Manipulation of DNA Flashcards
What are the physical properties of DNA are a direct consequence of its structural features?
double stranded helix
• complementary base pairing between strands
• hydrogen bonding that maintains helix
• length / diameter
• acidity due to negatively charged phosphate groups
Solubility of DNA
DNA is polyanionic due to its high phosphate content.
Thus it is:
• soluble in aqueous / low salt conditions
• a salt of Na+, Mg2+ etc. at neutral pH
• insoluble in alcohols
Viscosity of DNA
DNA is naturally very viscous (resists flow, thick and clinging like egg
white)
• long, rigid, rod-like shape
• large hydrodynamic volume - attracts a very large shell
of water (due to the phosphate groups)
• disruption of H-bonding decreases viscosity,
ie. dsDNA –>ssDNA
• mechanical shearing decreases viscosity
UV light Absorption and Fluorescence
The nitrogenous bases of DNA strongly absorb UV light maximally at 260nm • used to measure concentration • ssDNA absorption > dsDNA • double helix shields bases
Hoechst dye (H33342)
Dye bound to minor groove emits blue light
when illuminated with UV light (i.e. fluoresces)
Denaturation
H-bonding is disrupted
• caused by increase in temperature, extremes of pH
• on denaturation viscosity decreases, UV absorption increases
Tm
is temperature at which 50% of the DNA is melted
DNA melting curve steepness
Steepness of transition depends on the complexity of
DNA
steeper - melts over a narrower temperature range
and is less complex
ie. red is less complex than blue
Tm is a characteristic of DNA that depends on:
- the size of the molecule
- its GC content Tm µ G + C
- the pH and ionic strength
Hybridisation
Nucleic acids from different species can form
hybrids through complementary base pairing
1- Two DNA species are heat denatured, mixed and cooled.
2- In addition to renaturation, hybrid molecules can form where the species have complementary
Hybrids may also involve RNA species eg. DNA-RNA sequences
Probes
Labelled DNA or RNA fragments are used to detect
specific nucleic acid sequences by hybridisation.
Probes are among the most useful of molecular tools
it can be …
- Can be labelled with fluorescent tags
* Can be used on membranes, cells, tissues…
Separation of DNA by Gel Electrophoresis
1- DNA fragments Place mixture in the well of an
agarose or polyacrylamide gel. Apply electric field
Molecules move through pores in gel at a rate inversely proportional to their chain length
Separating DNA: agarose gel electrophoresis
DNA is negatively charged • DNA bands are invisible • Needs to be stained in some way • Ethidium bromide binds to DNA and fluoresces under UV light • Other stains visible under blue light (SyBr safe stain) • Or no light- eGel
Agarose gels
• Polysaccharide extracted from seaweed • coarse pore size – adjustable • used to separate fragments in the size range ~ 0.6 - 25 kb • discrimination of ~1% of length • Detection of bands: • autoradiography, or • staining e.g. with ethidium bromide and viewed under UV illumination (as here).
Vertical gel
Vertical gel to separate smaller fragments
• Made from
polyacrylamide
• Very thin
Polyacrylamide gels
synthetic • finer pore size - variable • can separate fragments differing by a single nucleotide - used for sequencing • used over size range 1-250b (up to ~1kb) • illustration of autoradiographic band detection
Two approaches can be used to determine DNA concentration:
DNA contains the bases adenine, cytosine, guanine and thymine whose molecular structure allows them to absorb light in the ultraviolet (UV) region of the spectrum (~260 nm). UV absorbance can be used to determine DNA concentration.
Assays that usefluorescenceto measure DNA concentration
- significantly more sensitive than those that rely on the intrinsic light absorbing properties of DNA.
Fluorescent molecules have properties such that, when excited by absorbing light at specific wavelengths, they emit light (i.e. fluoresce) at a wavelength longer than the excitation light.
DNA does not have a useful fluorescencebutcan be labelled with highly fluorescent chemicals such as ethidium bromide or Hoechst 33342 (H33342 for short).
When illuminated with UV light (~360 nm) H33342 fluoresces with blue light (~460 nm) but only when bound to double stranded DNA.
The fluorescence signal is proportional to the amount of DNA in solution, allowing us to quantify the amount present.
Using an Assay to Measure DNA
In order to determine the concentration of DNA in an unknown sample, we can utilise a series of references or standards. By incubating these standards with H33342 and measuring the fluorescence, we can prepare what is known as a calibration curve, also known as a standard curve. This is done by plotting the concentration of each standard against the emitted fluorescence and generating a ‘line of best fit’. This can then be used to estimate the relative concentration of an unknown sample based on its fluorescence.
