Q 2008 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION

Vol. 36, No. 2, pp. 139–146, 2008

Laboratory Exercises

A Laboratory Exercise to Determine Human ABO Blood Type by

Noninvasive Methods*
Received for publication, August 17, 2007, and in revised form, December 5, 2007

Michael P. Martin‡ and Stephen M. Detzel§

From the Biology Department, John Carroll University, University Heights, Ohio 44118

Analysis of single-nucleotide polymorphisms and their association with diseases and nondisease pheno-
types is of growing importance in human biology studies. In this laboratory exercise, students determine
the genetic basis for their ABO blood type; however, no blood is drawn. Students isolate genomic DNA
from buccal mucosa cells that are present in saliva and analyze the DNA on an agarose gel. Subse-
quently, this DNA is used as a PCR template to amplify exons 6 and 7 of the gene that determines the
human ABO phenotype. These PCR products are digested and run on agarose gels to examine the
restriction fragment length polymorphisms. A deletion in the O1 allele converts the BstE II site in exon 6
into a Kpn I site, and this feature is used to determine the presence of O1 alleles. The pattern of exon 7
digest products allows students to distinguish among four other common ABO alleles: A1, A2, B, and O2.
This exercise introduces students to commonly used molecular biology techniques, such as genomic
DNA isolation, PCR, gel electrophoresis, and restriction digests.
Keywords: PCR, ABO blood type, recombinant DNA technology, single-nucleotide polymorphism (SNP).

INTRODUCTION viduals inherit two ABO alleles that produce nonfunc-

The ABO blood group is the most important cell-sur-
face marker in transfusions and transplantations. ABO
incompatibility leads to red blood cell agglutination,
organ failure, and death. Although this medically relevant
system was discovered by Karl Landsteiner in 1900, the
enzymatic activity that produces the A and B antigens
was not elucidated until the 1970s [1, 2]. Subsequently,
the gene organization was determined near the end of
the century [3, 4]. The ABO gene encodes a glycosyl-
transferase that catalyzes the attachment of the terminal,
antigenic sugar to proteins, lipids, and soluble oligosac-
charides (Fig. 1A). Variation in the nucleotide sequence
or single-nucleotide polymorphisms (SNPs)1 produces
proteins with an altered specificity for their sugar sub-
strate [5]. The A glycosyltransferase protein attaches the
monosaccharide N-acetylgalactosamine, whereas the B
glycosyltransferase protein adds galactose to the H sub-
strate. If an individual can join only one of these sugars
to the H substrate, the A or B blood type is observed,
respectively. If an individual possesses both enzymatic
activities, then AB is the blood type. However, some indi-
* This work is supported by the John Carroll University
Faculty Instructional Grant
‡ To whom correspondence should be addressed. Tel.: 216-
397-4199 E-mail: mmartin@jcu.edu.
§ Current address: Ohio University College of Osteopathic
Medicine, Grosvenor Hall, Athens, OH 45701, USA.
1
The abbreviations used are: SNP, single-nucleotide polymor-
phism; NCBI, National Center for Biotechnology Information.
This paper is available on line at http://www.bambed.org
tional glycosyltransferases; these individuals have an O
phenotype.
The ABO gene is located on the long arm of chromo-
some 9 and is over 18 kilobases in size with 7 exons
(Fig. 1B). However, exons 6 and 7 harbor more than 75%
of the protein-encoding sequence, and these exons pos-
sess four SNPs that are sufficient to differentiate among
the five most common ABO alleles: A1, A2, B, O1, and
O2. A single base-pair deletion in exon 6 of the O1 allele
converts the BstE II restriction site to a Kpn I restriction
site. Therefore, all five alleles will be digested by either
Kpn I (for O1 alleles) or BstE II (non-O1 alleles). Three
SNPs in exon 7 allow for determination of A2, B, and O2
alleles. Because the O1 and A1 alleles produce the same
digestion pattern for exon 7, the exon 6 digestion is
essential for O1/A1 identification.
Olsson and Chester [6] developed a method of deter-
mining ABO blood type by using multiplex PCR, a tech-
nique in which multiple target sequences are amplified in
a single tube, followed by restriction digests. We have
modified this procedure to include two separate PCR
reactions, to be more appropriate for a midlevel under-
graduate laboratory course that emphasizes molecular
biology techniques. In this laboratory exercise, students
isolate their own genomic DNA from buccal epithelial
(cheek) cells and white blood cells found in saliva. PCR
of the gene that determines the ABO blood group pheno-
type and the subsequent digestion of these PCR prod-
ucts allow for determination of ABO blood type without
drawing blood.
139 DOI 10.1002/bmb.20165
140
FIG. 1. ABO blood type production. (A) Biochemistry of antigen production. H ¼ glycolipid or glycoprotein substrate; GalNAc ¼
N-acetylgalactosamine; Gal ¼ galactose. (B) ABO gene organization. Primers are indicated with arrowheads, and PCR product
sizes are shown between these arrowheads. Introns are represented by lines, whereas exons are filled boxes. Nucleotide 1096 is
located in the 30 untranslated region. A ‘‘þ’’ indicates Hpa II digestion, whereas a ‘‘2’’ indicates that no Hpa II digestion occurs.
EXPERIMENTAL PROCEDURES
Expected Background Skills
Students will need to be highly proficient in micropipettor
use; accurate measurement of small volumes will increase the
likelihood of generating quality results. Alternatively, students
may isolate DNA and add it to reactions that are prepared by
the instructor. A background in restriction digests, agarose gel
electrophoresis, and gel analysis would be useful, but it is not
absolutely required.
Web Sites
An overview of the principles of the ABO blood group is
present in the National Center for Biotechnology Information
(NCBI) web site: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?
rid¼rbcantigen.chapter.ch05ABO. The genetic, biochemical, and
immunological aspects of the ABO phenotypes are outlined.
There is also a comprehensive entry in the Online Mendelian In-
heritance in Men website: http://www.ncbi.nlm.nih.gov/entrez/
dispomim.cgi?id¼110300. NCBI also gives information about
the value and utility of SNPs at http://www.ncbi.nlm.nih.gov/
About/primer/snps.html. In addition, a database of human SNPs
can be accessed through this web site. A PCR animation that
requires Adobe Shockwave is maintained at the Dolan DNA
Learning Center: http://www.dnalc.org/ddnalc/resources/shock-
wave/pcranwhole.html. A discussion of personalized medicine
and pharmacogenomics is available from the Mayo Clinic: http://
www.mayoclinic.com/health/personalized-medicine/CA00078.
Materials
Laboratory chemicals were purchased from Research Organ-
ics (Cleveland, OH), Fisher (Pittsburgh, PA), and VWR (West
BAMBED, Vol. 36, No. 2, pp. 139–146, 2008
Chester, PA). Oragene DNA isolation kits were kindly provided
by Genotek (Ottawa, Ontario, Canada). Taq DNA polymerase
was purchased from USB (Cleveland, OH), whereas dNTPs, k
BstE II marker, and restriction enzymes were purchased from
Promega Corporation (Madison, WI). Primers used in PCR were
purchased from Operon Biotechnologies (Huntsville, AL). Aga-
rose and pUC18 Hae III marker were acquired from Amresco
(Solon, OH).
Equipment and Software
This exercise used the following equipments and software:
microcentrifuge models 5415C and 5415D (Eppendorf, Ham-
burg, Germany), Bio-Rad DNA Engine PTC200 (Hercules, CA),
BioMax QS710 and MP1015 gel electrophoresis systems,
DC290 camera, and 1D version 3.6 gel analysis software
(Kodak, Rochester, NY), Model 300 power supplies (VWR), and
EB-15 UV transilluminator (Ultra-Lum, Carlsbad, CA).
Safety Issues
Permission to conduct this research was approved by the
John Carroll University Institutional Review Board. Instructors
should contact their university committee that deals with human
studies for any special regulations. Collection of DNA from cells
present in saliva is noninvasive. However, students should wear
gloves whenever intact fluids are present. They should wash
their hands and arms up to the elbow prior to leaving lab.
Beakers containing 10% bleach should be used to decontami-
nate tips and tubes prior to disposal as biohazardous waste.
Ethidium bromide is a known carcinogen, and it should be
handled with care. Your institution may have special guidelines
to dispose of ethidium bromide waste. Ethidium bromide
decontamination protocols can be found in Sambrook et al. [7].
We disposed of the gels in a solid waste container, and the

contaminated buffer is stirred with a destaining bag from
Amresco. The destaining bag binds ethidium bromide from the
solution; subsequently, decontaminated buffer can be washed
down the sink. The destaining bag is discarded in the solid
waste container with gels. Finally, UV-light exposure should be
minimized when observing the gels that are stained with ethi-
dium bromide. UV-safe goggles or a UV shield should always
be used.
Scheduling
Isolation of Genomic DNA—For isolating genomic
DNA, 2–2.5 hours were needed, with 1 hour open during
the initial incubation at 508C.
Gel Electrophoresis of Genomic DNA—Two hours were
needed, for gel preparation, electrophoresis, and photog-
raphy, with 20–30 minutes open during electrophoresis.
Alternatively, gels may be prepared by the instructor prior
to class. Students can prepare samples and reduce the
necessary time to 1–1.5 hours.
PCR—For PCR 1 hour was needed, followed by a 2.5–
3-hour reaction that can be held overnight at 48C in most
PCR machines. The PCR reactions can be done on the
same day as gel electrophoresis of the genomic DNA to
save time.
Gel Electrophoresis of PCR Products—For gel electro-
phoresis of PCR products, 2 hours were needed, for gel
preparation, electrophoresis, and photography, with 20–
30 minutes open during electrophoresis.
Restriction Digests—Thirty minutes were needed to
prepare the digests and 2 hours for incubation.
Gel Electrophoresis of Restriction Digests—Two hours
were needed for gel preparation, electrophoresis, and
photography, with 20–30 minutes open during electropho-
resis. The 4% agarose (3:1 High Resolution Blend from
Amresco)-13 TBE gels are prepared prior to the lab, and
students load these gels immediately, as these have a
longer running time than the 1% agarose-13 TBE gels
that possess the exon 6 digests. Multiple groups can run
their samples on the same gel to conserve materials.
DNA Isolation
Prior to isolation of buccal mucosa cells, students are
instructed to be careful while working with bodily fluids.
Gloves are to be worn at all times during the collection
of cells and isolation of DNA. All tips, tubes, and vials
that come into contact with bodily fluids should be dis-
posed in biohazard bags for autoclaving, and all fluids
should be put into a 10% bleach solution to decontami-
nate them. Students should wash their hands and arms
up to the elbow prior to leaving lab. Groups of two stu-
dents are given reagents that have been aliquoted prior
to class in order to avoid contamination. However, each
student is responsible for handling and isolating their
own DNA sample. Students are given sample numbers
to identify their genomic DNA. Subsequently, the instruc-
tor can provide an aliquot of genomic DNA to use as an
unknown without students knowing the source of the
unknown. Oragene kits (DNA Genotek) are utilized to iso-
late genomic DNA, and the composition of the Oragene
141
Purifier is proprietary. An alternate method for isolating
DNA involves the binding of DNA to Chelex; protocols for
using Chelex are readily available. Both methods have
been shown to work for this exercise, and alternate DNA
isolation protocols are present in the Proceedings of the
27th Workshop/Conference of the Association for Biology
Laboratory Education.
1) Open an Oragene vial and spit into it until the
level of saliva reaches the 4-mL line on the side
of the vial.
2) Incubate the sample in the Oragene vial at 508C
for 1 hour.
3) Transfer 500 lL of sample into a 1.5-mL micro-
centrifuge tube. Label this sample with the sam-
ple number that is provided by the instructor.
Store the remaining sample at room temperature.
4) Add 20 lL of Oragene Purifier and mix by invert-
ing four times. The sample will become turbid.
5) Incubate on ice for 10 minutes.
6) Centrifuge at top speed (16,000 3 g) for 3
minutes at room temperature.
7) Remove supernatant using a pipettor (without dis-
turbing the pellet) and place in a new microcentri-
fuge tube.
8) Add 500 lL of room temperature 95% ethanol to
the supernatant and mix by inverting at least five
times.
9) Incubate for 10 minutes at room temperature.
10) Centrifuge for 1 minute at top speed at room
temperature; place the hinge of the microcentri-
fuge tube facing up. The pellet is not always visi-
ble at this point, but the DNA will be pelleted at
the bottom under the hinge. Remove the super-
natant by pipetting while being careful not to dis-
turb the pellet. If residual ethanol remains, centri-
fuge again for 10 seconds and remove the
remaining ethanol.
11) Place tube upside down on a Kimwipe to dry for
5 minutes.
12) Add 100 lL of 13 TE (10 mM Tris-HCl, 1 mM
EDTA (pH 8)) to dissolve the pellet. This volume
will typically yield genomic DNA that is 50–100
ng/lL. EDTA will chelate divalent cations such as
Mg2þ that are required by DNA-degrading
enzymes.
13) Vortex the sample for 30 seconds and incubate
overnight at room temperature. Alternatively, sam-
ples may be incubated at 508C for 1 hour.
14) Store the genomic DNA at 48C. Freezing and
thawing the genomic DNA samples can result in
DNA shearing.
Determination of Genomic DNA Concentration
Each group will have a single set of uncut lambda
DNA dilutions.
1) Prepare a 1% agarose/13 TBE (89 mM Tris, 89 mM
boric acid, 2 mM EDTA (pH 8)) gel that contains
0.5 lg/mL ethidium bromide.
142
2) Add 2 lL of genomic DNA, 8 lL of sterile water,
and 2 lL of 63 loading dye to a microcentrifuge
tube.
3) Thaw a tube of ‘‘uncut k DNA.’’ This tube contains
50 ng/lL uncut k DNA.
4) Make up the following three tubes:
Tube Uncut k DNA (lL) dH2O (lL) 63 loading dye (lL)
1 1 9 2
2 3 7 2
3 6 4 2
5) Load the gel with the three lambda dilutions (50,
150, and 300 ng) followed by the student DNA
samples.
6) Run the gel at a constant voltage of 10 V/cm for
45 minutes. Photograph as usual.
7) Estimate the concentration of the genomic DNA by
comparison to the known amounts in the lambda
dilution lanes. Alternatively, comparison of genomic
DNA intensity to a molecular weight standard can be
performed to estimate the concentration (see later).
PCR
Each two-person group will perform four PCR reac-
tions for both exon 6 and 7 (eight total reactions). The
four reactions for each exon contain genomic DNA from
each student in the group, an unknown DNA that is pro-
vided by the instructor, or water to act as a negative
control.
Amplification of exon 6 is performed using the follow-
ing conditions: 35 cycles at 948C for 30 seconds, 538C
for 30 seconds, and 728C for 60 seconds, followed by a
5-minute extension at 728C and a post-dwell at 48C.
Exon 7 is amplified using the same conditions as exon 6,
except the annealing temperature is 608C instead of
538C. Each PCR utilizes a 2-minute incubation at 948C to
ensure that DNA denatures prior to amplification.
Primers used in PCR are purchased from Operon Bio-
technologies (Huntsville, Alabama).
Exon 6-1: 50 -GGGCTGGGAATGATTTG-30
Exon 6-2: 50 -GGTGTCCCCCTCCTGCTATC-30
Exon 7-3: 50 -CCCCGTCCGCCTGCCTTGCAG-30
Exon 7-4: 50 -GGGCCTAGGCTTCAGTTACTC-30
1) Add 5 lL of template DNA or water (no DNA con-
trol) to labeled PCR tubes.
2) Prepare the exon 6 premix as follows: 153.75 lL of
sterile water, 25 lL of 103 Taq DNA polymerase
buffer (without MgCl2), 15 lL of 25 mM MgCl2,
12.5 lL of 10 pmol/lL primer exon 6-1, 12.5 lL of
10 pmol/lL primer exon 6-2, 5 lL of 10 mM
dNTPs, and 1.25 lL of Taq DNA polymerase (USB,
5 U/lL). Prepare the exon 7 premix; this premix is
identical to the exon 6 premix, except primers
exon 7-3 and exon 7-4 are used in place of exon
6-1 and exon 6-2. Gently mix both premixes.
BAMBED, Vol. 36, No. 2, pp. 139–146, 2008
3) Add 45 lL of exon 6 premix to the proper tubes
and place the reactions in the thermocycler.
4) Add 45 lL of exon 7 premix to the proper tubes
and place the reactions in the thermocycler.
Gel Electrophoresis of PCR Products
1) Prepare a 1% agarose-13 TBE gel that contains
0.5 lg/mL ethidium bromide.
2) While the gel is solidifying, students can prepare
their PCR products for loading. Remove 10 lL of
each PCR reaction and place in labeled microcen-
trifuge tubes.
3) Add 2 lL of 63 loading dye (0.4% bromophenol
blue, 30% glycerol) to each microcentrifuge tube.
4) Prepare lambda BstE II and pUC18 Hae III markers
(1 lL of marker, 9 lL of 13 TE, and 2 lL of 63
loading dye).
5) Load the entire 12 lL for each sample and record
the order.
6) Run the gel at 10 V/cm for
40 minutes. Gels are
visualized by exposure to UV light and photo-
graphed.
7) Estimate the concentration of DNA produced by
each of the PCR reactions of exons 6 and 7.
This estimation is done by comparison of the PCR
product to the pUC18 Hae III or lambda BstE II molecular
weight marker (500 ng each). The 587-bp band of
pUC18 Hae III represents
100 ng of DNA, whereas the
2,323-bp fragment in the lambda BstE II digest is

25 ng. If the PCR product and the 587-bp fragment are
of approximately equal intensities, then we have 100 ng
of PCR product. To determine the concentration of DNA
in the PCR reactions, divide the mass (in ng) by the vol-
ume of DNA that was loaded (10 lL).
Restriction Digests
Students will digest exon 6 PCR products with Kpn I
and BstE II in separate reactions, and exon 7 PCR prod-
ucts will be digested by Hpa II.
1) A premix for each restriction digest is prepped
(one set of three for each lab group). For the Kpn I
digest of exon 6, add 6 lL of 103 Promega buffer
J, 6 lL of 103 BSA, and 2 lL Kpn I (10 U/lL) to a
microcentrifuge tube. For the BstE II digest of
exon 6, add 6 lL of 103 Promega buffer D, 6 lL
of 103 BSA, and 2 lL BstE II (10 U/lL) to a micro-
centrifuge tube. For the Hpa II digest of exon 7,
add 6 lL of 103 Promega buffer A, 6 lL of 103
BSA, and 2 lL Hpa II (10 U/lL) to a microcentri-
fuge tube. Mix all the premixes by tapping the
tubes; briefly centrifuge if necessary.
2) Aliquot 3.5 lL of premix into three labeled micro-
centrifuge tubes.
3) Add 11.5 lL of the appropriate PCR product. Exon 6
products are added into the tubes containing Kpn I
and BstE II premixes, whereas exon 7 products are
added to the tube containing Hpa II premix.
4) Briefly centrifuge the reaction mixtures.

5) Place reactions exon 6-Kpn I and exon 7-Hpa II at
378C for 2 hours.
6) Place reaction exon 6-BstE II at 608C for 2 hours.
This reaction should be briefly centrifuged every
30–45 minutes, as evaporation and condensation
will occur under the cap.
7) Once completed, place all reactions at 2208C.
Gel Electrophoresis of Digests
A 4% agarose-13 TBE gel has been poured prior to
class. Two groups will share each gel; one group will
take the four lanes on the left and the other group will
take the four lanes on the right.
4% Agarose Gel for Exon 7 Hpa II Digests—Two
groups of students work together in this part of the exer-
cise. A 4% high resolution agarose (3:1 high resolution
blend, Amresco)-13 TBE gel with 0.5 lg/mL ethidium
bromide is poured prior to class for each group of four
students. It is advantageous to arrange student groups
such that each gel has a B allele on it; this result can be
achieved through the use of samples from previous
semesters, which are given to the students as unknowns,
or by polling students to determine who possesses B or
AB blood type.
1) Add 3 lL of 63 loading dye to each digest from
last lab meeting. Briefly centrifuge.
2) Prepare a sample of pUC18 Hae III as usual (1 lL
of DNA, 9 lL of 13 TE, and 2 lL of loading dye).
3) Once both groups are ready to load the gel, begin
loading. Load the entire 18 lL into a single well.
4) Run the gel at a constant voltage of 9.5 V/cm and
photograph as usual. The bromophenol blue track-
ing dye should travel 7 cm into the gel in order to
resolve the 223- and 204-bp bands and also the
145- and 137-bp bands. There is no xylene cyanol
in the loading dye, as it can obscure low-intensity
bands.
1% Agarose-1 TBE Gel for Exon 6 Digests—
1) Prepare a 1% agarose-13 TBE gel that contains
0.5 lg/mL ethidium bromide.
2) Prepare an uncut sample of exon 6 PCR product.
Add 5 lL of DNA from the exon 6 PCR reactions,
10 lL of sterile water, and 3 lL of 63 loading dye
to a microcentrifuge tube. Briefly centrifuge.
3) Add 3 lL of 63 loading dye to each of the exon 6
digests and briefly centrifuge.
4) Prepare a sample of pUC18 Hae III and lambda
BstE II as usual (1 lL of DNA, 9 lL of 13 TE, and
2 lL of loading dye).
5) Load the gel making sure to position the uncut
and digested PCR products for a given sample in
three consecutive lanes for ease of analysis.
6) Run the gel at 10 V/cm for 40 minutes and photo-
graph the gel.
RESULTS AND DISCUSSION
Following isolation of genomic DNA, the concentration,
intactness, and purity of the DNA are determined by gel
143
FIG. 2. Results of genomic DNA isolation. A 1% agarose/
13 TBE gel was run for 45 minutes prior to photographing the
gel. Lambda DNA is shown in lanes 1–3.
electrophoresis. Comparison of uncut lambda DNA of
known mass to an aliquot of genomic DNA is used to
estimate the genomic DNA concentration. Both student
samples exhibit an approximately equal mass to the
150 ng uncut lambda sample (Fig. 2, compare lane 2 to
lanes 4 and 5). Because 2 lL of genomic DNA is loaded,
a concentration of 75 ng/lL is estimated. Most genomic
DNA preparations have a concentration of 50–100 ng/lL.
In addition, the genomic DNA is not sheared during its
isolation, as streaking down the gel is not observed (Fig.
2, lanes 4 and 5), and fragments at least as large as
lambda DNA (
48,500 bp) are present. Rarely, a sample
will be less intact, but these samples do not act as
inferior templates for amplification. RNA may also be iso-
lated along with genomic DNA and appear as a fast-
migrating diffuse signal, but no RNA has been observed
when the DNA Genotek kit has been used.
A deletion of nt261 in the O1 allele is directly responsi-
ble for the production of a nonfunctional protein. This de-
letion also creates a restriction site for Kpn I in exon 6,
whereas the other four alleles possess a BstE II site (Fig.
1B). Therefore, digestion by Kpn I indicates the presence
of an O1 allele. There are three possible outcomes of
exon 6 digestion with Kpn I and BstE II: (i) half the PCR
products are digested by each enzyme (Fig. 3, lanes 4
and 5), (ii) all PCR products are digested by Kpn I and
none by BstE II (Fig. 3, lanes 7 and 8), and (iii) all PCR
products are digested by BstE II and none by Kpn I
(Fig. 3, lanes 10 and 11). Using these two restriction
digests eliminates the need to use a negative result,
such as Kpn I insensitivity, as a diagnostic measure. This
feature affords the instructor an opportunity to discuss
why a negative result can lead to incorrect conclusions
when a faulty reaction can easily be the culprit. Sources
of error that prevent DNA digestion include poor pipetting
or use of the wrong buffer.
144
FIG. 3. Results of exon 6 digestion. Exon 6 PCR products
were digested with Kpn I (K) or BstE II (B) for 2 hours, and a ‘‘-’’
indicates uncut DNA (no enzyme). A 1% agarose/13 TBE gel
was run for 40 minutes prior to photographing the gel. Geno-
types are indicated above each sample.
In contrast to the exon 6 SNP, most SNPs do not
cause a specific phenotype, but they can be associated
with a phenotype-causing SNP at a high frequency and
would be considered in linkage disequilibrium in this sce-
nario. Over many generations, DNA recombination that
occurs between the two SNPs can eventually erode this
uneven distribution. Two SNPs in exon 7 alter the amino
acids that are encoded at positions 266 and 268 in the
amino acid sequence and are sufficient to produce the
altered substrate-specificity [8]. The A alleles possess
C796 (Leu) and G803 (Gly), whereas the B allele has
A796 (Met) and C803 (Ala). In this exercise, students do
FIG. 4. Exon 7 sequence for the A1 allele. The primers that are used to amplify this sequence immediately flank this sequence.
Numbering is according to the nucleotides present in exons, beginning with the ‘‘a’’ in the start codon. Hpa II restriction sites are
underlined. SNPs in Hpa II sites are capitalized and are in bold. The two SNPs (C796 and G803) that alter sugar-specificity in the B
transferase are capitalized; these sites are not in Hpa II sites. The nucleotide that is deleted to produce the A2 allele (nt1061) is itali-
cized and capitalized.
BAMBED, Vol. 36, No. 2, pp. 139–146, 2008
not directly assay the exon 7 SNPs that alter substrate-
specificity, as inexpensive restriction enzymes are not
available to test these SNPs. Instead, SNPs that are in
linkage disequilibrium with the SNPs at nucleotides 796
and 803 are assayed. Therefore, students are detecting
the haplotype, the unique combination of genetic
markers, such as SNPs, that a chromosomal segment
possesses (Fig. 1B).
Three SNPs (nucleotides 467, 703, and 1,096) in exon
7 are present in Hpa II restriction sites (Fig. 4), and diges-
tion will produce a characteristic set of fragments for
each allele (Table I). Exon 7 PCR products are digested
with Hpa II and run on a high-resolution 4% agarose gel
to separate similarly sized fragments. Genotype BO1
exhibits both the 223- and 137-bp fragments that are
produced by digestion of the B allele (Fig. 5, lane 2). The
absence of the 223-bp fragment and the presence of the
137-bp fragment are observed in O1O2 (Fig. 5, lane 3);
this pattern is only produced by the O2 allele. Impor-
tantly, the 145-bp fragment that is diagnostic of the A2
allele is able to be separated from this 137-bp fragment
(Fig. 5, compare lanes 6 and 7 to lane 8). Genotypes
O1O1, A1O1, and A1A1 have the same pattern of frag-
ments (Fig. 5, lanes 4 and 5 and data not shown);
however, this result requires examination of the exon 6
digestion data to distinguish between A1 and O1 alleles.
TABLE I
Fragments produced by Hpa II digestion of exon 7
Exon 7 fragment (bp) A1 A2 B O1 O2
309 þ þ þ þ þ
223 þ
204 þ þ þ þ
145 þ
137 þ þ
119 þ þ þ þ
96 þ þ þ
Each sample will have a combination of these alleles present
unless the individual is homozygous for a particular allele. Also, the
A and O1 alleles cannot be distinguished by this method alone.
1
‘‘þ’’ indicates that the fragment will be present.

FIG. 5. Results of exon 7 digestion by Hpa II. Exon 7 PCR
products were digested by Hpa II for 2 hours, followed by
electrophoresis on a 4% high-resolution agarose/13 TBE gel.
Sample genotypes are shown above each lane. Fragment sizes
(in bps) are given on the right.
Interpretation of the exon 7 data is critical to deter-
mine all genotypes, except the O1O1 genotype. Stu-
dents can have difficulties if they have not given suffi-
cient thought to the possible fragment patterns that
can be observed, especially when the genotype is
heterozygous. They are required to construct a table in
which each heterozygous genotype (10 possible com-
binations) is present, and a plus symbol is used
whenever a particular fragment is expected (Table II).
Subsequently, students are easily able to eliminate
genotypes when the pattern of fragments does not
match their predictions.
Occasionally, students will get an insufficient amount
of exon 7 PCR product to observe fragments smaller
than 204 bp following digestion. In this case, gels can be
stained in 13 TBE supplemented with 0.5 lg/mL ethi-
dium bromide for 30 minutes. If this additional staining is
unsuccessful, students are asked to discuss the likeli-
hood of each allele being present based on their exon 6
data and by determining the allelic frequencies for all al-
leles that have not been ruled out. These calculations are
Exon 7 fragment pattern for heterozygous samples
Exon 7 fragment (bp) A1, A2 A1, B A1, O1
309 þþ þþ þþ
223 þ þ
204 þþ þ þþ
145 þ þ
137 þ þ
119 þ þþ þþ
96 þþ þ þþ
A single ‘‘þ’’ indicates that one allele will produce the fragment, whereas ‘‘þþ’’ indicates that both alleles will produce the fragment.
145
based on the data by Yip [9]. It has been suggested that
the use of a more sensitive DNA stain, such as SYBR
Green I, will allow for lower intensity fragments (96 and
119 bp, for example) to be more easily visualized. An
added advantage of using SYBR Green I would be the
omission of carcinogenic ethidium bromide from this lab
exercise.
One limitation of this exercise is the inability to identify
the large number of ABO alleles that exist. For example,
the A1v and A2 alleles have the same pattern of Hpa II
digestion, but A2 possesses a deletion of nt1061 that
results in a longer protein with lower galactosyltransfer-
ase activity. However, the phenotype of an individual
would not be altered in this case. For simplicity, these
two alleles are not considered separately in this exercise,
but DNA sequencing of exon 7 could easily reveal this
deletion.
Students perform this exercise at the end of a
semester-long writing-intensive course, and they write
a paper describing their work in a standard journal
format. Also, they are expected to generate figures
using PowerPoint. Special attention is given to the
introduction, with this section receiving multiple drafts
and reviews; previous reports have undergone scrutiny
of other sections. The most common mistake is that
students do not use technical terms to describe the
ABO phenotype; for example, ‘‘A sugar’’ is used in
place of N-acetylgalactosamine.
This exercise is a useful tool to teach the principles
of DNA isolation, gel electrophoresis, PCR, and restric-
tion digests. In addition, introduction to SNP analysis
in a laboratory setting is a valuable experience for stu-
dents, as SNPs are important in the field of pharmaco-
genomics in which cancer diagnosis and prognosis,
determination of drug efficacy, and genetic predisposi-
tion to certain diseases are correlated with SNPs [10].
For example, polymorphisms affect the ability of pro-
teins to catabolize medications, such as the leukemia
drug 6-mercaptopurine [11] and the chemotherapeutic
agent 5-fluorouracil [12]. Currently, there are tests to
determine the genotype of individuals who are candi-
dates to take these drugs. Physicians can more accu-
rately choose the appropriate dosage or consider alter-
native drugs if an incompatibility exists. The ABO
blood group is able to be tested with minimal concerns
about student privacy. If these methods are applied to
other genes, careful selection of genetic markers must
be done in order to maintain student privacy. However,
these techniques can be applied to other organisms in
TABLE II
A1, O2 A2, B A2, O1 A2, O2 B, O1 B, O2 O1, O2
þþ þþ þþ þþ þþ þþ þþ
þ þ
þþ þ þþ þþ þ þ þþ
þ þ
þ þ þ þþ þ
þþ þ þ þ þþ þþ þþ
þ þ þþ þ þ þ
146
which SNP information is available without the ethical
concerns.
STUDY QUESTIONS
1) Rh D-negative individuals generate an immune
response when they are exposed to the Rh D anti-
gen. However, incompatibility in the ABO blood
group system occurs naturally in the absence of
exposure to the A or B antigen in the blood from
another individual. Why do individuals produce an
immune response the first time they encounter the
A or B antigen as an adult?
2) What chromosome possesses the ABO blood
group gene? Is it on the long or short arm of this
chromosome?
3) What major advantage does using DNA polymer-
ase from a thermophilic bacterium provides during
a PCR reaction?
4) In rare cases, an individual can possess the A and/
or B transferase proteins (and their corresponding
alleles), but they possess an O phenotype. What is
this condition called and how does it occur?
5) The ABO blood group is a classic example of a
trait that exhibits codominance. The presence of
which phenotype allows one to conclude that
codominance exists?
6) Determine the frequencies of the major allele
classes: O, A, and B (find multiple populations).
Why might the O allele exhibit these frequencies in
comparison to the A and B allelic frequencies?
Why might the allelic frequencies exhibit differen-
ces among populations?
BAMBED, Vol. 36, No. 2, pp. 139–146, 2008
Acknowledgments— The authors thank DNA Genotek for pro-
viding Oragene DNA Isolation kits. Funding for this exercise
was provided by a John Carroll University Faculty Instructional
Grant to M.P.M. We thank James Lissemore and David Hall for
thoughtful comments and suggestions for this manuscript.
REFERENCES
[1] V. Ginsburg (1972) Enzymatic basis for blood groups in man, Adv.
Enzymol. Relat. Areas Mol. Biol. 36, 131–149.
[2] C. A. Tilley, M. C. Crookston, J. H. Crookston, J. Shindman, H.
Schachter (1978) Human blood-group A- and H-specified glycosyl-
transferase levels in the sera of newborn infants and their mothers,
Vox. Sang. 34, 8–13.
[3] F. Yamamoto, P. D. McNeil, S. Hakamori (1995) Genomic organization
of human histo-blood group ABO genes, Glycobiology 5, 51–58.
[4] E. P. Bennett, R. Steffensen, H. Clausen, D. O. Weghuis, A. Geurts
van Kessel (1995) Genomic cloning of the human histo-blood group
ABO locus, Biochem. Biophys. Res. Commun. 206, 318–325.
[5] F. Yamamoto (2004) Review: ABO blood group system-ABH oligo-
saccharide antigens, anti-A and anti-B, A and B glycosyltransfer-
ases, ABO genes, Immunohematology 20, 3–22.
[6] M. L. Olsson, M. A. Chester (1995) A rapid and simple ABO genotype
screening method using a novel B/O2 versus A/O1 discriminating
nucleotide substitution at the ABO locus, Vox. Sang. 69, 242–247.
[7] J. Sambrook, E. F. Fritsch, T. Maniatis (1989) Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
New York.
[8] F. Yamamoto, S. Hakomori (1990) Sugar-nucleotide donor specific-
ity of histo-blood group A and B transferases is based on amino
acid substitutions, J. Biol. Chem. 265, 19257–19262.
[9] S. P. Yip (2000) Single-tube multiplex PCR-SSCP analysis distin-
guishes 7 common ABO alleles and readily identifies new alleles,
Blood 95, 1487–1492.
[10] W. Lee, C. Lockhart, R. B. Kim, M. L. Rothenberg (2005) Cancer
pharmacogenomics: Powerful tools in cancer chemotherapy and
drug development, The Oncologist 10, 104–111.
[11] R. M. Weinshilboum, S. L. Sladek (1980) Mercaptopurine pharma-
cogenetics: Monogenic inheritance of erythrocyte thiopurine meth-
tyltransferase activity, Am. J. Hum. Genet. 32, 651–662.
[12] G. D. Heggie, J. P. Sommadossi, D. S. Cross, W. J. Huster, R. B.
Diasio (1987) Clinical pharmacokinetics of 5-fluorouracil and its
metabolites in plasma, urine, and bile, Cancer Res. 47, 2203–2206.