Download Gel Electrophoresis

Gel Electrophoresis, Gel
Loading Practice, and
Polymerase Chain
Reaction (PCR)
October 15th – October 19th, 2012
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.
What you
should
already
know…
Wells
Molecule
Charge
Behavior
DNA
Negative
Moves to positive
RNA
Negative
Moves to positive
Proteins
Positive
Negative
Neutral
Moves to negative
Moves to positive
No 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)
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
Methylene Blue
•
Ethidium Bromide
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
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
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
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
Summary
 Denaturation: Strand Separation
REPEAT!!!
 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
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
http://www.cnpg.com/video/flatfiles/539/
http://www.youtube.com/watch?v=7uafU
VNkuzg
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)
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
•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
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
 The simplest case of a single locus (location of gene on
chromosome) with two alleles
Hardy Weinberg Formula
 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 HardyWeinberg 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)
Males
a (q)
A (p)
AA (p2)
Aa (pq)
a (q)
Aa (pq)
aa (q2)
Bioinformatics
A discipline that integrates mathematical, statistical, and
computer tools to collect and process biological data
Overview Timeline: Prep
 Before Labs
 Aliquot InstaGene matrix, orange G DNA loading dye, molecular mass
ruler, control samples
 Prepare & aliquot 10mL .9% saline solution
 Prepare TAE buffer
 Set up student workstations
 Before isolation and PCR (Tuesday)






Get ice and bucket
Prepare complete master mix and aliquot
Set up control PCR reactions
Prepare molten agarose
Program thermalcycler
Set up student workstations
 Before Running Gels (Thursday)
 Pour Gels with EtBr
 Set up student workstations
Overview Timeline: Lab
Day 1
 Lesson 1: Cheek Cell DNA Template Preparation
 Isolate cheek cells
 Prepare genomic DNA from cheek cells/hair follicles
 Lesson 2: PCR Amplification
 Set up and perform PCR reactions
Lab Service: Pour agarose gels
Day 2
 Lesson 3: Gel Electrophoresis of Amplified PCR Samples
 Load and run gels
 View gels
 Lesson 4: Analysis and Interpretation of Results
 Record the results
 Analyze results
8 stations will be used, station 9 will run the controls!
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
 Then 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 PCR today
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 doublestranded 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.
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
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 2 con’t: Overview and Tips
 Lesson 4: Analysis and Interpretation of Results
 Be sure to figure out a way to get everyone’s data
 Alleles: Basic characteristics that population geneticists use to
describe and analyze populations