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:

- 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

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
samples 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 mms 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