12 Separation
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Transcript of 12 Separation
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DNA Separation Methods
Chapter 12
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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
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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
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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
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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
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DNA Backbone:OH
O-CH2P
=
O
O
O
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N
N
O
O-CH2P
=
O
O
O
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N
N
O-CH2P
=
O
O
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N
N
Nucleotide DNA Chain
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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
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Types of Separation Matrixes
Gels Agarose gels Polyacrylamide gels Denaturing or native
Capillaries Narrow silica capillary with polymer matrix
inside
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Separation Methods
Agarose
Capillary
Acrylamide
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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
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Horizontal Gels
Anode + - Cathode
- Cathode
Anode +
Loading Wells
Buffer
Gel
Side View of Gel and Gel Box Top view of gel
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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
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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
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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
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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
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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
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Acrylamide monomer
Bisacrylamide cross-linker
Figure 12.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press
Acrylamide
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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
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Vertical Gels
Anode +
- Cathode- Cathode
Anode +
Loading Wells
BufferGel
Side View of Gel and Gel Box Front view of gel
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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
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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
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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
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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
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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!
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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
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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
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Capillary Electrophoresis
- Cathode + Anode
Sample Tray
Capillaryfilled with polymer
Laser Detection
Buffer
Buffer
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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
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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
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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
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DNA Separation
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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
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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
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(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
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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
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Any Questions?
Read Chapter 13
DNA Separation MethodsDNA moleculesSeparationElectrophoresisDNA is chargedDNA Backbone:Electrical ChargeTypes of Separation MatrixesSeparation MethodsSlab GelsHorizontal GelsSlab GelsAgaroseAgaroseAgaroseAcrylamideAcrylamideVertical GelsCombsHeatDenaturing GelsDenaturing ConditionsProblems with GelsCapillary ElectrophoresisCapillariesCapillary ElectrophoresisAdvantages of CapillariesDisadvantages to CapillariesDNA separationDNA SeparationOgston SievingReptationSize StandardsAny Questions?