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Determination of STR Genotypes by Fluorescent Multiplex PCR
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CSI: Fredonia – Determination of STR Genotypes by Fluorescent Multiplex PCR.
Objectives: 1. Isolate genomic DNA from your own buccal cells using a DNA swab
tandem repeat (STR) loci and one gender determination locus.
PCR products.
assess the molecular weight of unknowns.
scene.
region of DNA can be synthesized from a minute amount of DNA, as little as a single molecule, to
yield quantities of DNA sufficient for detailed analyses such as gel electrophoresis or sequencing.
Today, you will collect your buccal cells using a DNA swab and isolate your own genomic DNA
from these cells. You will use your DNA preparation to set up a PCR reaction specific for 11
different loci. Your samples will be amplified, and the PCR products will be analyzed by
polyacrylamide gel electrophoresis. The goal is to determine the identity of a criminal from a
slate of suspects and assess the likelihood that you have the correct perpetrator.
Fictional Premise of CSI Fredonia: A crime has been committed in the new Science Center.
Over the winter break a trespasser broke into the science center and walked across the freshly
poured floors in their dirty boots. As a result the floors in the second floor hallway has been
ruined and must be replaced. University police arived on the scene in time to see a single person
running from the worksite. While the perpetrator was not apprehended, they did loose their hat
on the fence as they made their escape. Several hairs were found in the hat with the follicles
intact. The police have provided us with DNA extracted from these follicles in hopes that we can
identify the perpetrator and bring them to justice. It is up to you to solve this hairy crime and
identify the person responsible for delaying the completion of the Biology department’s new
home.
The COmbined DNA Index System (CODIS) is a set of 13 loci, each with multiple
tetranucleotide (4 bp) repeat alleles. This system is used by the FBI to match evidence collected
at crime scenes with potential suspects. It also serves as a mechanism to resolve paternity
disputes by comparing children with alleged fathers. It is extremely robust and is capable of
generating genotypes that are able to identify individuals with high probability. The figure to the
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right shows the 13 loci and their location in the human
genome. An interactive version of this figure is available
at http://www.cstl.nist.gov/div831/strbase/fbicore.htm
These loci are analyzed by designing primers that flank
(are on either side of) the variable region. This allows the
PCR product to vary in size proportionally to the number
of repeats. The greater the number of repeats at that
locus, the larger the PCR product will be. This can be
ascertained by gel electrophoresis.
Multiplex PCR: The locus that will be amplified by PCR
is determined by the sequence of the primers that are
used. PCR primers have several important features that you should be familiar with.
amplified (also called an amplicon) and is called the forward primer and one is
downstream and referred to as the reverse primer.
bottom strand while the reverse primer binds to the top strand.
each other. Because DNA polymerase extends the 3’ end it is necessary that it extend into
the amplicon and not away from it. During the extension phase each primer must be able
to extend across the amplicon and synthesize the complement for the other primer.
Figure 2: The basis of STR allele discrimination when amplified by PCR. Increased numbers of repeats result in a longer PCR product. In this example at the vWA locus, the 10 repeat allele is 123 bp while the 13 repeat allele is 135 bp. It is important to appreciate that molecular alleles are codominant. A heterozygote for the 10 and 13 alleles would produce two bands of 123 and 135 bp that could both be detected. The discriminating feature is size.
Figure 1: The 13 CODIS Loci used for forensic identity testing.
http://www.cstl.nist.gov/div831/strbase/fbicore.htm
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Because the primers dictate the amplicon, it is possible to design different size amplicons by
simply placing the primers closer or further apart from each other. This allows researchers to
specify the range of sizes produced when amplifying an STR locus (or any locus). It is also possible
to amplify multiple loci simultaneously in the same tube by designing the primers such that the
resulting products do not overlap in size. We will use this premise to amplify multiple different
loci in the same PCR reaction. Consider the set of 6 loci in Figure 3. The primers for these 6 loci
have been designed to
generate PCR products
that do not overlap.
They range within the
region specified for
each locus, therefore if
a band appears in that
size range it can be
attributed to that locus
rather than one of the
other loci.
Further Multiplexing with Fluorescence: We have just learned about multiplexing by size,
however it is possible to further discriminate between amplicons of similar size by tagging each
with a fluorescent molecule. In PCR this is as simple as adding a fluorescent tag to the 5’ end of
one of the primers (see figure 2). Modifications at the 5’ end do not interfere with extension of
the 3’ end of the primer and therefore don’t affect their efficiency in PCR. It does however allow
Figure 4: Fluorescent Multiplex amplicons. The amplicon in the PowerPlex 16 kit from Promega amplify 16 loci, grouped into three colors (the three rows). The loci bound by the blue box are the CODIS loci, while the orange box denotes two supplementary pentanucleotide STRs. The 11 loci to be studied in our lab are bound by the green box. The IRD700 and IRD800 labeled loci are designated. Figure modified from Promega corp.
Figure 3: Multiplex amplicons. The amplicon size for each of six loci do not overlap, thereby permitting simultaneous amplification and allele discrimination. Figure from www.nfstc.org
4
researchers to uniquely identify two products that are tagged with fluorophores that emit
different wavelengths (colors) of light. Using this technology “real forensic labs” can
simultaneously detect four (or more) different colored PCR products that overlap in size. In our
lab we will be using two dyes called IRD700 and IRD800. These dyes emit infrared light that
cannot be detected by the human eye, but can be resolved with our electrophoresis system, the
Li-Cor Genetic Analyzer 4300. Figure 4 shows the 16 loci contained in the PowerPlex 16 system by
Promega corporation. PowerPlex 16 contains all 13 CODIS loci plus two pentanucleotide repeat
loci and the Amelogenin locus which can be used to distinguish gender. We will be using a subset
of these loci that we’ll refer to (tongue-in-cheek) as FergPlex 11.
Allele Frequencies and Genotype Frequencies: All of the loci that we are amplifying are
unlinked and thereby segregate independently. Only D5S818 and CXF1PO are located on the
same chromosome, however they are far enough apart that they too segregate independently.
These loci are also not associated with any phenotypic characteristics that could result in
selective pressure on any of their alleles. These conditions allow us to make predictions about
genotype frequencies based on Hardy-Weinberg Equilibrium (HWE) assumptions. Essentially
there are several frequencies that are important for this exercise:
that match the allele in question. In other words, the number of a specific allele divided
by the total alleles in the population. As with all frequencies, this number will be between
1 and 0. An allele that is fixed has a frequency of 1. An allele that is lost has a frequency of
They can be the same (homozygous) or different (heterozygous).
where p is the allele frequency for the allele in question.
where p is the frequency of one allele and q is the frequency of the other.
of a genotype at one locus is an independent event from genotypes at other loci.
Therefore to calculate an overall genotype frequency, you must multiply the genotype
frequencies at each individual locus. This statistic is the probability of a specific genotype
arising at random in a population with the allele frequencies specified. If evidence is
matched with a suspect, this is the probability that the match is due to chance and that
you have the incorrect suspect. By determining the genotype at additional loci it is
possible to increase the confidence of establishing a correct identity by reducing the
probability that the match has occurred by chance. These frequencies are often reported
5
as odds (chance of 1 in 1,600,000 for example). Simply take the reciprocal of the
genotype frequency to convert this statistic to odds ((6.25E-7) -1
= 1,600,000).
Experimental Procedures:
equal number of samples, including their own by following the procedure described below.
Check each box as you complete the steps:
¨ Each person should rinse her/his mouth thoroughly with water before collecting her/his
buccal cells. Walk to the water fountain and rinse your mouth twice. Lick the insides of
your cheeks to rinse off any bacteria. PLEASE DON’T SPIT IN THE FOUNTAIN!!
¨ Obtain a Catchall DNA swab and a clear tube containing Quick-Extract DNA solution from
the side bench.
¨ Collect your buccal cells by rolling the swab firmly against the inside of your cheek.
¨ Roll the swab about 20 times against the inside of each cheek, making sure you move it
over your entire cheek. The more cells you collect, the higher your yield of DNA will be.
¨ Place the swab into the tube containing Quick-Extract DNA extraction solution.
¨ Rotate the brush in this solution a minimum of 5 times. Rotating the brush between 5 and
10 times dislodges the cells from the brush.
¨ Press the swab against the side of the tube and rotate the swab while removing it from
the tube. This ensures that most of the liquid and cells remain in the tube.
¨ Tightly screw the tube cap closed and vortex the tube for 10 seconds. This ensures that
the cells and the solution are well mixed.
¨ Label this tube with your 4 digit ID number (the numbers in your e-mail address) or the
forensic ID for that sample using a black marker.
¨ Place your samples in the 65° C hot block for 10 minutes.
¨ Vortex your tube for 15 seconds.
¨ Boil the tube for 2 minutes to inactivate enzymes that could degrade the DNA or inhibit
the PCR reaction.
¨ Vortex your tube for 15 seconds.
¨ Place your tube on ice until you are ready to set up your PCR reactions.
¨ Congratulations! You have now isolated a small quantity of genomic DNA suitable for
PCR amplification.
for the 11 loci described above using the buccal cell genomic DNA you just isolated.
¨ You will need to dilute the template DNA 1:4 with water prior to adding it to the reaction.
Set up an eppi (this is shorthand for eppendorf microcentrifuge tube and will be used
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henceforth) for each sample and label it with
the sample number. Pipette 15 mL of water
into each eppi and add 5 mL of genomic DNA.
Repeat this for each sample. Set the diluted
DNA on ice until it is called for.
¨ Obtain one PCR tube for each sample that you
will be genotyping. Do not separate the tubes. Keep them in strips of 8 if possible.
¨ Label the tubes with your ID number. Make sure to mark the tube on the neck rather than
on the top or conical portion. This will prevent the markings from coming off.
¨ You have been provided with 5x primer mix which contains all 22 primers necessary to
amplify the 11 loci in the FergPlex 11 reaction. Detailed primer information is posted on
ANGEL. You will need to dilute the 5x concentrate to working strength (1x) prior to adding
it to your PCR tubes.
¨ Calculate the number of reactions to be performed. This is the variable n in the following
formula.
¨ Dilute the primers by adding mL of water to mL of primer
mix. The 0.5 that is introduced into this formula is to compensate for pipetting error
and thereby ensure that you will have adequate primer working solution for each
sample.
¨ Transfer 23 ml of 1x primer mix from the step above into each PCR tube. DO NOT
touch the bead with the pipette tip.
¨ Add 2 ml of your DILUTED genomic DNA to the tube with the matching ID and cap them.
BE SURE TO USE A NEW PIPETTE FOR EACH INDIVIDUAL!
¨ Vortex the tubes briefly (1-2 sec).
Figure 5: Proper labeling of PCR tubes. Write on the neck, NOT the top or conical portion.
Figure 6: The temperature profile used to amplify the PCR products in the FergPlex 11 multiplex. Figure and conditions modified from the PowerPlex 16 manual (Promega Corp.)
7
¨ Pulse spin your tubes in the purple mini centrifuge.
¨ Place your tubes in the thermalcycler.
separated on an extremely high resolution polyacrylamide gel. This gel is different from the
agarose gels that you have run in the past in a few important ways. First, it is made of a
polymer of acrylamide and N,N’-methylene-bisacrylamide (colloquially “bis”). The monomeric
form of acrylamide is a neurotoxin. Therefore always wear gloves when handling acrylamide
products. The polymerization of acrylamide and bis is initiated by the addition of ammonium
persulfate and TEMED. These initiators generate free radicals that convert acrylamide to a
free radical that reacts with other monomers to form a polymer. The bis acrylamide is bi-
functional and forms crosslinks between adjacent acrylamide chains. The second difference
between our gel and agarose
gels is that the denaturant
urea is incorporated into the
gel. The urea prevents the
two strands of a DNA duplex
from coming together. This
ensures that there is no
secondary structure in the
sample (a source of
heterogeneity) and facilitates
higher resolution. The gel is
also run at 40 °C to further
prevent annealing. The final
difference is that this gel is
only 0.25 mm thick and is run
at over 1,000 volts.
¨ Remove the 6.5 % acrylamide solution from the fridge and measure 20 mL in a graduated
cylinder. Pour this solution into a small beaker with a stir bar and stir slowly. This will
allow the solution to warm to room temperature.
¨ Clean the plates for the gel well to ensure that there are no pieces of dry acrylamide or
dust. Clean the plates one last time with 70% ethanol and a large kimwipe. Also clean a
comb and set it aside.
¨ Place two 0.25 mm spacers between the plates. The notched side of the plates should
face each other.
¨ Add the side rails to the plates and stand it up in the casting rig.
Figure 7: Acrylamide polymerization reaction. Acrylamide monomers are converted to free radicals by ammonium persulfate (APS) and react with other monomers or the end of an existing polymer. Bis acrylamide is bifunctional and acts as a crosslink between adjacent chains. Figure from http://sdspage123.blogspot.com/
http://sdspage123.blogspot.com/
8
¨ Ensure that the plates both rest on the bottom of the casting rig and then tighten the rails
well.
¨ Lay the plates horizontal with the cutout plate on top.
¨ Mix a solution of 10% ammonium persulfate in water by weighing out 100 mg of APS and
dissolving it in 1 mL of water in an eppi. This solution should be made fresh right before
casting the gel.
¨ Add 150 mL of 10% APS and 15 mL of TEMED to the acrylamide solution. Allow the solution
to mix for 15-20 seconds. You must work quickly now because the polymerization
reaction has begun.
¨ Draw up the acrylamide in a syringe and begin injecting the solution into the top of the
gel. Do not inject too quickly or air bubbles will form in the gel. Prevent bubbles by
knocking on the front plate (like knocking on a door).
¨ Once the entire gel area is full, lay the plates flat and insert the flat side of the comb.
¨ Put the pressure plate in place and GENTLY tighten the screws. This piece is easily broken
by over tightening. Only moderate pressure is necessary.
¨ Allow the gel to solidify for about an hour.
with the buffer, and to run out any residual initiators (APS and TEMED).
¨ Clean the back plate of the gel to ensure that the laser has an unobstructed view of the
gel through the glass plate. This is very important for the best gel image.
¨ Replace the pressure plate with the upper buffer reservoir and place the lower reservoir
on the genetic analyzer. Don’t fill them yet.
¨ Hang the gel plate on the genetic analyzer.
¨ Make 1 L of 1x TBE electrophoresis buffer from the 5x (or 10x) concentrate.
¨ Add 1x TBE buffer to the upper reservoir up to the fill line, then pour the remainder of the
liter of buffer into the lower buffer reservoir.
¨ Remove the comb and scrape away any acrylamide scraps from the top of the gel.
¨ Install the electrodes and the cathode wire and close the cover of the genetic analyzer.
¨ Select “Run” from the computer. Select the microsatellite program and then prerun.
them prior to loading on the gel. While the gel is prerunning, prep your samples for loading
as follows:
¨ Dilute a portion of the PCR reaction 1:20 by pipetting 95 mL of water into a clean eppi and
then adding 5 mL of the PCR reaction. This is because the concentration of the PCR
products is too high and will result in overexposed and smeary bands.
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¨ Transfer 10 mL of the diluted PCR reaction to an empty PCR tube (no bead) and add 5 mL
of stop solution. The stop solution is analogous to loading dye that you have used in the
past with the exception that it contains the denaturant formamide.
¨ Mix and briefly spin the tubes.
¨ Denature for 3 minutes at 95°C in the PCR machine and then immediately transfer the
samples to ice (called quenching). The fast quench prevents the denatured strands from
having time to renature.
think. Follow these instructions to ensure a clean looking gel.
¨ Remove the cathode and rinse out the top of the gel to ensure that no scraps of
acrylamide have become lodged between the plates at the top of the gel. This is a major
source of frustration for beginners.
¨ Insert the comb, tooth side down until the teeth just contact the top of the gel (about 2-3
mm below the top of the plate).
¨ Urea will leach out of the gel and fill the wells, therefore it is necessary to periodically
rinse them out with a syringe filled with 1x TBE. Do this several lanes ahead of where you
are loading.
¨ Load 2 lanes with 1 mL of blank stop solution following the directions in figure 8. This
prevents curvature at the edge of the gel (smiling) due to ion imbalance.
¨ Add 1 mL of molecular weight ladder. This will be placed every 4 lanes to permit accurate
Figure 8: Polyacrylamide Gel Loading. When loading, place the sample into the void between the teeth of the comb. The pipette tip will not fit into this space (it is only 0.25 mm wide), so it is necessary to allow the sample to run down into the void. Because the buffer is warm, it will cause the air in the tip above your sample to expand and gently push the sample into the well. Don’t use the pipette plunger to push the sample is as bubbles will result causing sample mixing.
10
interpolation of unknown bands in our samples.
¨ Add 1 mL of sample to each well. Freeze the remaining sample in case the gel needs to be
repeated.
¨ Run the gel for 2.5 hours. Data is acquired in real time and will be posted to ANGEL for
subsequent analysis.
Gel analysis using Image J from the NIH
mode which means that each color (red and green in our data) is treated separately. You
will notice that turning the wheel on your mouse
or sliding the scroll bar at the bottom of the
image will change the channel at the top of the
window.
should be somewhat narrower than the entire lane.
that you selected as the first lane in your gel.
anywhere else or you’ll have to start over! Drag this rectangle over to the center of the
adjacent lane.
lane.
at the bottom until it reads 1/3 (Red)
peaks option is NOT selected.
channel from your gel will appear.
to black. This will be important for the overlaid image that we will
work with shortly.
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not work.
Image -> Stacks -> Images to Stack. Then click Image -> Color -> Stack to RGB.
time to pause if you need to return to your
analysis later.
that corresponds to your standards.
information about the position of the point selector (and the
position of the peak) in a new window called “Results”.
peaks by moving the point selector and pressing Ctrl + M at each
peak (there should be 10 peaks).
mouse and copy it with Ctrl + C.
Macros to run.
spreadsheet, then copy the X position data into the “Observed
Position” area under Molecular Weight Standards.
curve which will allow us to interpolate between the known
positions of the ladder to determine the molecular weight of an
unknown band.
record the position of each of the red peaks in the lane that you
are analyzing. Make a note of any loci that only have a single
band and are therefore homozygous. This may be easier when looking at the gel.
the corresponding column labeled “Observed Position” under the “Red Unknown Bands”
heading. If a locus was homozygous, then enter that value twice at that locus.
return the allele with the closest molecular weight.
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Collaborative Analysis of Allele Frequencies
The genotypes that you have generated above will need to be pooled across the class. We will be
using Google Drive to achieve this goal. The genotypes of each individual will be entered into the
Google spreadsheet called “Allele Frequency Analysis”. To get to your Google Drive, click on the
“Drive” link at the top of your Fredonia e-mail page (you must be logged into your Fredonia e-
mail). Once there, click on the “Shared with me” link on the left side of the Drive page. Under
that link, you should see the “Molecular Genetics 2013” folder. The “Allele Frequency Analysis”
sheet should be visible therein.
Data Analysis” spreadsheet into this spreadsheet. DO
NOT INCLUDE AMEL.
can see the allele frequencies at all of the loci and then
calculate the probability of the perpetrator’s genotype
arising at random in the population.
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