DNA Separation Methods
Chapter 12
DNA molecules
• After PCR reaction produces many copies
of DNA molecules
• Need a way to separate the DNA
molecules from similar sized molecules
• Only way to genotype samples
• Multiplex PCR may produce:
– More than 20 different products
– Some only 1 or 2 base pairs apart
Separation
• Need to pull DNA molecules apart from
each other in their solutions
• Separation based on size differences
– Also by color of dye, more on that later
• Electrophoresis:
– Using electricity and different sized pores
– Gel techniques
– Capillary techniques
Electrophoresis
•
Means – electricity (or charge) bearer
• Two key components:
1. Electric charge
1. Pull on the DNA molecules
2. Matrix with pores
1. Separate the molecules based on the size of
the DNA and the size of the pores
DNA is charged
• Nucleic acid is an acid = drops off its H+
• One phosphorous component on each
nucleotide is an acid
– Other two are taken up with covalent bonds
• Acids are negatively charged in solution
– Because H+ has been stripped off
• Backbone of DNA has negative charge
• Is attracted to positive charge
DNA Backbone:
–
N
- O P – O-CH
2
–
N
=
O
N
N
OP – O-CH2
=
-O P – O-CH
2
=
O
N
–
–
O
–
O
OH
Nucleotide
O
–
-O
DNA Chain
N
Electrical Charge
Electrophoresis uses two charges:
• Anode
– Positive charge
– Attracts DNA molecules
• Cathode
– Negative charge
– DNA will migrate away
• Voltage = amount of charge
– Higher voltage – faster DNA will move
Types of Separation Matrixes
Gels
• Agarose gels
• Polyacrylamide gels
• Denaturing or “native”
Capillaries
• Narrow silica capillary with polymer matrix
inside
Separation Methods
Acrylamide
Agarose
Capillary
Slab Gels
• Solid matrix with pores
• Buffer solution goes through pores
• DNA is separated as it tries to pass
through pores
• Matrix is mixed with buffer solution
• Poured into a mold
• A “comb” is inserted – makes holes for the
“wells” – where the sample will be added
Horizontal Gels
Loading Wells
Anode +
- Cathode
- Cathode
Gel
Buffer
Side View of Gel and Gel Box
Anode +
Top view of gel
Slab Gels
• Agarose gels
– Sugar from seaweed
– Large pores – quicker travel time
– ~ 2000 angstroms in diameter
• Acrylamide gels
– Polymerization of acrylamide subunits
– Small pores – finer resolution of samples
– ~200 angstroms in diameter
Agarose
•
•
•
•
Large pores ~2000 angstroms
Useful for RFLP or DNA quantification
Not useful for STRs
Weigh out appropriate amount of agarose
powder – add buffer
• Heat until agarose goes into solution
• Pour into gel box – define shape and
thickness of gel
Agarose
• Add comb before agarose cools
• Comb is removed after agarose has “set”
• Leaving behind loading wells
– Usually hold around 10 uL of sample
– Depends on size and depth of comb
• Number of teeth in comb define number of
wells per gel
• Molecular weight standards and controls
are loaded into wells adjacent to samples
Agarose
• Loading dye is added to samples
– Contains a dark blue dye so that you can see
the sample while you load it
– Also contains something to increase the
sample’s viscosity so that it will stay in well
• Have to be very careful not to spill sample
out of well or place into wrong well
• Smaller DNA moves faster through matrix
– Separating the samples based on size
Acrylamide
• Smaller pores ~ 200 angstroms
• Useful to separate STRs
– Resolution down to 1 base pair difference
• Acrylamide mixture is “activated” by
adding TEMED
– Starts the polymerization
• Must pour gel immediately after adding
TEMED – before it hardens
Acrylamide
Acrylamide
monomer
Bisacrylamide
cross-linker
Figure 12.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Acrylamide
• Usually vertical gels
• “Pouring” gel is actually sliding two glass
plates over gel material
• Making very thin sheet of gel matrix
– Few mm’s thick between glass
• Bubbles are a huge problem
– Introduced when sliding plates together
– Cannot run a sample through a bubble
– Will push sample into surrounding lanes
Vertical Gels
Loading Wells
- Cathode
- Cathode
Buffer
Gel
Anode +
Side View of Gel and Gel Box
Anode +
Front view of gel
Combs
• Shape of wells depends on the combs
used
• Square tooth combs
– Have square teeth – form thick square wells
• Shark tooth combs
– Arched divisions between lanes
– Keep comb in the gel while running samples
– More often used with vertical acrylamide gels
Heat
• Movement of electrons generates heat
• Heat must be dissipated while running
– Buffer is liquid to help absorb heat
• Excessive heat will cause gel to “smile”
– Bands will curve up at each end
– Makes difficult to correctly call allele size
• Too much heat will cause gel to melt
completely
Denaturing Gels
In order to get better resolution:
• Remove any secondary structure between
DNA strands
• Make DNA single stranded
– Denatured
• Single stranded DNA is more flexible
• Secondary structure can stop DNA from
traveling through the matrix at all
Denaturing Conditions
Ways to denature DNA:
• Chemicals that keep the strands of DNA
from forming H-bonds
– Formamide or urea
• Heat
– Opens up DNA just like with 1st step of PCR
– Heat sample to 95° immediately before
loading gel
Problems with Gels
• Labor intensive
– And mundane
• Bubbles waste time and materials
– Especially if you waste evidence DNA
• Acrylamide is a neurotoxin
– Therefore dangerous to work with
• Have to be careful when loading
– Cannot spill sample or load into wrong lane!
Capillary Electrophoresis
• Narrow flexible glass capillary
– Filled with polymer liquid
• Capillary sucks sample up and through the
polymer matrix based on high voltage
• Buffer held at beginning and end of
capillary – also sucked through polymer
• Larger DNA molecules are retarded by the
polymer chains – travel slower through
capillary than smaller DNA molecules
Capillaries
• Polymer is “poured” by filling capillary
• Capillary can be thought of as long and
narrow gel box
• Polymer is like liquid gel matrix
• Voltage can be much higher with capillaries
than with a standard gel
– Because heat is dissipated quickly
• A laser read the “bands” as they travel past
Capillary Electrophoresis
Capillary
filled with polymer
Laser Detection
- Cathode
+ Anode
Buffer
Buffer
Sample Tray
Advantages of Capillaries
• No gels to pour
– Saves time, money and sample
• Can be fully automated
– Injection, separation and detection
• Less sample is used
• Detection of bands is done immediately
• Separation can be completed within
minutes rather than hours
– Because can run at a higher voltage
Disadvantages to Capillaries
• Throughput
– Idea is that one capillary can only run one
sample at a time
– Whereas a gel runs 20 or more samples
– No longer an issue
– 96 Capillary machines
• Cost
– Machines cost more than $ 100,000
– All reagents cost more as well
DNA separation
Two main ideas for how DNA separates as it
goes through matrixes
1. Ogston Sieving
– Behavior of molecules smaller than pores
2. Reptation
– Behavior of molecules larger than pores
Both based on the idea that the larger a
molecule is the slower it will travel
through matrix
DNA Separation
Ogston Sieving
• Regards the DNA molecule like a tangle of
thread
• Or a small sphere
• Tumbling through the pores
• Travel as fast as they can find the next
pore they can fit through
• Smaller molecules fit into more pores
• Therefore travel faster
Reptation
• Regards the long DNA molecule as a
snake
• Slithering through the matrix by stretching
out fairly straight without tangles
• As the DNA winds its way through the
pores the longer the DNA strand the
longer it takes because its route is more
complicated
DNA Separation
(b)
Gel
Long DNA
molecules
Small DNA
molecules
Ogston Sieving
Reptation
Figure 12.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Size Standards
• Electrophoresis and how long it takes DNA
to travel through matrix is relative
• Therefore there must be a size standard
run at the same time
• In a gel
– Run the size standard in an adjacent lane
• In a capillary
– Run the size standard with the sample
– With a different color florescent dye
Any Questions?
Read Chapter 13