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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
Q

What is denaturing of DNA?

A

Denaturing is the disruption of Hydrogen bonds within the double helix

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26
Q

When does DNA denaturing occur?

A
  • DNA in solution heated to energise bonds
  • Or induced by strong alkali / urea, forms randomly
    structured coil
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27
Q

How is denaturing measured?

A

Denaturation can be measured optically by absorbance at 260nm

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28
Q

What is hyperchromicity?

A

Increase of absorbance (optical density) of a material

Increased absorption of light at 260nm On denaturation

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29
Q

Which type of DNA (ss/dsDNA) absorbs more UV light?

A

Single stranded DNA absorbs more UV light than double stranded DNA (hyperchromicity)

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30
Q

What determines how well DNA denatures?

A

The denaturing of a specific DNA depends upon the stability of the specific structure

31
Q

What is the Tm?

A

Point at which 50% of all strands separate is called the melting temperature or Tm

32
Q

What is the significance of Tm?

A

The Tm is specific to individual double helices and is used to control its formation

33
Q

What factors determine the Tm of a DNA molecule?

A

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
Q

How does the GC content affect Tm?

A

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
Q

How would you calculate the % of GC base pairs in a DNA strand?

A

%GC = (G+C / G+C+A+T) x 100

36
Q

How does molecule length affect the Tm?

A

Longer the contiguous duplex, the higher Tm
More Hydrogen bonds within the molecule greater stability

However little further contribution beyond 300 bp

37
Q

How does the salt concentration affect Tm?

A

Salt stabilises DNA duplexes

High [Na+] = High Tm

38
Q

How does increasing [salt] overcome base pairing mismatches?

A

Increasing salt concentration stabilises the structure, increases the Tm and thus overcomes the destabilising effect of mismatched base pairing

39
Q

What is the effect of a high [salt] of DNA?

A

High salt reduces the specificity of base pairing at a given temperature

40
Q

How does [salt] affect duplex stability?

A

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
Q

How does pH affect the Tm?

A

Chemical denaturants disrupt hydrogen bonds e.g. Alkali, formamide, urea

NaOH ⇆ Na+ + OH-
OH- disrupts H bond pairing

Fewer hydrogen bonds = Lower Tm

42
Q

What is the effect of high pH on stability of DNA duplexes?

A

High pH (alkalinity) destabilises DNA duplexes

43
Q

What is a mismatch?

A

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

44
Q

What effect does mismatches have on the Tm of DNA?

A

Reduces Number of Hydrogen bonds, Fewer = lower Tm

45
Q

What effect does mismatching have on the structure of a DNA duplex?

A

Mismatches also distorts the structure and destabilises adjacent base pairing

46
Q

What is renaturation?

A

The reversal of denaturation

47
Q

Explain renaturation in terms of free energy

A

Formation of structure favours energy minimisation driven by change in free energy DG

48
Q

What is renaturation facilitated by?

A

Slow Cooling

Neutralisation

49
Q

Explain how renaturation and hybridisation differ?

A

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
Q

What determines the specificity of base pairing?

A

Complementarity and Tm is the basis of specificity

51
Q

Outline what a perfect match would involve

A
  • Have a higher Tm
  • Thermodynamically favoured over Mismatches
  • Property can be used to form a complementary molecule
    with no mismatches
52
Q

How can we prevent mismatches forming between two molecules?

A

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
Q

What is stringency?

A

Stringency is the concept of manipulating the conditions to select duplexes with a perfect match only

54
Q

What does manipulating conditions of hybridisation allow us to do?

A

Limiting hybridisation between imperfectly matched sequences allows us to manipulate (increase) specificity

55
Q

Explain the outcome of hybridisation under high stringency

A

Only complementary sequences are stable (no mismatches) determined by a

  • Temperature near Tm
  • Low salt concentration
56
Q

What is the effect of hybridisation under low stringency?

A

multiple duplexes form containing different mismatch pairings

57
Q

Which methods use Complementarity and hybridisation techniques?

A 
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
Q

What is the significance of Nucleic Acid Hybridisation Techniques?

A

Identifies presence of nucleic acids containing a specific sequence of bases
Allows the absolute / relative quantitation of these sequences in a mixture

59
Q

Why are nucleic acids so important for hybridisation?

A

Hybridisation uses the ability of NA to form specific duplexes

60
Q

What is a probe?

A 
A ssDNA (or RNA) molecule
Typically 20 – 1000 bases in length
61
Q

How are probes identifiable?

A

Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
In some techniques thousands or millions of probes are used simultaneously

62
Q

Explain how probes are used to identify specific base sequences

A

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
Q

How are probes able to identify specific sequences?

A

Uses the complementarity and hybridisation of labelled nucleic acids

64
Q

What is Northern Blotting?

A

Northern Blotting is a technique adapted from Southern Blotting for analysing the genes that are expressed by a cell tissue or organ

65
Q

Outline the technique of Northern Blotting

A
  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
Q

What is the use of Northern Blotting?

A

Analysis of mRNA or DNA

67
Q

What are the drawbacks of northern blotting technique?

A

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
Q

What is a microarray?

A

An ordered assembly of thousands nucleic acid probes

can also assess millions of SNPs

69
Q

Explain how microarrays work

A

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
Q

What are microarrays used for?

A

microarray might be used for gene expression profiling for example a comparison of drug treated cells and untreated cells (compares gene expression)

71
Q

Outline the process of microarrays

A
  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
Q

How many SNPs can be detected from 1 persons DNA?

A

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
Q

What is an SNP?

A

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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