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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
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
DNA Backbone:
- O P O-CH
2
=
O
N
N
OP O-CH2
-O P O-CH
2
=
OH
Nucleotide
-O
DNA Chain
Electrical Charge
Electrophoresis uses two charges:
Anode
Positive charge
Attracts DNA molecules
Cathode
Negative charge
DNA will migrate away
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
Anode +
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
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
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
samples viscosity so that it will stay in well
Acrylamide
Smaller pores ~ 200 angstroms
Useful to separate STRs
Resolution down to 1 base pair difference
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 mms thick between glass
Vertical Gels
Loading Wells
- Cathode
- Cathode
Buffer
Gel
Anode +
Anode +
Combs
Shape of wells depends on the combs
used
Square tooth combs
Have square teeth form thick square wells
Heat
Movement of electrons generates heat
Heat must be dissipated while running
Buffer is liquid to help absorb heat
Denaturing Gels
In order to get better resolution:
Remove any secondary structure between
DNA strands
Make DNA single stranded
Denatured
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
Acrylamide is a neurotoxin
Therefore dangerous to work with
Capillary Electrophoresis
Narrow flexible glass capillary
Filled with polymer liquid
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
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
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
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