DNA Separation Methods. Chapter 12

Transcription

1 DNA Separation Methods Chapter 12

2 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

3 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

4 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

5 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

6 DNA Backbone: N - O P O-CH 2 N -O O - P = O O-CH 2 N N = O -O O P O-CH 2 = O N N OH Nucleotide O DNA Chain

7 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

8 Types of Separation Matrixes Gels Agarose gels Polyacrylamide gels Denaturing or native Capillaries Narrow silica capillary with polymer matrix inside

9 Separation Methods Acrylamide Agarose Capillary

10 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

11 Horizontal Gels Loading Wells - Cathode Anode + - Cathode Gel Buffer Side View of Gel and Gel Box Anode + Top view of gel

12 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

13 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

14 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

15 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

16 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

17 Acrylamide Acrylamide monomer Bisacrylamide cross-linker Figure 12.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition 2005 Elsevier Academic Press

18 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

19 Vertical Gels - Cathode Loading Wells - Cathode Buffer Gel Anode + Anode + Side View of Gel and Gel Box Front view of gel

20 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

21 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

22 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

23 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 1 st step of PCR Heat sample to 95 immediately before loading gel

24 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!

25 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

26 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

27 Capillary Electrophoresis Capillary filled with polymer Laser Detection - Cathode + Anode Buffer Sample Tray Buffer

28 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

29 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

30 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

31 DNA Separation

32 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

33 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

34 DNA Separation (b) Gel Long DNA molecules Small DNA molecules Ogston Sieving Reptation Figure 12.4, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition 2005 Elsevier Academic Press

35 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

36 Any Questions? Read Chapter 13