule length? Does this effect last forever?

Answer 27

The longer the contiguous (touching) DNA duplex, the more H bonds are present, the higher the Tm

This effect diminishes beyond about 300 BPs, where there is no more addition to stability

Question 28

How does Tm relate to salt concentration?

What can be overcome by increasing salt concentrations?

Answer 28

Salt stabilizes the molecule so the higher the sodium ion concentration, the higher the Tm.

Increasing salt concentrations can overcome the effect of mismatched base pairs as it reduces the specificity of base pair formation at a given temperature.

A duplex containing mismatches can form and be stable at a given temperature in the presence of high salt concentration whilst the same duplex would be unstable and dissociate at the same temperature in low salt.

Question 29

What happens to adjacent base pairing as a result of mismatched base pairs?

Answer 29

Adjacent base pairs get destabilized by the mismatches, encouraging zipping and unzipping. There consequently forms an equilibrium between the 2 states.

Question 30

How is Tm related to pH?

Answer 30

Having an alkali present provides OH- ions which disrupt the hydrogen bonds and lowers Tm (i.e. a high pH would lower the Tm and destabilize)

Question 31

What is a mismatch?

What is it’s effect on the Tm?

Answer 31

A potential base pair combination that is unable to form hydrogen bonds

With lower H bonds the Tm is lowered too. This is because there are shorter contiguous stretches of DNA + destabilization of adjacent base pairs

Question 32

What is renaturation?

How can it be done?

Answer 32

It is the opposite of denaturation i.e. forming a duplex

It is done by decreasing temp, increasing salt conc., decreasing alkali (neutralising it/ removing urea) etc.

Question 33

How are renaturation and hybridisation different?

Answer 33

Renaturation uses a molecule that was originally a duplex to form the duplex again where as hybridisation uses the introduction of a 2nd molecule to form the duplex

Question 34

How do you manipulate the formation of a duplex?

Answer 34

You manipulate the environment in which the molecule resides e.g. if you had a molecule with mismatches at Tm 69 and another with no mismatches at Tm 85, if you hybridise at 85 degrees then only the non-mismatched duplex will be promoted to form

Question 35

What does stringency mean in context of hybridisation?

Answer 35

The strictness of conditions to form the hybridized molecules

Question 36

How is stringency altered in hybridisation?

Answer 36

You can change the environmental conditions and temperature.

Question 37

What are the conditions for low stringency and what do they favour?

Answer 37

Low stringency are less strict conditions. This means that they favour H bond formation between duplexes, so consists of low temp and high salt conc. This means they favour the formation of multiple duplexes, some with mismatches

Question 38

What are the conditions for high stringency and what do they favour?

Answer 38

High stringency are more strict conditions. This means they do not favour H bond formation between duplexes, so consists of high temperature (i.e. a temperature near the molecules Tm) and low salt conc. This means they favour formation of only duplex with non mismatch BPs i.e. only complimentary sequences form the duplexes)

Question 39

What are some nucleic acid based techniques that use complementarity and hybridisation?

Answer 39

Northern blotting
Southern blotting
Microarrays
Dideoxy and Next Gen Sequencing 
PCR
Cloning

Question 40

What are nucleic acid hybridisation techniques?

Answer 40

Techniques that use nucleic acids to identify complimentary nucleic acids (i.e. DNA sequences to find other DNA sequences) + quantify them

Question 41

What are nucleic acids used to carry out hybridisations known as? What are their features?

Answer 41

Probes

• Sequences of bases, can vary from 20-1000 bases long
• Usually ssDNA/ RNA
• Usually synthetic
• Labelled with fluorescent/ luminescent molecules or radioactive isotopes
• Used to identify specific sequences in a population
• Some techniques use millions at the same time

Question 42

What is a probe?

Answer 42

A sequence that is complimentary to and so uniquely identifies specific parts of the genome (exons/ genes)

Question 43

What condition must the target gene meet?

Answer 43

It must form a duplex under high stringency conditions

Question 44

Which is an adaptation of which technique, northern or southern blotting?

Answer 44

Northern blotting is an adaptation of southern blotting

Question 45

What are northern and southern blotting used for?

Answer 45

Analysis of mRNA and DNA

Question 46

What are blotting techniques limitations?

Answer 46

• Detects one gene at a time
• Analyses a small number of samples
• Time consuming
• Messy
• They are gel based btw

Question 47

Which technique has overtaken the blotting techniques?

Answer 47

Quantitative PCR

Question 48

How are the blotting techniques done?

Answer 48

1. DNA/ RNA is extracted
2. Gel electrophoresis is performed to separate the nucleic acid strands
3. The strands are transferred to a nylon membrane
4. The probes are added in order to hybridize to the target sequences and thus identify their presence, location and quantity
5. The probes are visualized/ measured (i.e. the fluorescence/ radionuclei decay)

Question 49

What are microarrays? What is a benefit of it?

Answer 49

An assembly of thousands of probes on a solid surface which act to hybridize a sample when it is added
It can simultaneously measure 50,000 different transcripts in a cell/ tissue/ organ

Question 50

What are microarrays used for?

Answer 50

They are used to compare gene expression (quantity and location) e.g. in a sample of cells that have/ have not had drug added to them, you see if there is more/ less hybridisation occurring in the sample with the drug added to specific probes (indicating more/ less expression of genes in the samples)

Question 51

See diagram in notes - what would happen in equal/ greater levels of hybridisation?

Answer 51

Equal - stays on line

Greater - positive deviation from line

Question 52

How can microarrays be used to assess SNPs?

Answer 52

The DNA from 1 person can be put on a microarray surface and be used to detect upto 2.5 million SNPs simultaneously. This is done in GWAS and can help in finding if a SNP is homozygous/ heterozygous.