Manipulation of DNA

Question 1

What are the physical properties of DNA are a direct consequence of its structural features?

Answer 1

double stranded helix
• complementary base pairing between strands
• hydrogen bonding that maintains helix
• length / diameter
• acidity due to negatively charged phosphate groups

Question 2

Solubility of DNA

Answer 2

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

Question 3

Viscosity of DNA

Answer 3

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

Question 4

UV light Absorption and Fluorescence

Answer 4

The nitrogenous bases of DNA strongly absorb UV
light maximally at 260nm
• used to measure concentration
• ssDNA absorption > dsDNA
• double helix shields bases

Question 5

Hoechst dye (H33342)

Answer 5

Dye bound to minor groove emits blue light

when illuminated with UV light (i.e. fluoresces)

Question 6

Denaturation

Answer 6

H-bonding is disrupted
• caused by increase in temperature, extremes of pH
• on denaturation viscosity decreases, UV absorption increases ­

Question 7

Tm

Answer 7

is temperature at which 50% of the DNA is melted

Question 8

DNA melting curve steepness

Answer 8

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

Question 9

Tm is a characteristic of DNA that depends on:

Answer 9

• the size of the molecule
• its GC content Tm µ G + C
• the pH and ionic strength

Question 10

Hybridisation

Answer 10

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

Question 11

Probes

Answer 11

Labelled DNA or RNA fragments are used to detect

specific nucleic acid sequences by hybridisation.

Question 12

Probes are among the most useful of molecular tools

it can be …

Answer 12

• Can be labelled with fluorescent tags

* Can be used on membranes, cells, tissues…

Question 13

Separation of DNA by Gel Electrophoresis

Answer 13

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

Question 14

Separating DNA: agarose gel electrophoresis

Answer 14

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

Question 15

Agarose gels

Answer 15

• 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).

Question 16

Vertical gel

Answer 16

Vertical gel to separate smaller fragments
• Made from
polyacrylamide
• Very thin

Question 17

Polyacrylamide gels

Answer 17

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

Question 18

Two approaches can be used to determine DNA concentration:

Answer 18

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.

Question 19

Assays that usefluorescenceto measure DNA concentration

Answer 19

• 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.

Question 20

Using an Assay to Measure DNA

Answer 20

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.