Laboratory 4: Setting up PCR Reactions Specific for D1S80, a VNTR

Part I (Wednesday Morning)

Objective of Laboratory 4, Part I:

Set up a PCR reaction using the buccal cell DNA you isolated Monday as the template for
amplification of your D1S80 alleles

I. INTRODUCTION: Monday, you isolated your genomic DNA from your own buccal cells
and set up a PCR reaction specific for your mitochondrial (mt) DNA. Your DNA was the
template for amplifying the constant region of your mtDNA by PCR. Today, you will use the
same sample of your buccal cell DNA to set up a different PCR reaction that is specific for a
region or locus within your genome known as D1S80. This region is highly variable or
polymorphic within the human population. The variability at D1S80 is caused by the presence of
Variable Numbers of Tandem Repeats or VNTRs, which are DNA sequences composed of
different numbers of a repeated “core” DNA sequence arranged in tandem. The size of the core
sequence can vary from 7 to 100 bp in different VNTRs. The number of repeats present at a
VNTR locus can also vary widely depending on which allele you have inherited. Although many
different VNTRs have been identified in the human genome, their function is not currently known.

The D1S80 locus is located on chromosome 1, the largest human chromosome, and the
repeating sequence at this locus is 16 bp in length. An example of one D1S80 sequence, including
the primer sites flanking the repeats, is shown on p. 58 (Kasai et. al., J. Forensic Science 35: 1196.
1990). To date, twenty-nine different alleles of D1S80, which range in size from 200 bp to 700 bp,
have been identified. Thus, 435 different allelic combinations are theoretically possible. Each of us
has two copies of the D1S80 locus, one inherited from each of our parents. Approximately 86% of
the population is heterozygous at this particular locus because they have inherited a different
VNTR allele from each parent, as illustrated below. Following agarose gel electrophoresis, the
amplified PCR products of the D1S80 locus will appear as one band in a homozygous individual
or two bands in a heterozygous individual. How this great diversity is generated at the D1S80
locus among different members of a population is still unknown, but could be caused by genetic
recombination or errors in DNA replication or repair. The technique we will use in the lab today is
a modification of a procedure used by the FBI for human identification. (We thank Ms. Judy
Brown, Edison Career Center, Wheaton, MD, for making a previous version of this procedure
available to us.)

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 Maternal D1S80 DNA
D1S80 Repeat
5’ 3’

3’ 5’

Primer 1

Primer 2

5’ 3’

3’ 5’ Paternal D1S80 DNA

Because of the polymorphic nature of loci like the VNTR D1S80 and others, it is theoretically
highly unlikely that more than a few people in the world have the same profile of a few different
polymorphic loci. Thus, several of these loci can be used as the “DNA fingerprint” or “DNA
profile” of an individual. DNA fingerprints can be used to identify individuals or to prove
relationships among individuals. You will understand and appreciate this concept better when you
see the class gel containing the D1S80 samples of everyone in the workshop.

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II. Setting up a PCR Reaction Specific for your
D1S80 Alleles: Each person will set up her/his own PCR
reaction specific for D1S80 using the genomic DNA she/he
isolated on Monday.

1. Put on gloves before working with the solution of your DNA. > Continue to use ARTs each time
you pipette to minimize
contamination.
2. Obtain a PCR tube rack and a pink thin walled 0.2 ml PCR
tube from the front bench.

3. Each person should write her/his number she/he has been > Use a fine-tipped black marker.
assigned on the side of a pink thin-walled 0.2 ml PCR tube near
the top (and on the hinge if possible).

4. Using your P20 with a 20 µl ART, transfer 10 µl of your

buccal cell DNA preparation to the pink PCR tube.

5. Flick this tube with your fingers to mix and keep this tube in
ice.

6. Pulse spin your tube using a black adaptor then take your
PCR tube in the PCR rack to the front bench.

7. Using an ART and the "PCR ONLY" P200 Pipetman on the > This solution contains Taq
buffer, 1 µM of each D1S80
front bench, add 40 µl of the prepared PCR reaction solution to primer, the 4 dNTPs and Taq
your PCR tube. polymerase.

8. Flick this tube gently with your fingers to mix.

LEAVE THE “PCR ONLY” P200 AT THE FRONT BENCH.

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9. Spin your tube for 10 sec in the microfuge using a black
adapter.

10. Give your tube to Lorraine or a member of the lab staff. It

will be put into the thermal cycler that has been programmed for
the conditions outlined below:

Soak: 94o C 1 min > This step brings the samples to

the correct temperature.

30 Cycles: 94o C 15 sec > This temperature denatures the

DNA.

68o C 15 sec > Primers anneal to the template

during this incubation.

72o C 15 sec > New DNA is synthesized during

this incubation.

Termination: 72o C 10 min

4o C Until sample removal > The low temperature ensures the
stability of the PCR products.

The sequences of the two primers used in the D1S80 PCR

reaction are:

5'-GAAACTGGCCTCCAAACACTGCCCGCCG-3' > Primer 1

5'-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3' > Primer 2

You are welcome to observe our incredibly fast 9600 thermal

cycler in action toward the end of today’s lab in 104 Schultz.

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Laboratory 4: Analyzing the PCR Products of Your D1S80 Alleles
Using Agarose Gel Electrophoresis
Part II (Wednesday Afternoon)

Objectives of Laboratory 4, Part II:

1. Analyze your amplified D1S80 alleles by electrophoresis in a 2% E-Gel
2. Determine the number of repeats in your D1S80 alleles
3. Make stabs of six bacterial strains that you will use in your classroom

Flow Chart of Laboratory 4, Part II:

Load your D1S80 PCR Perform gel Calculate the number of Make Stabs
product into a gel electrophoresis repeats in your D1S80 alleles of Bacteria

I. EXPERIMENTAL PROCEDURES:

A. Preparation of your Sample for Electrophoresis:

1. Obtain a small grey ice bucket (an Isotherm) and some ice. > Both the Isotherm and ice are
near the large sink in the back of
the lab.

2. Obtain the tube containing your amplified D1S80 alleles and

keep this tube on ice at all times.

3. Transfer 20 µl of your amplified D1S80 alleles to a fresh,

clear 0.65 ml tube.

4. Add 2.0 µl of loading dye to this tube. > Loading dye is in the clear tube
with the purple dot.

5. Flick the tube with your fingers to mix in the dye and spin > This pulse spin will bring the
sample to the bottom of the tube.
your tube in the microfuge for a few seconds.

6. Leave your sample in ice until you have prepared your gel as
described next.

B. Analysis of Your Amplified D1S80 Alleles using E-Gel > Ethidium bromide is a mild
carcinogen, so you should always
Electrophoresis: As discussed earlier, e-gels contain ethidium wear gloves when working with
bromide, which intercalates into DNA and fluoresces under e-gels.
ultraviolet illumination. Groups of 6 people will use one gel.
The procedure described below for preparing your gel is the
same procedure you used on Tuesday morning.

1. Use scissors to open the package containing the e-gel.

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2. With the comb in place, insert the gel into the apparatus,
inserting the right edge first.

3. Press firmly at the top and bottom to seat the gel in the base. > You should hear a snap when the
gel is seated correctly.
4. The Invitrogen logo should be located at the bottom of the
base, close to the positive pole, as shown in the diagram below.

5. Connect electric leads from the base unit to the power supply > Connect the red lead to the
positive pole and the black lead
as shown in the diagram above. to the negative pole.

6. Pre-run the gel (with the comb in place) for 1-2 minutes at 60- > Do not exceed 2 minutes.
70V (or 40-50 mA).

7. Turn off the power supply.

8. Remove the comb by using both hands to gently lift the comb
and roll the comb slowly towards you.

9. Make a solution for testing your gel by mixing 10 µl loading

dye (clear tube with purple dot) and 100 µl TE buffer (clear tube
with red dot).

10. Add 10 µl of this mixture to one well. Do not discard the > You may later load a sample in
this lane if needed.
remaining solution.

11. Run the gel for a few minutes until you see that the dye is > If the dye is not moving after 5-
10 minutes, discard the gel and
moving out of the well. insert a new e-gel.

12. Shut off the power to the gel.

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13. Load 20 µl of the 123 BP Ladder (green tube) in one lane. > The 123 Base Pair Ladder is a
mixture of fragments that differ
in size by 123 base pairs.

14. Each person should load 20 µl of her/his sample in one of > Be sure to record the order in
which the samples are loaded.
the other wells.

15. Be careful not to introduce bubbles while loading, as they > You can avoid introducing
bubbles into your sample by
will cause bands to distort. setting your Pipetman to the
exact volume you wish to
pipette, which is 20 µl in this
case.

16. Add 20 µl of your TE buffer + loading dye mixture that you

prepared earlier to all empty wells.

17. Run your gel at 60 to 70 volts for approximately 30 to 40 > Do not run longer than 60
minutes because longer run times
minutes until the blue dye touches the black label at the bottom will damage the gel.
of the gel.

18. Remove the gel cassette from the apparatus at the end of the
electrophoresis.

19. Place the cassette on top of the UV transilluminator and The camera should be set to on
camera 4.5 (f stop) and 2 (an
photograph your gel. exposure time of 1/2 second). If
you want a lighter exposure, set
the camera to 4.5 and 1 (an
exposure time of 1 second).
References:
Kasai et al. J. Forensic Science 35:1196-1200 (1990).
Budowle et al. J. Human Genetics 48:137-144 (1991).
Morell. Science 260:1422-1423 (4 June 1993).

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 C. Preparation of Stab Cultures: You will now make stab cultures > These stabs will serve as your
own stocks after the Workshop.
of six bacterial strains (DH5α, MM294, XL1-Blu/pUC19, XL1-Blu You will use them for
/pRAS2, MM294/pGFP, and MM294/pBFP). Each person should experiments with your class.
make a set of six stabs as described below. Tightly capped stabs are an
excellent way to store bacterial
strains and can be maintained for
a. Obtain six stab vials and, using a black marker, write your several years at moderate room
temperatures (or in the refrigerator
initials, the month and year, and the name of one strain (e.g., if air conditioning is not
DH5α) on the white label of each vial. available)

b. Loosen, but do not remove, the cap of each vial. > Be careful not to touch the rim of
the vial with your fingers.

c. Flame your loop and then cool it by touching it to a clear area of > What is the purpose of flaming
the loop?
the DH5α stock plate.
d. Use the tip of your loop to scrape up one DH5α colony from
the plate.
e. Open the vial you labeled “DH5α” by grasping the bottom of
the vial with your free hand and removing the vial cap using the
little finger of the hand holding the loop.

f. Flame the mouth of the vial by passing the mouth through the
flame once or twice.
g. Insert the loop partway into the stab agar, being careful not to > Do not stab the loop all the
way to the bottom of the agar,
insert it all the way through the agar. because this could cause the agar
to dry out quickly.

h. Without removing the loop from the vial, stab your loop several
times into different areas of the agar.
i. Flame the mouth of the vial by passing it through the burner > Flaming the vial removes any
microorganisms or dust particles
flame a couple of times. that might have landed on it from
the air.

j. Screw the cap onto the vial tightly.

k. Repeat this process to make stabs of the other five strains.

l. Be sure to save these six stock plates.

m. Turn off the gas valve > Make sure that the four handles
all point in the same direction
when you turn off the gas valve.

Your stabs can incubate at room temperature instead of 37o C.

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5’ACCGGCCCCT CACGGTGCCA AGGAAACAGC CCCACCATGA GGCGCTGAGA
GAAACTGGCC TCCAAACACT GCCCGCCGTC CACGGCCGGC CGGTCCTGCG
TGTGAATGAC TCAGGAGCGT ATTCCCCACG CGCCAGCACT GCATTCAGAT
AAGCGCTGGC TCAGT
GTCAGCCCAA GGAAGA
CAGACCACAG GCAAGG
AGGACCACCG GAAAGG
AAGACCACCG GAAAGG
AAGACCACCG GAAAGG
AAGACCACAG GCAAGG
AGGACCACCG GAAAGG
AAGACCACCG GCAAGG
AGGACCACCG GCAAGG
AGGACCACCA GGAAGG
AGGACCACCA GCAAGG
AGGACCACCA GCAAGG
AGGACCACCA GGAAGG
AGGACCACCA GGAAGG
AGGACCACCG GCAAGG
AGGACCACCA GGAAGG
AGGACCACCA GGAAGG
AGGACCACCG GCAAGG
AGGACCACCA GGAAGG
AGAACCACCA GGAAGG
AGGACCACCA GGAAGG
AGGACCACCA GGAAGG
AGGACCACTG GCAAGG
AAGACCACCG GCAAGC
CTGCAAGGGG CACGTGCATC TCCAACAAGA CAAAATAAAC AAGCCAGAGA GGGCTTGTGA
CCAGTGTGGC ATTTGTCAC 3’

Figure 1. DNA Sequence of a Typical D1S80 Locus. The 16 base pair tandem repeats are aligned in
the vertical column on the left to emphasize their sequence similarities. The locations of the PCR
primers are underlined. [Kasai, Nakamura and White. J. Forensic Sci. 35 (5): 1196 – 1200. 1990;
Sekiguchi et. al. Thesis, Nat. Res. Institute of Police Science. 1994].

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III. Questions and Analysis of Your Amplified D1S80 Alleles
1. As you will remember, the migration of DNA molecules in agarose gels is roughly proportional
to the inverse of the log of the molecular weight (size). From the photograph of your gel,
measure the distance (in mm) that all fragments in the molecular weight standard (123 bp ladder)
have migrated from the wells. Do the same for all the fragments in your other samples.
Manually plot out a standard curve from your known samples and then determine the sizes of
the fragments in your amplified samples.

2. Having plotted a standard curve using the 123 bp DNA Ladder as the standard and having
determined the sizes of all DNA bands in the PCR products, calculate the number of the tandem
repeats in your D1S80 alleles. We will pool the data from the entire class and discuss it or
provide you with the data.

3. Knowing that there are 29 different alleles of D1S80, how many different classes of
homozygotes exist? How many different classes of heterozygotes exist? It was stated earlier
that 435 different D1S80 genotypes are possible. How was this number obtained?

4. The sizes of the PCR products of the D1S80 locus in one family were 531 and 643 bp in the
mother and 435 and 531 bp in the father. What are all possible fingerprints their children could
have?

5. The percentage of GC base pairs is an important factor when designing PCR primers. Should the
percentage of GC base pairs be high or low? Explain your answer briefly. What is the
percentage of GC base pairs in D1S80 primer 1 (p. 49)? What is the percentage of GC base
pairs in D1S80 primer 2 (p. 49)? Is there another region of approximately 20 bp in the sequence
shown in Figure 1 (p. 54) that has a percentage of GC base pairs that is approximately equal to
that of the primers? If there is such a region, what might be some reasons why it was not
chosen as a primer sequence?

The 123 Base Pair Ladder

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