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Genomics > DNA Hybridisation, Complementarity and Its Application > Flashcards

DNA Hybridisation, Complementarity and Its Application Flashcards

1
Q

What is RNA and DNA made up of?

A

Nucleotides

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2
Q

What is DNA?

A

DNA is a polymer / polynucleotide comprising of 4 nucleotides

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3
Q

Describe the structure of a nucleotide

A

Nitrogenous Base:
Phosphate group
Pentose Sugar

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4
Q

What is the structure of the nitrogenous base?

A

a ring structure composed of carbon and nitrogen

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5
Q

What is a pentose sugar?

A 
5 carbons (numbered 1 - 5) that form a cyclical structure with oxygen bridge
Hydroxyl OH group carbon 3
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6
Q

Where does the nitrogenous base join the pentose sugar in a nucleotide?

A

Nitrogenous base joined to carbon 1

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7
Q

Where does the phosphate group lie in relation to the pentose sugar in a nucleotide molecule?

A

Phosphate group joined to carbon 5 - forms phosphodiester bond with OH on adjacent carbon on next base

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8
Q

What is the difference between RNA and DNA structure?

A

RNA also has 2nd OH group at carbon 2

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9
Q

What are purine bases?

A

double ringed e.g.

guanine, adenine (G,A)

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10
Q

What are pyrimidine bases?

A

single ringed bases e.g.

thymine, cytosine (T,C)

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11
Q

How do bases form specific pairs?

A

Charged polar groups provide the specificity of base pairing

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12
Q

How does base pairing differ between RNA and DNA?

A

In RNA Uracil substitutes Thymine and base pairs with Adenine in RNA to form duplex structure

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13
Q

How does base pairing form a double helix?

A

Hydrogen bonding between oppositely charged groups forms basis of Watson and Crick base pairing forming the double helix

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14
Q

Where on the bases does the Hydrogen bond form?

A

Hydrogen bonding involves amine and carboxyl groups between Adenine and Thymine or between Guanine and Cytosine

In each pair there is a single purine and pyrimidine

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15
Q

Why is Cytosine-guanine pairing stronger than adenine- thymine/uracil pairing?

A

There is an extra Hydrogen bond between C-G than A-T/U

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16
Q

How do nucleotides join to one another to form the sugar-phosphate backbone?

A

linked by phosphodiester bonds C3

O bonds to phosphate group

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17
Q

How does base stacking occur in a double helix?

A

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

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18
Q

What is the significance of Van der Waals forces in DNA?

A

individually small but contributes to the stability

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19
Q

What conformations does the DNA double helix form?

A

A, B and Z forms

B form is most common

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20
Q

What forms the DNA backbone?

A

The DNA backbone is formed from phosphodiester linkages connecting 3 and 5 prime carbons of the DNA deoxyribose sugar

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21
Q

What determines DNA stability?

A

The structure stability is determined by the free energy of the molecule and energy minimisation just as in protein structure

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22
Q

What directions do the two DNA strands forming the double helix run along?

A

Double stranded helix is formed from 2 antiparallel strands

5’-3’and 3’-5’

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23
Q

Why does DNA have a negative charge?

A

The bases are on the inside forming stacked bases and the negatively charged phosphates are external giving DNA an overall negative charge

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24
Q

What is meant by denaturing DNA?

A

Conversion of a double stranded molecule → single stranded molecules

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25
What is denaturing of DNA?
Denaturing is the disruption of Hydrogen bonds within the double helix
26
When does DNA denaturing occur?
- DNA in solution heated to energise bonds - Or induced by strong alkali / urea, forms randomly structured coil
27
How is denaturing measured?
Denaturation can be measured optically by absorbance at 260nm
28
What is hyperchromicity?
Increase of absorbance (optical density) of a material Increased absorption of light at 260nm On denaturation
29
Which type of DNA (ss/dsDNA) absorbs more UV light?
Single stranded DNA absorbs more UV light than double stranded DNA (hyperchromicity)
30
What determines how well DNA denatures?
The denaturing of a specific DNA depends upon the stability of the specific structure
31
What is the Tm?
Point at which 50% of all strands separate is called the melting temperature or Tm
32
What is the significance of Tm?
The Tm is specific to individual double helices and is used to control its formation
33
What factors determine the Tm of a DNA molecule?
Tm depends largely on hydrogen bonds affected by: - GC content - Length of DNA molecule - Salt concentration (environment) - pH (alkali is a denaturant) - Mismatches (unmatched base pairs)
34
How does the GC content affect Tm?
Higher GC content = more H bonds = higher Tm 3 H+ bonds in G:C vs 2 in A:T ∴ more G:C pairing = more H+ bonds
35
How would you calculate the % of GC base pairs in a DNA strand?
%GC = (G+C / G+C+A+T) x 100
36
How does molecule length affect the Tm?
Longer the contiguous duplex, the higher Tm More Hydrogen bonds within the molecule greater stability However little further contribution beyond 300 bp
37
How does the salt concentration affect Tm?
Salt stabilises DNA duplexes | High [Na+] = High Tm
38
How does increasing [salt] overcome base pairing mismatches?
Increasing salt concentration stabilises the structure, increases the Tm and thus overcomes the destabilising effect of mismatched base pairing
39
What is the effect of a high [salt] of DNA?
High salt reduces the specificity of base pairing at a given temperature
40
How does [salt] affect duplex stability?
A duplex containing mismatches can form and be stable at a given temperature in the presence of high [salt] whilst the same duplex would be unstable and dissociate at the same temperature in low salt
41
How does pH affect the Tm?
Chemical denaturants disrupt hydrogen bonds e.g. Alkali, formamide, urea NaOH ⇆ Na+ + OH- OH- disrupts H bond pairing Fewer hydrogen bonds = Lower Tm
42
What is the effect of high pH on stability of DNA duplexes?
High pH (alkalinity) destabilises DNA duplexes
43
What is a mismatch?
A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
44
What effect does mismatches have on the Tm of DNA?
Reduces Number of Hydrogen bonds, Fewer = lower Tm
45
What effect does mismatching have on the structure of a DNA duplex?
Mismatches also distorts the structure and destabilises adjacent base pairing
46
What is renaturation?
The reversal of denaturation
47
Explain renaturation in terms of free energy
Formation of structure favours energy minimisation driven by change in free energy DG
48
What is renaturation facilitated by?
Slow Cooling | Neutralisation
49
Explain how renaturation and hybridisation differ?
Renaturation : forming a duplex from single strands that were once already a double helix Hybridisation : formation of a duplex structure of 2 DNA molecules that have been introduced to one another e.g., a short synthetic DNA (or primer) and genomic DNA
50
What determines the specificity of base pairing?
Complementarity and Tm is the basis of specificity
51
Outline what a perfect match would involve
- Have a higher Tm - Thermodynamically favoured over Mismatches - Property can be used to form a complementary molecule with no mismatches
52
How can we prevent mismatches forming between two molecules?
Since mis-matches destabilise DNA and reduce the Tm preventing mismatches forming between two molecules can be achieved by performing a hybridisation at the Tm of the duplex molecule
53
What is stringency?
Stringency is the concept of manipulating the conditions to select duplexes with a perfect match only
54
What does manipulating conditions of hybridisation allow us to do?
Limiting hybridisation between imperfectly matched sequences allows us to manipulate (increase) specificity
55
Explain the outcome of hybridisation under high stringency
Only complementary sequences are stable (no mismatches) determined by a - Temperature near Tm - Low salt concentration
56
What is the effect of hybridisation under low stringency?
multiple duplexes form containing different mismatch pairings
57
Which methods use Complementarity and hybridisation techniques?
``` Northern blotting Southern blotting Microarrays Dideoxy and Next Gen Sequencing PCR Cloning ``` All rely on complementary base pairing specificity and avoidance of mismatches using Tm + manipulating conditions of hybridisation
58
What is the significance of Nucleic Acid Hybridisation Techniques?
Identifies presence of nucleic acids containing a specific sequence of bases Allows the absolute / relative quantitation of these sequences in a mixture
59
Why are nucleic acids so important for hybridisation?
Hybridisation uses the ability of NA to form specific duplexes
60
What is a probe?
``` A ssDNA (or RNA) molecule Typically 20 – 1000 bases in length ```
61
How are probes identifiable?
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope) In some techniques thousands or millions of probes are used simultaneously
62
Explain how probes are used to identify specific base sequences
Probes used to detect nucleic acids are designed to be complementary to a specific target gene sequence region which is unique to that gene, which under high stringency conditions form a duplex
63
How are probes able to identify specific sequences?
Uses the complementarity and hybridisation of labelled nucleic acids
64
What is Northern Blotting?
Northern Blotting is a technique adapted from Southern Blotting for analysing the genes that are expressed by a cell tissue or organ
65
Outline the technique of Northern Blotting
1. Southern / Northern blotting uses DNA / RNA which is separated by gel electrophoresis 2. Transferred by mass capillary flow to a nylon membrane 3. Covalently bond to membrane and then hybridised with a labelled probe 4. The probe can be visualised by some means
66
What is the use of Northern Blotting?
Analysis of mRNA or DNA
67
What are the drawbacks of northern blotting technique?
Limited technique: only detects one gene at a time and small numbers of samples Gel based techniques are time consuming and messy Largely Superseded by quantitative PCR
68
What is a microarray?
An ordered assembly of thousands nucleic acid probes | can also assess millions of SNPs
69
Explain how microarrays work
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
70
What are microarrays used for?
microarray might be used for gene expression profiling for example a comparison of drug treated cells and untreated cells (compares gene expression)
71
Outline the process of microarrays
1. RNA is extracted 2. Labelled 3. Hybridised to array and amount + location of label is measured 4. This tells us how much of each and everyone of the transcripts in the human genome are being expressed
72
How many SNPs can be detected from 1 persons DNA?
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)
73
What is an SNP?
Single-nucleotide polymorphism most common type of genetic variation among people Each SNP represents a difference in a single nucleotide

Genomics (20 decks)

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