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 molecules2. 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:OH

O-CH2P –

–

=

–

O

O

O

-

-

N

N

O

O-CH2P –

–

=

–

O

O

O

-

N

N

O-CH2P –

–

=–

O

O

-

N

N

Nucleotide 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

Agarose

Capillary

Acrylamide

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

Anode + - Cathode

- Cathode

Anode +

Loading Wells

Buffer

Gel

Side View of Gel and Gel Box 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 monomer

Bisacrylamide cross-linker

Figure 12.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Acrylamide

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

Anode +

- Cathode- Cathode

Anode +

Loading Wells

BufferGel

Side View of Gel and Gel Box 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

- Cathode + Anode

Sample Tray

Capillaryfilled with polymer

Laser Detection

Buffer

Buffer

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

(b)

Ogston Sieving Reptation

Small DNA molecules

Long DNA molecules

Gel

Figure 12.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

DNA Separation

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