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DNA hybridisation Flashcards

1
Q

what is a pentose sugar in DNA

A

5 carbon (1-5) that form a cyclical structure with an oxygen bridge and a hydroxyl OH group on carbon 3

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2
Q

what is the structure of a nitrogenous base

A

a ring structure composed of carbon and nitrogen

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3
Q

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

A

carbon 1

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4
Q

where does phosphate group lie on pentose sugar

A

carbon 5

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5
Q

what are the purines (double ringed)

A

guanine adenine

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6
Q

what are the pyrimidines (single ringed)

A

thymine cytosine

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7
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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8
Q

what is base stacking and what does it do

A

base stacking has hydrophobic interactions - arrangement of bases set above each other internalised to the structure and excludes water

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9
Q

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

A

individually small but contributes to the stability

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10
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.Stability is derived from the H bonding and internal arrangement of bases gaining additional stability by base stacking
VDW forces also impacts stability

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11
Q

Why does DNA have a negative charge?

A

the negatively charged phosphates are external giving DNA an overall negative charge

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12
Q

What is meant by denaturing DNA?

A

Conversion of a double stranded molecule → single stranded molecules by disruption of Hydrogen bonds within the double helix

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13
Q

When does DNA denaturing occur?

A

DNA in solution heated to energise bonds

- Or induced by strong alkali / urea, formamide forms randomly structured coil

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14
Q

How is denaturing measured?

A

Denaturation can be measured optically by absorbance at 260nm

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15
Q

What is hyperchromicity?

A

As temp increases, duplex melts and optical density (absorption) increases. This is because single stranded DNA absorbs UV light at a greater extent than double stranded DNA = hyperchromicity - absorption of UV light increases on denaturation

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16
Q

what does denaturation of a DNA duplex depend on

A

duplex stability determined by its sequence of bases.

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17
Q

What is the Tm?

A

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

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18
Q

What is the significance of Tm?

A

Tm is specific to a duplex with a given sequence - can use this to control the formation of a short duplex used as a primer or a probe

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19
Q

What factors determine the Tm of a DNA molecule?

A

Stability and thus Tm of a molecule is determined by 5 factors:
GC content
Length of a molecule
Salt concentration (molecular environment)
pH (alkali is a denaturant)
Mismatches (unmatched base pairs)

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20
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

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21
Q

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

A

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

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22
Q

How does molecule length affect the Tm?

A

Longer the contiguous duplex, the higher Tm and stability
More H bonds, so more stable.
However a length beyond 300bp contributes little to no more to the Tm and stability.

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23
Q

How does the salt concentration affect Tm?

A

Salt stabilises DNA duplexes, so higher the NA+ (sodium ion) concentration, the higher the Tm

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24
Q

How does increasing [salt] overcome base pairing mismatches?

A

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

25
What is the effect of a high [salt] of DNA?
High salt reduces the specificity of base pairing at a given temperature.
26
x
x
27
How does (high) pH affect the Tm?
Chemical denaturants disrupt hydrogen bonds e.g. Alkali, formamide, urea NaOH ⇆ Na+ + OH- = high pH OH- disrupts H bond pairing Fewer hydrogen bonds = Lower Tm high pH destabilises DNA
28
What is a mismatch?
a base pair combination that is unable to form hydrogen bonds
29
What effect does mismatches have on the Tm of DNA?
Reduces Number of Hydrogen bonds, Fewer = lower Tm
30
What effect does mismatching have on the structure of a DNA duplex?
Mismatches also distorts the structure and destabilises adjacent base pairing
31
What is renaturation?
The reversal of denaturation
32
Explain renaturation in terms of free energy
Formation of structure favours energy minimisation driven by change in free energy DG
33
What is renaturation facilitated by?
Slow Cooling | Neutralisation
34
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
35
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
36
How can we prevent mismatches forming between two molecules?
by performing a hybridisation at the Tm of the duplex by hybridising at a temperature near to the perfectly matched duplex.
37
What is stringency and what does it allow us to do?
Stringency is the concept of manipulating the conditions to select duplexes with a perfect match only allows us to manipulate (increase) specificity
38
Explain the outcome of hybridisation under high stringency
Only complementary sequences are stable (no mismatches) determined by a - Temperature near Tm - Low salt concentration
39
What is the effect of hybridisation under low stringency?
multiple duplexes form containing different mismatch pairings kinetics of hybridisation are much faster under low stringency conditions, high salt conc
40
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
41
What is the significance of Nucleic Acid Hybridisation Techniques?
to indicate the presence or absence of specific sequences of bases in a mixture of nucleic acids. So essentially the nucleic acid hybridisation techniques Identify the presence on nucleic acids containing a specific sequence of bases to capture specific sequences of DNA Allows the absolute or relative quantitation (quantifying the amount) of these sequences in a mixture
42
Why are nucleic acids so important for hybridisation?
Hybridisation uses the ability of NA to form specific duplexes
43
What is a probe?
``` Labelled short DNA molecules (oligonucleotides) that are used to detect unique sequences and are part of a gene A ssDNA (or RNA) molecule Typically 20 – 1000 bases in length ```
44
How are probes identifiable?
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
45
what is a primer
Used in an enzymatic reaction to prime the reaction | Short single stranded polynucleotide, between 16-30 nucleotides
46
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
47
How are probes able to identify specific sequences?
Uses the complementarity and hybridisation of labelled nucleic acids
48
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
49
northern blotting limitation
it can only detect one gene at a time and small numbers of samples The gel based techniques are time consuming and messy and as a consequence is superseded by quantitative PCR or other techniques
50
Outline the technique of Northern Blotting
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 fluorescence
51
What is the use of Northern Blotting?
Analysis of mRNA or DNA
52
What is a microarray and what kind of method is it?
An ordered assembly of thousands nucleic acid probes can also assess millions of SNPs A comparative methodology - cannot find an absolute measurement of gene expression.
53
Explain how microarrays work
Probes are fixed to a solid surface such as silicone or glass matrix, then sample of interest is hybridised to the probes Simultaneously measuring 50,000 different transcripts in a Cell, Tissue or Organ
54
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)
55
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
56
compare microarrays and blotting techniques
Microarrays compare many different conditions simultaneously. Have advantage over blotting techniques in that it can simultaneously measure the expression of many different transcripts and compare multiple experimental conditions whereas northern blotting can only measure single gene targets.
57
How many SNPs can be detected from 1 persons DNA?
DNA from 1 person on 1 microarray | Detects 2.5 million SNPs simultaneously
58
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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