Extraction, Analysis And Hybridisation Techniques

Question 1

What is DNA extraction and what does it involve?

Answer 1

A routine procedure used to isolate DNA from the nucleus of cells.
This is performed in the following ways:

Break open cells
physical (grind/vortex) or solubilise membranes
e.g. detergent

Separate DNA from proteins
Filter or denature proteins e.g. enzymatic digestion

Purify nucleic acid
e.g. precipitation with alcohol (ethanol or isopropanol - DNA is soluble in water but insoluble in salt and alcohol)

Protect nucleic acid
enzyme inhibitors e.g. EDTA, RNAsin (DNA issoluble in water but insoluble in salt and water - can solubilise it later)

Confirm quantity and quality of DNA
Optical density readings taken by a spectrophotometer (DNA absorbs UV light maximally at 260nm and proteins absorb light maximally at 280nm)
Gel electrophoresis

Question 2

What methods can be used to perform this extraction?

Answer 2

Boiling

Detergent lysis - break open membranes (different amount depending on the type of cell)

Protease digestion and denaturation

Phenol-chloroform extraction and ethanol precipitation

Guanidinium isothiocyanate lysis and alcohol precipitation (harsh reagents)

Commercial kits – formulated for specific sample use - incorporates controls and standardises the procedure, unlike previous methods.
often incorporate affinity/exclusion chromatography
E.g. Qiagen kits

Question 3

What is QIAprep Miniprep?

Answer 3

It is used for purification of molecular biology grade DNA. Do you want plasma or nuclear genomic DNA? If plasma you don’t want to contaminate it with genomic DNA so have to use subtler approach such as this.

It involves alkaline lysis of bacterial cells followed by adsorption of DNA onto silica (solid phase surface) in the presence of high salt detergent lysis.

Three basic steps:

1. Preparation and clearing of a bacterial lysate
   - Lysis in NaOH/SDS in the presence of RNase A
   
   • SDS solubilizes the phospholipid and protein components of the cell membrane
   • Alkaline conditions denature the chromosomal and plasmid DNAs, as well as proteins.
2. Adsorption of DNA onto the QIAprep membrane
   - RNA, cellular proteins, and metabolites are not retained on the membrane - separation of other cellular components
3. Washing and elution of plasmid DNA
   - Washing removes endonucleases and salt

The spin procedure can be performed using micro-centrifuges or vacuum manifolds, but either way involves; resuspending, lysing and neutralising the bacterial pellet, followed by centrifugation and binding to a surface, washing, elution and a final spin to give pure plasmid DNA. The only difference is that in microcentrifuges there are further spins between each step.

All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation - good as all these are toxic and bad for the environment.

Question 4

How is automation used in the nuclei come acid uextraction process?

Answer 4

Equipment such as the Qiagen QIAcube has been developed for use on a wide range of clinical samples e.g. blood, urine faeces, sputum, RNA and DNA targets. It is able to remove any human error in the process by performing automated DNA and RNA extraction using commercial kits.
Thee are also alternatives such as the Roche MagnaPure Sample
Preparation System.

Question 5

What is Restriction endonuclease analysis as a technique for extraction?

Answer 5

Restriction endonucleases are enzymes that recognise a particular base sequence (usually 4-6 bases) in DNA and cut the molecule at specific sites where the appropriate base sequence occurs (Cuts between the A’s - can cut and re anneal DNA from different sources using ligases).

This unique precision evolved to protect bacteria against viral invasion.

Some cut with ‘sticky ends’ - helpful as it gives information on the orientation of the DNA. Others just cut and produce ‘blunt ends’.

Question 6

What are the uses of Restriction Endonuclease Analysis (REA) as an extraction technique?

Answer 6

Identification of genes and their function
- Deletion then reinsertion of fragments may determine gene function
DNA from any source , when digested with the same restriction enzyme, will produce DNA fragments with identical complementary ends.
- Construction of RE maps allows localisation of specific genes
- DNA fragments visualised by agarose gel electrophoresis.

Epidemiology: Determine source and relationship of isolates

Aetiology: Identification of causative agents/genes

Question 7

How is Restriction Fragment Length Polymorphism (RFLP) used as an extraction technique?

Answer 7

RFLP examines the size variation of restriction fragments, this allows it to

• illustrate genotypic differences: e.g. restriction enzyme activity modified by single nucleotide polymorphisms (SNPs may modify activity of enzymes, causing DNA lengths to be different sizes - these can be correlated with the particular polymorphism).
• determine resistance mechanisms
• elucidate the association with pathogenesis

Question 8

What is the principle of Hybridisation?

Answer 8

Hybridisation is the annealing of a labelled probe to target nucleic acid, which is possible due to the complementarity of the base pairs.

DNA-DNA
DNA-RNA
RNA-RNA

• Requires both a single-stranded target and probe. The probe is made up of specific oligonucleotides labelled with:-
  radioactive markers ( traditionally due to v high sensitivity and specificity, though there are inherent risks)
  - 32P
  - 35S
  non-radioactive markers
  - biotin (binds to avidin)
  - digoxygenin (forms a hapten with high immunogenicity causing an immune response)
  - fluorescein
  - phycoerythrin
• the double-stranded nucleic acid must be denatured
  • heat/alkaline disruption

Question 9

What does stringency mean in Hybridisation?

Answer 9

The specificity of hybridization reactions is controlled by the stringency of conditions, i.e. adopting conditions which favour hybridization only to specific targets (control conditions to get specific base pairing, otherwise will bind to an unwanted place. High stringency = perfect match).

Conditions of high stringency require all bases of one polynucleotide to be paired with complementary bases on the other; conditions of low stringency allow some bases to be unpaired (slight mismatches allow you to sequence related genes based on a known sequence. Hence occasionally, allowing a probe to bind non selectively may be favourable sometimes).

Tm is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability, which is dependent on the base composition (ie CG has 3 H bonds so is more difficult to break that AT).

The temperature at which double-stranded nucleic acid will denature is determined by the base composition, i.e.

Tm = 0.41 (%G+C) + 69.3

Must know an estimated melting temp of probe interaction. More than one formula for this. More GC mean duplex needs more energy to disrupt it.Therefore, the melting temperature of nucleic acid duplexes during hybridisation reactions is dependent upon base composition, denaturing agents (such as formaldehyde and DMSO) and ionic strength (amount of salt - sodium ions - in solution), eg:

Tm = 81.5 + 16.6 (log[Na+]) + 0.41 (%G+C)
- 0.72 (% formamide)

Specificity of reaction may be altered by adjusting the temperature and ionic strength to levels where only specific hybrids are stable

Can be achieved during or after hybridisation

Question 10

Work out these stringency examples:

1. Hybridisation in 0.375M NaCl
2. Hybridisation in 0.375M NaCl with 50% formamide

Post Hybridisation stringency examples and state whether the answer is low or high stringency:

3. Washing in 0.125M NaCl, at RT (22 deg)
4. Washing in 0.0125M NaCl, at RT (22 deg and 56 deg)

Answer 10

1. Tm = 81.5 + 16.6(-0.43) + 0.41 (60)
   = 81.5 - 7.138 + 24.6
   =98.96oC
2. Tm = 81.5 + 16.6(-0.43) + 0.41 (60) - 0.72(50)
   = 81.5 - 7.138 + 24.6 - 36
   = 62.96oC
   Favour an interaction based on what you add. Disfavour it - so probe dissociates by adding a denaturing agent.

3. Tm = 81.5 + 16.6(-0.9) + 0.41 (60)
		= 81.5 – 14.94 + 24.6
		= 91.16oC   
		@ RT (22oC) = Tm – 69.16oC 
        (low stringency)  
Probe could bind to non-specific sites - if you raise the temp only the specific binding sites will be able to survive.

4. Tm = 81.5 + 16.6(-1.9) + 0.41 (60)
		   = 81.5 – 31.54 + 24.6
		   =74.56oC    
	@ RT (22oC) = Tm – 52.56oC 
    (low stringency)  
	@ 56oC = Tm –18.56oC
    (high stringency)  
Much closer to melting temperature so better stringency.

Question 11

What is the dot blot test?

Answer 11

The principle of the dot blot test is:

• Extract nucleic acid
• Denature with alkali then neutralise (Make it single stranded so probe can bind).
• Apply to membrane
• Hybridisation (Add probe that has been pre-labelled (can layer the labelling methods))
• detection

The applications are direct detection and genotyping.

An example is using a radioactive probe against photographic paper, giving a bind where you specifically put in sample - specific hybridisation.

Question 12

What is Southern/northern blotand what are the applications?

Answer 12

Southern blot involves hybridising DNA and transferring to a membrane for subsequent hybridisation to a probe. It uses a tray with a stack consisting top down of a weight, paper towels, a nylon membrane with the hybridised DNA samples annealed to, gel, a salt solution and a slab of glass.

Northern blot involves Hybridisation of RNA, it uses electrophoresis to separate RNA by size and detection with a Hybridisation probe complementary to part of or the entire target sequence. The process is:

• RNA is separated by size using gel electrophoresis. Since probes are unable to enter this matrix the samples are then transferred to a nylon membrane through capillary or vacuum blotting.
• the nylon membrane is positively charged as negatively charged nucleic acids have an affinity for it.
• The transfer buffer for blotting contains formaldehyde to lower the annealing temperature of the probe-RNA interaction (high temp degrades RNA).
• RNA is immobilised to the membrane by covalent linkage using UV light or heat.
• the labelled probe is hybridised to the RNA, and the membrane is washed to ensure the probe is bound specifically and prevent background signals.
• the hybrid signals are detected by x-ray film are can be quantified by densitometry.
• to create controls for comparison, samples not displaying the gene product of interest can be used after determination by microarray or RT-PCR.

The applications of both blotting methods are:

• localisation of gene fragments following RE analysis
• detection with increased specificity
• confirmation of PCR results

Question 13

What is in-situ Hybridisation (ISH)?

Answer 13

ISH allows for precise localization of a specific segment of nucleic acid within a histologic section. It combines specific detection with precise localisation.

The underlying basis of ISH: nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached.

Useful for

• Comparison of presence of specific target with pathology
• Determination of structures and cell types affected
• Developmental biology (gene expression profiling in embryonic tissues)
• Karyotyping and phylogenetic analysis (unique FISH patterns on individual chromosomes, chromosomal aberrations)

Potential problems

• fragility of tissue and condition of sections
• damage to tissue during processing
• specificity of probe
• endogenous reactants

Question 14

What are microarrays and what does the process involve?

Answer 14

Hybridization of a labelled nucleic acid sample (target = mRNA/cDNA/PCR product) to a very large set of oligonucleotide probes, where probes are attached to a solid support.

• mRNA is extracted from each sample, it is reverse transcribed to cDNA.
• one sample is labelled red while the other is labelled green.
• The combined sample of fluorescently labelled cDNA is applied to the microarray chip.
• the sample cDNA binds to the complementary sequence of DNA bases in the chip wells.
• after the Hybridisation step the chip is washed to remove any that are unbound.
• the chip is put in a laser scanner to electronically capture the data - the laser activated the fluorescent dyes.
  The computer calculates the ratio of red to green to determine and compare which genes are expressed in each sample.

Two major applications

• gene expression analysis
• genetic variation analysis.)