2. DNA Hybridisation Flashcards
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
RECAP: Which nucleotides are pyrimidine and purines?
pYrimidines:
• cYtosine
• thYmine
pUrines:
• gUanine
• adenine
RECAP: What about the structure provides specificity of base pairing?
The charged or polar groups
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
RECAP: How are sugar phosphates linked?
Backbone of DNA is formed by phosphodiester linkage. – Connects the 3 and 5 prime carbons of deoxyribose sugar
RECAP: What is the stability of the DNA structure determined by?
Free energy of the molecule and energy minimisation.
RECAP: What is base stacking?
A form of hydrophobic interactions, arrangement of bases set above each other, which excludes water from the internal structure.
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).
RECAP: What give DNA an overall negative charge?
The negatively charged phosphates external
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
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
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
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)
What is the relationship between GC content and Tm?
• Higher GC content= more hydrogen bonds = higher Tm
How can you measure the %GC?
(G+C)/(G+C+A+T) X 100
What is the relationship between molecule length and Tm?
Longer contiguous duplex = more hydrogen bonds = higher Tm
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
What is the relationship between Tm and pH?
• Chemical denaturants disrupt hydrogen bonds:
Alkali, formamide, urea
- Dissociated -OH- group disrupts H bonds.
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
What is renaturation?
• The reversal of denaturation
• This occurs as a result of a change in free energy upon:
– Slow cooling
– Neutralisation
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
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
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
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
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
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.
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.
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
