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

October 15th – October 19th, 2012

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

What you should already know…

Molecule Charge Behavior

DNA Negative Moves to positive

RNA Negative Moves to positive

Proteins PositiveNegativeNeutral

Moves to negativeMoves to positiveNo movement

Carbohydrates Neutral No movement

Lipids Neutral No movement

• Remember that opposite charges attract so if a molecule is negatively charged it will move towards the area with a positive charge

• DNA (negative charge) runs to red (red=positive charge)Wells

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

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.

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

• 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

Methylene Blue

Ethidium Bromide

Contents of Gel

DNA Samples

•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

Concept Check! Where, on this gel are the largest molecules (in this case

DNA fragments)? The smallest?

Largest

Smallest

Gel Loading

Loading Samples: Good

Loading Samples: BAD

Micropipette tip punched through the

gel

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

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

RE

PE

AT

!!!

Temperatures Denaturation: 94 degrees Celsius Annealing: 56 degrees Celsius Elongation: 72 degrees Celsius

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

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

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)

http://www.cnpg.com/video/flatfiles/539/

http://www.youtube.com/watch?v=7uafUVNkuzg

PV92 PCR Informatics Kit: PCR, Gel Electrophoresis, Hardy-Weinberg, Bioinformatics, and mtDNA Isolation

October 29th – November 2nd, 2012

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

Gel Electrophoresis Review

DNA Samples

•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

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

Hardy Weinberg Formula

The simplest case of a single locus (location of gene on chromosome) with two alleles Dominant allele: A  Recessive allele: 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 aa homozygotes 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.

Table 1: Punnett square for Hardy–Weinberg equilibrium

Females

A (p) a (q)

MalesA (p) AA (p2) Aa (pq)

a (q) Aa (pq) aa (q2)

BioinformaticsA discipline that integrates mathematical, statistical, and computer tools to collect and process biological data

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 MyCycler thermalcycler 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

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

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

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.

Day 1: Overview and Tips

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

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 Alu insert 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

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

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 Alu polymorphism in your class population and compare them with the genotypic frequencies of other populations.