Gel Electrophoresis, Gel Loading Practice, and Polymerase Chain Reaction (PCR)

1. Gel Electrophoresis, Gel Loading Practice, and Polymerase Chain Reaction (PCR) October 15th – October 19th, 2012

2. Gel Electrophoresis The process by which electricity is used to separate charged molecules (DNA fragments, RNA, and proteins) based on there size, shape, and charge.

3. What you should already know… • Remember that opposite charges attract so if a molecule is negatively charged it will move towards the area with a positive charge • DNA (negativecharge) runs to red (red=positive charge) Wells

4. How it Works • Molecules are separated by an electrical current moving the molecules through an agarose gel • Electrical Current: Establishes electric field between the positive and negative electrodes • Causes molecules to move from well (where samples are loaded) through the gel • Positive molecules move toward negative end • Negative molecules move toward positive end • Agarose Gel: Acts as a “Molecular Strainer” • Creates a gel matrix • Smaller molecules pass more easily through the tiny spaces in the gel matrix and therefore run faster and farther than larger molecules Well

5. HINT!!! • Picture all of your friends running through a jungle. Your tall friends will probably get caught more easily on low hanging vines or branches and may struggle to get through particularly dense areas. Your short friends, however, will easily elude low hanging vines or branches and will be able to get through the areas with the dense vegetation that your taller friends struggled with. Your smaller friends (smaller molecules) will be able to travel farther and faster than your taller friends (larger molecules) because they won’t be caught by the dense vines and trees (gel matrix) that your tall friends will be slowed down by.

6. How We See the Results • Methylene blue – a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color • Used to see where samples are when loading them in a gel Methylene Blue • Ethidium bromide – a DNA stain (indicator); glows orange when it is mixed with DNA and exposed to UV light; abbreviated EtBr • Used to see how molecules were separated after running gel Ethidium Bromide

7. Contents of Gel • Ladder, or sample containing DNA fragments of known length/size (in base pairs) • Used to estimate size of/base pair length of isolated DNA fragments or other DNA samples run on the same gel DNA Samples

8. Concept Check! Largest • Where, on this gel are the largest molecules (in this case DNA fragments)? The smallest? Smallest

9. Gel Loading

10. Loading Samples: Good

11. Loading Samples: BAD Micropipette tip punched through the gel

12. PCR: Polymerase Chain Reaction A process by which a fragment of DNA is copied and recopied to produce millions of identical DNA fragments in a short amount of time

13. Summary • Denaturation: Strand Separation • The double-stranded DNA Template (DNA to be copied or amplified) is split into individual DNA strands with heat • Annealing: Primer binding • Primers bind to the separated DNA strands • Forward and Reverse primers • Primers: Short pieces of single-stranded DNA that are complementary to the target sequence of DNA • Target Sequence: Section you want to copy • Extension: New DNA synthesis • Taq polymerase synthesizes new DNA from the end of the primer • Heat Resistant!!! • Uses dNTPs (dATP, dCTP, dGTP, dTTP) • Single units of bases A, C, G, and T which act as the building blocks for the new DNA strands REPEAT!!!

14. Temperatures • Denaturation: 94 degrees Celsius • Annealing: 56 degrees Celsius • Elongation: 72 degrees Celsius

15. VIDEO!!! • http://www.hhmi.org/biointeractive/media/DNAi_PCR-lg.mov

16. PCR and Gel Electrophoresis • Run PCR samples through a Gel to see if you successfully copied the target sequence • Based on base pair length of samples

17. http://www.cnpg.com/video/flatfiles/539/ http://www.youtube.com/watch?v=7uafUVNkuzg The PCR Song There was a time when to amplify DNA, You had to grow tons and tons of tiny cells. Then along came a guy named Dr. Kary Mullis, Said you can amplify in vitro just as well. Just mix your template with a buffer and some primers, Nucleotides and polymerases, too. Denaturing, annealing, and extending. Well it’s amazing what heating and cooling and heating will do. PCR, when you need to detect mutations. PCR, when you need to recombine. PCR, when you need to find out who the daddy is. PCR, when you need to solve a crime. (repeat chorus)

18. PV92 PCR Informatics Kit: PCR, Gel Electrophoresis, Hardy-Weinberg, Bioinformatics, and mtDNA Isolation October 29th – November 2nd, 2012

19. PCR Review: Video • http://www.hhmi.org/biointeractive/media/DNAi_PCR-lg.mov

20. Gel Electrophoresis Review • Ladder, or sample containing DNA fragments of known length/size (in base pairs) • Used to estimate size of/base pair length of isolated DNA fragments or other DNA samples run on the same gel DNA Samples

21. Hardy-Weinberg • As G. H. Hardy stated in 1908, 'There is not the slightest foundation for the idea that a dominant trait should show a tendency to spread over a whole population, or that a recessive trait should die out.'  • A population maintains its genetic frequencies in the right conditions • Recessive traits do not die out, dominant traits are not taking over the world • Conditions: large population, random mating, no immigration or emigration, no mutations, and no natural selection • Hardy-Weinberg does not apply • Mutation, gene flow, genetic drift, assortative mating (marriage between close relatives) • Another explanation: • http://www.woodrow.org/teachers/bi/1994/hwintro.html

22. Hardy Weinberg Formula • The simplest case of a single locus (location of gene on chromosome) with two alleles • Dominant allele:A • Recessiveallele: a • Frequencies (how often alleles appear in a population): p and q • freq(A) = p • freq(a) = q • p + q = 1 • If mating is random then new individuals will have Hardy-Weinberg frequencies • freq(AA) = p2 for the AA homozygotes in the population • freq(aa) = q2 for the aahomozygotes • freq(Aa) = 2pq for the heterozygotes • The different ways to form new genotypes can be derived using a Punnett square • The formula is sometimes written as (p2) + (2pq) + (q2) = 1 • Probabilities must add up to one.

23. Bioinformatics A discipline that integrates mathematical, statistical, and computer tools to collect and process biological data

24. Overview Timeline: Prep • Before Lesson 1 (29th) • Aliquot InstaGene matrix • Set up student workstations • Before Lesson 2 (29th) • Prepare complete master mix and aliquot • Set up control PCR reactions • Prepare TAE buffer • Prepare molten agarose • Program MyCyclerthermalcycler • Set up student workstations • Before Lesson 3 (31st) • Prepare Fast Blast DNA stain • Set up student workstations • Before Lesson 4 (1st, FIRE) • Set up student workstations

25. Overview Timeline: Lab • Day 1 • Lesson 1: Cheek Cell DNA Template Preparation OR Hair Follicle DNA Template Preparation • Isolate cheek cells OR hairs • Prepare genomic DNA from cheek cells/hair follicles • Day 1 • Lesson 2: PCR Amplification • Set up and perform PCR reactions • Pour agarose gels (can be done before lab) • Day 2 • Lesson 3: Gel Electrophoresis of Amplified PCR Samples and Staining of Agarose Gels • Load and run gels • Stain gels • Day 3 • Lesson 4: Analysis and Interpretation of Results • Record the results and dry the gels • Analyze results • Day 4 • Lesson 5: Interpretation of Results: Bioinformatics • Enter classroom data into PV 92 Allele Server and analyze data

26. Day 1: Overview and Tips • Lesson 1: Cheek Cell DNA Template Preparation OR Hair Follicle DNA Template Preparation • Isolate DNA from epithelial cells that line the inside of your cheek by rinsing your mouth with a saline (salt) solution, and collect the cells using a centrifuge • The boil the cells to rupture them and release the DNA they contain • You will use the extracted genomic DNA as the target template for PCR amplification • Proceed to step 2 of Lesson 2

27. Day 1: Overview and Tips • Lesson 2: PCR Amplification • To replicate a piece of DNA, the reaction mixture requires the following components • 1. DNA template — containing the intact sequence of DNA to be amplified • 2. Individual deoxynucleotides (A, T, G, and C) — raw material of DNA (dNTPs) • 3. DNA polymerase — an enzyme that assembles the nucleotides into a new DNA chain • 4. Magnesium ions — a cofactor (catalyst) required by DNA polymerase to create the DNA chain • 5. Oligonucleotide primers — pieces of DNA complementary to the template that tell DNA polymerase exactly where to start making copies • 6. Salt buffer — provides the optimum ionic environment and pH for the PCR reaction • When combined under the right conditions, a copy of the original double-stranded template DNA molecule is made — doubling the number of template strands. Each time this cycle is repeated, copies are made from copies and the number of template strands doubles —from 2 to 4 to 8 to 16 and so on — until after 20 cycles there are 1,048,576 exact copies of the target sequence.

28. PCR Review: Video • http://www.hhmi.org/biointeractive/media/DNAi_PCR-lg.mov

29. Day 2: Overview and Tips • Lesson 3: Gel Electrophoresis of Amplified PCR Samples and Staining of Agarose Gels • Amplifying Alu element found in the PV92 region of chromosome 16 • Contained within an intron: region of DNA is never really used • Primers: Designed to bracket a sequence within the PV92 region that is 641 base pairs long if the intron does not contain the Alu insertion, or 941 base pairs long if Alu is present. • Neither chromosome contains the insert: each amplified PCR product will be 641 base pairs • Aluinsert on one chromosome but not the other: PCR product of 641 base pairs and one of 941 base pairs. The gel will reveal two bands for such a sample

30. Day 3: Overview and Tips • Lesson 4: Analysis and Interpretation of Results • Go in during FIRE to see results of gel • Be sure to figure out a way to get everyone’s data • Alleles: Basic characteristics that population geneticists use to describe and analyze populations

31. Day 4: Overview and Tips • Lesson 5: Interpretation of Results: Bioinformatics • Enter classroom data into PV 92 Allele Server and analyze data • You will perform a bioinformatics exercise to investigate the genotypic frequencies for the Alupolymorphism in your class population and compare them with the genotypic frequencies of other populations.