2. DNA Hybridisation Flashcards Preview

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Flashcards in 2. DNA Hybridisation Deck (28):
1

RECAP: Where do the nitrogenous base, phosphate group and hydroxyl group join onto?

• Nitrogenous base joined to carbon 1
• Phosphate group joined to carbon 5
• Hydroxyl group carbon 3

2

RECAP: Which nucleotides are pyrimidine and purines?

pYrimidines:
• cYtosine
• thYmine

pUrines:
• gUanine
• adenine

3

RECAP: What about the structure provides specificity of base pairing?

The charged or polar groups

4

RECAP:What type on bonding determines Watson and Crick base pairing? and how many between each base pair?

Hydrogen bonds between oppositely charged groups

AT = 2
GC = 3 – So this is a stronger bond

5

RECAP: How are sugar phosphates linked?

Backbone of DNA is formed by phosphodiester linkage. – Connects the 3 and 5 prime carbons of deoxyribose sugar

6

RECAP: What is the stability of the DNA structure determined by?

Free energy of the molecule and energy minimisation.

7

RECAP: What is base stacking?

A form of hydrophobic interactions, arrangement of bases set above each other, which excludes water from the internal structure.

8

RECAP: What provides structural stability to DNA?

Hydrogen bonding of the bases, and internal arrangement and additional stability by base stacking and van der Waals (individually small).

9

RECAP: What give DNA an overall negative charge?

The negatively charged phosphates external

10

What happens when DNA is denatured? And what causes this to happen?

• Conversion of ds molecule to ss molecules – which forms randomly structured coil.
• Caused by disruption of the H bonds
o Occurs when DNA in solution is heated
o Can be induced by strong alkali or urea

11

How can denaturation be optically measured?

Ss DNA absorbs UV light to a greater extent than ds DNA
Hyperchromicity: Increased absorption of light at 260nm on denaturation

12

What is Tm?

• The melting temperature. The point at which 50% of all strands separate.
• This characteristic is specific to an individual double helical structure and we can use this knowledge to control formation of the duplex

13

What does the stability and Tm of a DNA molecule depend on?

• GC content
• Length of molecule
• Salt conc
• pH (alkali is a denaturant)
• No of mismatches (unmatched base pairs)

14

What is the relationship between GC content and Tm?

• Higher GC content= more hydrogen bonds = higher Tm

15

How can you measure the %GC?

(G+C)/(G+C+A+T) X 100

16

What is the relationship between molecule length and Tm?

Longer contiguous duplex = more hydrogen bonds = higher Tm

17

What is the relationship between Tm and salt conc [Na+]?

High [Na+] = High Tm
• Increasing the salt concentration overcomes the destabilising effect of mismatched base pairing – reducing specificity of base pairing at a given temperature

18

What is the relationship between Tm and pH?

• Chemical denaturants disrupt hydrogen bonds:
Alkali, formamide, urea
- Dissociated -OH- group disrupts H bonds.

19

What is the relationship between Tm and mismatches?

• A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
• Reduces Number of Hydrogen bonds, Fewer = lower Tm
• Shorter contiguous stretches of double stranded sequence = lower Tm
• Mismatches also distorts the structure and destabilises adjacent base pairing

20

What is renaturation?

• The reversal of denaturation
• This occurs as a result of a change in free energy upon:
– Slow cooling
– Neutralisation

21

What is hybridisation? and how is this used to form a stable DNA molecule?

Formation of duplex structure of two DNA molecules that have been introduced to one another, for example a short synthetic DNA (or primer) and genomic DNA.
• Perfect matches have a higher Tm
• Are thermodynamically favoured over Mismatches
• Preventing mismatches forming between two molecules can be achieved by performing a hybridisation at the Tm of the duplex molecule

22

What is stringency? what happens under high stringency?

Stringency is the concept of manipulating the conditions to select duplexes with a perfect match only.
Only complementary sequences are stable determined by a
• Temperature near Tm
• Low salt concentration

23

Hybridisation is important in which of the many nucleic acid based techniques? And how?

• Northern blotting
• Southern blotting
• Microarrays
• Dideoxy and Next Gen Sequencing
• PCR
• Cloning
These all rely upon the avoidance of mis-matches using Tm and manipulating the conditions under which hybridisation is carried out.
involves use of probes

24

What is a probe?

Probes that are used to detect nucleic acids are designed to be complementary to a specific region of a target gene sequence which is unique to that gene
- 20-1000 bps
- labelled
- complementary

25

Describe the process of northern or southern blotting.

• The technique of Southern or Northern blotting uses DNA or RNA respectively that is separated by gel electrophoresis
• which is then transferred by mass capillary flow to a nylon membrane
• It is covalently bond to the membrane and then hybridised with a labelled probe
• The probe can be visualised by some means

26

What is a microarray used for? And how?

• A microarray might be used for gene expression profiling for example a comparison of drug treated cells and untreated cells.
• Also, assess the presence or absence of millions of individual SNPs simply through hybridisation of genomic DNA to an array. ~ Result: homozygous or heterozygous for each SNP. Used in GWAS.

- RNA is extracted
- Labelled
- Hybridised to the array and the amount and location of the label measured
- This tells us how much of each and every one of the transcripts in the human genome that are being expressed
• The intensity of the colour in a microarray shows us the level of hybridisation.

27

What is a microarray used for? And how?

• A microarray might be used for gene expression profiling for example a comparison of drug treated cells and untreated cells.
• Also, assess the presence or absence of millions of individual SNPs simply through hybridisation of genomic DNA to an array. ~ Result: homozygous or heterozygous for each SNP. Used in GWAS.

- RNA is extracted
- Labelled
- Hybridised to the array and the amount and location of the label measured
- This tells us how much of each and every one of the transcripts in the human genome that are being expressed
• The intensity of the colour in a microarray shows us the level of hybridisation.

28

What is a microarray?

• An ordered assembly of thousands nucleic acid probes
• 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