Introduction to Electrophoresis

1
Introduction to Electrophoresis
2
.
What is Electrophoresis?     A method of
separating large molecules (such as DNA fragments
or Proteins ) from a mixture of similar
molecules. An electric current is passed through
a medium containing the mixture, and each kind of
molecule travels through the medium at a
different rate, depending on its electrical
charge and size. Separation is based on these
differences. Agarose and acrylamide gels are the
media commonly used for electrophoresis of
proteins and nucleic acids
3
Gel Electrophoresis

• The pH and other buffer conditions are arranged
  so that the molecules being separated carry a net
  (negative) charge so that they will me moved by
  the electric field toward the positive pole. As
  they move through the gel, the larger molecules
  will be held up as they try to pass through the
  pores of the gel, while the smaller molecules
  will be impeded less and move faster. This
  results in a separation by size, with the larger
  molecules nearer the well and the smaller
  molecules farther away.

4
Electrophoresis of DNA

• The Phosphate groups on the backbone of the DNA
  molecule readily give up their H ions, therefore
  nucleic acids are negatively charged in most
  buffer systems.
• DNA molecules will migrate away from the negative
  electrode (cathode), and migrate towards the
  positive electrode (anode).
• The higher the voltage, the greater the force
  felt by the DNA molecule, and the faster they
  will migrate in an electric field.

5
Electrophoretic Separation of DNA

• Agarose Gel Electrophoresis
• Acrylamide Gel Electrophoresis (Native versus
  Denaturing Conditions)
• Capillary Electrophoresis

6
Gel Matrices Used for Electrophoresis of DNA

• Agarose Gels have fairly large pore sizes and are
  used for separating larger DNA molecules
  (Restriction Fragment Length Polymorphism
  Analysis)
• Polyacrylamide Gels are used to obtain high
  resolution separations for smaller DNA molecules
  (STR analysis and DNA sequence analysis)

7
Introduction to Agarose Gel Electrophoresis
8
Agarose Gel Electrophoresis

• Yield Gel Semiquantitative and qualitative
  analysis of isolated DNA
• Separation of DNA restricted with Hae III (RFLP
  analysis) followed by a Southern Blot and
  Hybridization with a labeled probe
• Post Amplification confirmation and qualitative
  assessment of PCR product

9
Assessing DNA Quality
Experiment

• 100 ng K562 DNA
• Digest with DNAse

23Kbp
2kbp
10
Agarose Gel Electrophoresis

• An electrophoresis chamber and power supply
• Gel casting trays, which are available in a
  variety of sizes and composed of UV-transparent
  plastic.
• Sample combs, around which molten agarose is
  poured to form sample wells in the gel.
• Electrophoresis buffer, usually Tris-acetate-EDTA
  (TAE) or Tris-borate-EDTA (TBE).

11
Agarose Gel Electrophoresis

• Loading buffer, which contains something dense
  (e.g. glycerol) to allow the sample to "fall"
  into the sample wells, and one or two tracking
  dyes, which migrate in the gel and allow
  monitoring or how far electrophoresis has
  proceeded.
• A fluorescent dye used for staining nucleic
  acids, such as Ethidium bromide, Sybr Green, or
  Sybr Gold.
• Transilluminator or Fluorescent Gel Scanner for
  photodocumentation

12
Migration of DNA Fragments in Agarose

• Fragments of linear DNA migrate through agarose
  gels with a mobility that is inversely
  proportional to the log10 of their molecular
  weight

13
Agarose Concentration

• By using gels with different concentrations of
  agarose, one can resolve different sizes of DNA
  fragments. Higher concentrations of agarose
  facilite separation of small DNAs, while low
  agarose concentrations allow resolution of larger
  DNAs.

14
Agarose Concentration
15
(No Transcript)
16
Agarose ElectrophoresisVoltage

• As the voltage applied to a gel is increased,
  larger fragments migrate proportionally faster
  that small fragments. For that reason, the best
  resolution of fragments larger than about 2 kb is
  attained by applying no more than 5 volts per cm
  to the gel (the cm value is the distance between
  the two electrodes, not the length of the gel).

17
Electrophoresis Buffer

• Several different buffers have been recommended
  for electrophoresis of DNA. The most commonly
  used for duplex DNA are TAE (Tris-acetate-EDTA)
  and TBE (Tris-borate-EDTA). DNA fragments will
  migrate at somewhat different rates in these two
  buffers due to differences in ionic strength.
  Buffers not only establish a pH, but provide ions
  to support conductivity. If you mistakenly use
  water instead of buffer, there will be
  essentially no migration of DNA in the gel!
  Similarly, if you use concentrated buffer (e.g. a
  10X stock solution), enough heat may be generated
  in the gel to melt it.

18
Agarose Gel Electrophoresis System
19
Agarose Gel Tips

• When preparing agarose for electrophoresis, it is
  best to sprinkle the agarose into
  room-temperature buffer, swirl, and let sit at
  least 1 min before microwaving. This allows the
  agarose to hydrate first, which minimizes foaming
  during heating.
• Electrophoresis buffer can affect the resolution
  of DNA. TAE (Tris-Acetate-EDTA) buffer provides
  better resolution of fragments gt4 kb, while TBE
  (Tris-Borate-EDTA) buffer provides better
  resolution of 0.1- to 3-kb fragments. In
  addition, use TBE buffer when electrophoresing
  gt150 V and use TAE buffer with supercoiled DNA
  for best results.

20
Agarose Gel Tips

• Migration of DNA is retarded and band distortion
  can occur when too much buffer covers the gel.
  The slower migration results from a reduced
  voltage gradient across the gel..
• Loading DNA in the smallest volume possible will
  result in sharper bands.
• Electrophoresing a gel too "hot" can cause the
  DNA to denature in the gel. It can also cause the
  agarose gel to deform.

21
Weigh out a gram of agarose.
22

• Mix the agarose with 50- 100 ml of buffer.

23

• Heat to dissolve the agarose.

24

• Assemble the gel tray and comb.

25
Pour the gel.
26

• Load one DNA sample into each well on the gel.

27
Connect the gel to a low voltage power supply.
28
Agarose Gel Electrophoresis

• After the samples are loaded, slowly fill the gel
  box with the 1X buffer. Make sure the gel is
  completely covered.
• Alternatively gels can be covered with buffer
  first, and then samples in dye buffer are loaded
  into each well.

29
Agarose Gel Electrophoresis

• Turn the switch on the power supply to "Off"
  before connecting the electrophoresis chamber.
• Place the lid tightly on the chamber and plug the
  electrical leads into the recessed output jacks
  of the power supply.
• Plug the red () lead into the red jack, and the
  black (-) lead into the black jack.

30
Agarose Gel Electrophoresis

• Select the desired voltage on the power supply. A
  voltage of 150 will permit the electrophoresis
  run to be completed in about an hour.

31
Agarose Gel Electrophoresis

• Turn the power supply switch "ON." The blue
  migration dye should move toward the positive
  electrode (red). If it is migrating toward the
  negative electrode (black), turn off the power
  supply, remove the lid, turn the gel tray 180o,
  replace the lid, and turn the power supply "ON."

32
After completion of the run, add a DNA staining
material and visualize the DNA under UV light.
33
Ethidium Bromide
34
Ethidium Bromide

• This compound contains a planar group that
  intercalates between the stacked bases of DNA.
• The orientation and proximity of ethidium with
  the stacked bases causes the dye to display an
  increased flourescence compared to free dye (in
  solution).
• U.V. radiation at 254 nm is absorbed by the DNA
  and transmitted to the bound dye.
• The energy is re-emitted at 590 nm in the
  red-orange region of the spectrum.

35
Ethidium Bromide

• Ethidium bromide is usually prepared as a stock
  solution of 10 mg/ml in water, stored at room
  temp and protected from light.
• The dye is usually incorporated into the gel and
  running buffer, or conversely, the gel is stained
  after running by soaking in a solution of
  ethidium bromide (0.5 ug/ml for 30 min).
• The stain is visualized by irradiating with a UV
  light source (i.e. using a transiluminator) and
  photographing with Polaroid film.
• The usual sensitivity of detection is better than
  0.1 ug of DNA.

36
Gel Staining
37
Introduction to Polyacrylamide Gel Electrophoresis
38
Polyacrylamide Gel Electrophoresis Monomeric
acrylamide (which is neurotoxic) is polymerized
in the presence of free radicals to form
polyacrylamide. The free radicals are provided by
ammonium persulphate and stabilized by TEMED
(N'N'N'N'-tetramethylethylene-diamine). The
chains of polyacrylamide are cross-linked by the
addition of methylenebisacrylamide ( bis) to form
a gel whose porosity is determined by the length
of chains and the degree of crosslinking.
39
Polyacrylamide Gel Electrophoresis

• The chain length is proportional to the
  acrylamide concentration usually between 3.5
  and 20. Cross-linking BIS-acrylamide is usually
  added at a ratio of 2g BIS 38g acrylamide (1
  20).

40
Polyacrylamide Gel Electrophoresis
Polyacrylamide gels are poured between two glass
plates held apart by spacers of 0.4 - 1.0 mm and
sealed with tape. Most of the acrylamide solution
is shielded from oxygen so that inhibition of
polymerization is confined to the very top
portion of the gel. The length of the gel can
vary between 10 cm and 1m depending on the
separation required. They are always run
vertically with 0.5x or 1x TBE as a buffer.
41
Polyacrylamide Gel Electrophoresis

• Polyacrylamide gels have enough resolving power
  to separate fragments differing by only one base
  pair in size, but their range is 5 to 1000 bp.
  They are much more difficult to handle than
  agarose gels.

42
Types of Polyacrylamide gels

• Non-denaturing gels these are run at low
  voltages - 8V/cm - and 1 x TBE to prevent
  denaturation of small fragments of DNA by the
  heat generated in the gel during electrophoresis.
  The rate of migration is approximately inversely
  proportional to log10 of their size. However, the
  base sequence composition can alter the
  electrophoretic mobility of DNAs such that two
  DNAs of the same size may show up to a 10
  difference in electrophoretic mobility

43
Types of Polyacrylamide gels

• Denaturing gels these gels are polymerized with
  a denaturant that suppresses base pairing in
  nucleic acids - this is usually urea but can be
  formamide. Denatured DNA migrates through the gel
  at a rate which is almost completely independent
  of its composition or sequence.

44
Acrylamide Gel Electrophoresis Effect of Gel
Percentage and Size Separation
45
Dye Migration in Different Polyacrylamide Gels
46
SEPARATION OF PCR PRODUCTS DENATURING
ACRYLAMIDEGEL ELECTROPHORESIS
47
Digital Imaging Hardware
FMBIO II Fluorescence Imaging System
48
PowerPlexTM 1.1
CSF1PO
D16S539
D7820
TPOX
D13S317
TH01
vWA
D5S818
P-41411 P-41414
P-41411 P-41414
49
Introduction to Capillary Electrophoresis
50
Electrophoresis

• Electrophoresis refers to the migration of
  charged electrical species when dissolved, or
  suspended, in an electrolyte through which an
  electric current is passed. Cations migrate
  toward the negatively charged electrode (cathode)
  and anions are attracted toward the positively
  charged electrode (anode). Neutral solutes are
  not attracted to either electrode. The
  traditional electrophoresis equipment offered a
  low level of automation and long analysis times.

51
Electrophoresis

• Detection of the separated bands was performed by
  post-separation visualization.
• The analysis times were long as only relatively
  low voltages could be applied before excessive
  heat formation caused loss of separation.

52
Heat Dissipation

• In conventional slab gel electrophoresis the
  Joule heat associated with the generation of
  current during separation can cause problems of
  peak dispersion. This Joule heat causes the
  formation of convention currents within the gel
  which mixes the zones during separation and
  results in band broadening and peak dispersion.
  Heat generation therefore restricts the operating
  voltages that can be used in slab gel
  electrophoresis which produces longer analysis
  times.

53
Heat Dissipation

• Performing electrophoresis in a capillary allows
  the heat to be effectively dissipated through the
  capillary walls which reduces any convection
  related band broadening. This improved heat
  dissipation means that higher operating voltages
  can be used in CE which can produce significantly
  faster analysis times.

54
Capillary Electrophoresis

• The advantages of conducting electrophoresis in
  capillaries was highlighted in the early 1980's
  by the work of Jorgenson and Lukacs who
  popularized the use of CE. Performing
  electrophoretic separations in capillaries was
  shown to offer the possibility of automated
  analytical equipment, fast analysis times and
  on-line detection of the separated peaks.

55
Capillary Electrophoresis

• Heat generated inside the capillary was
  effectively dissipated through the walls of the
  capillary which allowed high voltages to be used
  to achieve rapid separations. The capillary was
  inserted through the optical center of a detector
  which allowed real time capillary detection.

56
Capillary Electrophoresis

• Operation of a CE system involves application of
  a high voltage (typically 10-30kV) across a
  narrow bore (25-100mm) capillary. The capillary
  is filled with electrolyte solution which
  conducts current through the inside of the
  capillary. The ends of the capillary are dipped
  into reservoirs filled with the electrolyte.

57
Capillary Electrophoresis

• Electrodes made of an inert material such as
  platinum are also inserted into the electrolyte
  reservoirs to complete the electrical circuit. A
  small volume of sample is injected into one end
  of the capillary. The capillary passes through a
  detector, usually a UV absorbance detector, at
  the opposite end of the capillary.

58
Capillary Electrophoresis

• Application of a voltage causes movement of
  sample ions towards their appropriate electrode
  usually passing through the detector. The plot of
  detector response with time is generated which is
  termed an electropherogram.

59
Raw Data from the ABI Prism 310
(prior to separation of fluorescent dye colors)
60
Capillaries

• The capillaries used are normally fused silica
  capillaries covered with an external polyimide
  protective coating to give them increased
  mechanical strength as bare fused silica is
  extremely fragile. A small portion of this
  coating is removed to form a window for detection
  purposes. The window is aligned in the optical
  centre of the detector.

61
Capillaries

• Capillaries are typically 25-75 cm long with 50
  and 75 micron being the most commonly employed
  inner diameters. On standard commercial CE
  instruments the capillary is often held in a
  housing device such as a cartridge to facilitate
  ease of capillary insertion into the instrument
  and to protect the delicate detection window
  area.

62
Capillaries

• The inner surface of the capillary can be
  chemically modified by covalently binding
  (coating) different substances onto the capillary
  wall. These coatings are used for a variety of
  purposes such as to reduce sample adsorption or
  to change the ionic charge on the capillary wall.

63
Capillary Gel Electrophoresis

• The capillaries we typically use in CE are
  commercially available in single or multiple
  arrays. We use capillaries that range about 30 to
  50 centimeters in length, 0.150 to 0.375
  millimeters in outer diameter, and a 0.010 to
  0.075 millimeter diameter channel down the
  center.

64
(No Transcript)
65
Capillary Electrophoresis Process
66
Capillary Electrophoresis
67
(No Transcript)
68
ABI Prism 310 Genetic Analyzer
69
ABI Prism 310 Genetic Analyzer
70
Chemistry Involved

• Injection
• electrokinetic injection process
• importance of sample preparation (formamide)
• Separation
• capillary
• POP-4 polymer
• buffer
• Detection
• fluorescent dyes with excitation and emission
  traits
• virtual filters (hardware/software issues)

71
Principles of Sample Separation and Detection
Labeled DNA fragments (PCR products)
Capillary or Gel Lane
72
Sample Application

• The standard sample injection procedure is to dip
  the capillary and electrode into the sample
  solution vial and to apply a voltage. If the
  sample is ionized and the appropriate voltage
  polarity is used then sample ions will migrate
  into the capillary. This type of injection is
  known as electrokinetic sampling.

73
SAMPLE APPLICATION
74
Electrokinetic Injection Process
Capillary
Electrode
75
Comments on Sample Preparation

• Use high quality formamide (lt100 ?S/cm)!
• ABI sells Hi-Di formamide
• regular formamide can be made more pure with ion
  exchange resin
• Deionized water vs. formamide
• Biega and Duceman (1999) J. Forensic Sci. 44
  1029-1031
• water works fine but samples are not stable as
  long as with formamide
• Denaturation with heating and snap cooling
• use thermocycler for heating and wet ice bath to
  snap cool
• heat/cool denaturation step is not always
  necessary...

76
Separation Issues

• Run temperature -- 60 oC helps reduce secondary
  structure on DNA and improves precision
• Electrophoresis buffer -- urea in running buffer
  helps keep DNA strands denatured
• Capillary wall coating -- dynamic coating with
  polymer
• Polymer solution -- POP-4, POP-6

77
DNA Separation Mechanism

• Size based separation due to interaction of DNA
  molecules with entangled polymer strands
• Polymers are not cross-linked (as in slab gels)
• Gel is not attached to the capillary wall
• Pumpable -- can be replaced after each run
• Polymer length and concentration determine the
  separation characteristics

78
Detection Issues

• Fluorescent dyes
• spectral emission overlap
• relative levels on primers used to label PCR
  products
• dye blobs (free dye)
• Virtual filters
• hardware (CCD camera)
• software (color matrix)

Filters determine which wavelengths of light are
collected onto the CCD camera
79
Laser Used in ABI 310

• Argon Ion Laser
• 488 nm and 514.5 nm for excitation of dyes
• 10 mW power
• Lifetime 5,000 hours (1 year of full-time use)
• Cost to replace 5,500
• Leads to highest degree of variability between
  instruments and is most replaced part
• Color separation matrix is specific to laser used
  on the instrument