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Laboratory Exercises
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).
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 Chester, PA). Oragene DNA isolation kits were kindly provided
Expected Background Skills by Genotek (Ottawa, Ontario, Canada). Taq DNA polymerase
was purchased from USB (Cleveland, OH), whereas dNTPs, k
Students will need to be highly proficient in micropipettor BstE II marker, and restriction enzymes were purchased from
use; accurate measurement of small volumes will increase the Promega Corporation (Madison, WI). Primers used in PCR were
likelihood of generating quality results. Alternatively, students purchased from Operon Biotechnologies (Huntsville, AL). Aga-
may isolate DNA and add it to reactions that are prepared by rose and pUC18 Hae III marker were acquired from Amresco
the instructor. A background in restriction digests, agarose gel (Solon, OH).
electrophoresis, and gel analysis would be useful, but it is not
absolutely required.
Equipment and Software
This exercise used the following equipments and software:
Web Sites microcentrifuge models 5415C and 5415D (Eppendorf, Ham-
burg, Germany), Bio-Rad DNA Engine PTC200 (Hercules, CA),
An overview of the principles of the ABO blood group is
BioMax QS710 and MP1015 gel electrophoresis systems,
present in the National Center for Biotechnology Information
DC290 camera, and 1D version 3.6 gel analysis software
(NCBI) web site: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?
(Kodak, Rochester, NY), Model 300 power supplies (VWR), and
rid¼rbcantigen.chapter.ch05ABO. The genetic, biochemical, and
EB-15 UV transilluminator (Ultra-Lum, Carlsbad, CA).
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/ Safety Issues
dispomim.cgi?id¼110300. NCBI also gives information about
the value and utility of SNPs at http://www.ncbi.nlm.nih.gov/ Permission to conduct this research was approved by the
About/primer/snps.html. In addition, a database of human SNPs John Carroll University Institutional Review Board. Instructors
can be accessed through this web site. A PCR animation that should contact their university committee that deals with human
requires Adobe Shockwave is maintained at the Dolan DNA studies for any special regulations. Collection of DNA from cells
Learning Center: http://www.dnalc.org/ddnalc/resources/shock- present in saliva is noninvasive. However, students should wear
wave/pcranwhole.html. A discussion of personalized medicine gloves whenever intact fluids are present. They should wash
and pharmacogenomics is available from the Mayo Clinic: http:// their hands and arms up to the elbow prior to leaving lab.
www.mayoclinic.com/health/personalized-medicine/CA00078. 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
Materials handled with care. Your institution may have special guidelines
to dispose of ethidium bromide waste. Ethidium bromide
Laboratory chemicals were purchased from Research Organ- decontamination protocols can be found in Sambrook et al. [7].
ics (Cleveland, OH), Fisher (Pittsburgh, PA), and VWR (West We disposed of the gels in a solid waste container, and the
141
contaminated buffer is stirred with a destaining bag from Purifier is proprietary. An alternate method for isolating
Amresco. The destaining bag binds ethidium bromide from the DNA involves the binding of DNA to Chelex; protocols for
solution; subsequently, decontaminated buffer can be washed using Chelex are readily available. Both methods have
down the sink. The destaining bag is discarded in the solid
been shown to work for this exercise, and alternate DNA
waste container with gels. Finally, UV-light exposure should be
minimized when observing the gels that are stained with ethi- isolation protocols are present in the Proceedings of the
dium bromide. UV-safe goggles or a UV shield should always 27th Workshop/Conference of the Association for Biology
be used. Laboratory Education.
1) Open an Oragene vial and spit into it until the
level of saliva reaches the 4-mL line on the side
Scheduling
of the vial.
Isolation of Genomic DNA—For isolating genomic 2) Incubate the sample in the Oragene vial at 508C
DNA, 2–2.5 hours were needed, with 1 hour open during for 1 hour.
the initial incubation at 508C. 3) Transfer 500 lL of sample into a 1.5-mL micro-
Gel Electrophoresis of Genomic DNA—Two hours were centrifuge tube. Label this sample with the sam-
needed, for gel preparation, electrophoresis, and photog- ple number that is provided by the instructor.
raphy, with 20–30 minutes open during electrophoresis. Store the remaining sample at room temperature.
Alternatively, gels may be prepared by the instructor prior 4) Add 20 lL of Oragene Purifier and mix by invert-
to class. Students can prepare samples and reduce the ing four times. The sample will become turbid.
necessary time to 1–1.5 hours. 5) Incubate on ice for 10 minutes.
PCR—For PCR 1 hour was needed, followed by a 2.5– 6) Centrifuge at top speed (16,000 3 g) for 3
3-hour reaction that can be held overnight at 48C in most minutes at room temperature.
PCR machines. The PCR reactions can be done on the 7) Remove supernatant using a pipettor (without dis-
same day as gel electrophoresis of the genomic DNA to turbing the pellet) and place in a new microcentri-
save time. fuge tube.
Gel Electrophoresis of PCR Products—For gel electro- 8) Add 500 lL of room temperature 95% ethanol to
phoresis of PCR products, 2 hours were needed, for gel the supernatant and mix by inverting at least five
preparation, electrophoresis, and photography, with 20– times.
30 minutes open during electrophoresis. 9) Incubate for 10 minutes at room temperature.
Restriction Digests—Thirty minutes were needed to 10) Centrifuge for 1 minute at top speed at room
prepare the digests and 2 hours for incubation. temperature; place the hinge of the microcentri-
Gel Electrophoresis of Restriction Digests—Two hours fuge tube facing up. The pellet is not always visi-
were needed for gel preparation, electrophoresis, and ble at this point, but the DNA will be pelleted at
photography, with 20–30 minutes open during electropho- the bottom under the hinge. Remove the super-
resis. The 4% agarose (3:1 High Resolution Blend from natant by pipetting while being careful not to dis-
Amresco)-13 TBE gels are prepared prior to the lab, and turb the pellet. If residual ethanol remains, centri-
students load these gels immediately, as these have a fuge again for 10 seconds and remove the
longer running time than the 1% agarose-13 TBE gels remaining ethanol.
that possess the exon 6 digests. Multiple groups can run 11) Place tube upside down on a Kimwipe to dry for
their samples on the same gel to conserve materials. 5 minutes.
12) Add 100 lL of 13 TE (10 mM Tris-HCl, 1 mM
EDTA (pH 8)) to dissolve the pellet. This volume
DNA Isolation will typically yield genomic DNA that is 50–100
ng/lL. EDTA will chelate divalent cations such as
Prior to isolation of buccal mucosa cells, students are
Mg2þ that are required by DNA-degrading
instructed to be careful while working with bodily fluids.
enzymes.
Gloves are to be worn at all times during the collection
13) Vortex the sample for 30 seconds and incubate
of cells and isolation of DNA. All tips, tubes, and vials
overnight at room temperature. Alternatively, sam-
that come into contact with bodily fluids should be dis-
ples may be incubated at 508C for 1 hour.
posed in biohazard bags for autoclaving, and all fluids
14) Store the genomic DNA at 48C. Freezing and
should be put into a 10% bleach solution to decontami-
thawing the genomic DNA samples can result in
nate them. Students should wash their hands and arms
DNA shearing.
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 Determination of Genomic DNA Concentration
own DNA sample. Students are given sample numbers
Each group will have a single set of uncut lambda
to identify their genomic DNA. Subsequently, the instruc-
DNA dilutions.
tor can provide an aliquot of genomic DNA to use as an
unknown without students knowing the source of the 1) Prepare a 1% agarose/13 TBE (89 mM Tris, 89 mM
unknown. Oragene kits (DNA Genotek) are utilized to iso- boric acid, 2 mM EDTA (pH 8)) gel that contains
late genomic DNA, and the composition of the Oragene 0.5 lg/mL ethidium bromide.
142 BAMBED, Vol. 36, No. 2, pp. 139–146, 2008
2) Add 2 lL of genomic DNA, 8 lL of sterile water, 3) Add 45 lL of exon 6 premix to the proper tubes
and 2 lL of 63 loading dye to a microcentrifuge and place the reactions in the thermocycler.
tube. 4) Add 45 lL of exon 7 premix to the proper tubes
3) Thaw a tube of ‘‘uncut k DNA.’’ This tube contains and place the reactions in the thermocycler.
50 ng/lL uncut k DNA.
4) Make up the following three tubes: Gel Electrophoresis of PCR Products
1) Prepare a 1% agarose-13 TBE gel that contains
0.5 lg/mL ethidium bromide.
Tube Uncut k DNA (lL) dH2O (lL) 63 loading dye (lL)
2) While the gel is solidifying, students can prepare
1 1 9 2 their PCR products for loading. Remove 10 lL of
2 3 7 2 each PCR reaction and place in labeled microcen-
3 6 4 2
trifuge tubes.
3) Add 2 lL of 63 loading dye (0.4% bromophenol
blue, 30% glycerol) to each microcentrifuge tube.
5) Load the gel with the three lambda dilutions (50, 4) Prepare lambda BstE II and pUC18 Hae III markers
150, and 300 ng) followed by the student DNA (1 lL of marker, 9 lL of 13 TE, and 2 lL of 63
samples. loading dye).
6) Run the gel at a constant voltage of 10 V/cm for 5) Load the entire 12 lL for each sample and record
45 minutes. Photograph as usual. the order.
7) Estimate the concentration of the genomic DNA by 6) Run the gel at 10 V/cm for 40 minutes. Gels are
comparison to the known amounts in the lambda visualized by exposure to UV light and photo-
dilution lanes. Alternatively, comparison of genomic graphed.
DNA intensity to a molecular weight standard can be 7) Estimate the concentration of DNA produced by
performed to estimate the concentration (see later). each of the PCR reactions of exons 6 and 7.
This estimation is done by comparison of the PCR
PCR product to the pUC18 Hae III or lambda BstE II molecular
Each two-person group will perform four PCR reac- weight marker (500 ng each). The 587-bp band of
tions for both exon 6 and 7 (eight total reactions). The pUC18 Hae III represents 100 ng of DNA, whereas the
four reactions for each exon contain genomic DNA from 2,323-bp fragment in the lambda BstE II digest is
each student in the group, an unknown DNA that is pro- 25 ng. If the PCR product and the 587-bp fragment are
vided by the instructor, or water to act as a negative of approximately equal intensities, then we have 100 ng
control. of PCR product. To determine the concentration of DNA
Amplification of exon 6 is performed using the follow- in the PCR reactions, divide the mass (in ng) by the vol-
ing conditions: 35 cycles at 948C for 30 seconds, 538C ume of DNA that was loaded (10 lL).
for 30 seconds, and 728C for 60 seconds, followed by a
5-minute extension at 728C and a post-dwell at 48C.
Restriction Digests
Exon 7 is amplified using the same conditions as exon 6,
except the annealing temperature is 608C instead of Students will digest exon 6 PCR products with Kpn I
538C. Each PCR utilizes a 2-minute incubation at 948C to and BstE II in separate reactions, and exon 7 PCR prod-
ensure that DNA denatures prior to amplification. ucts will be digested by Hpa II.
Primers used in PCR are purchased from Operon Bio-
1) A premix for each restriction digest is prepped
technologies (Huntsville, Alabama).
(one set of three for each lab group). For the Kpn I
Exon 6-1: 50 -GGGCTGGGAATGATTTG-30 digest of exon 6, add 6 lL of 103 Promega buffer
Exon 6-2: 50 -GGTGTCCCCCTCCTGCTATC-30 J, 6 lL of 103 BSA, and 2 lL Kpn I (10 U/lL) to a
Exon 7-3: 50 -CCCCGTCCGCCTGCCTTGCAG-30 microcentrifuge tube. For the BstE II digest of
Exon 7-4: 50 -GGGCCTAGGCTTCAGTTACTC-30 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-
1) Add 5 lL of template DNA or water (no DNA con- centrifuge tube. For the Hpa II digest of exon 7,
trol) to labeled PCR tubes. add 6 lL of 103 Promega buffer A, 6 lL of 103
2) Prepare the exon 6 premix as follows: 153.75 lL of BSA, and 2 lL Hpa II (10 U/lL) to a microcentri-
sterile water, 25 lL of 103 Taq DNA polymerase fuge tube. Mix all the premixes by tapping the
buffer (without MgCl2), 15 lL of 25 mM MgCl2, tubes; briefly centrifuge if necessary.
12.5 lL of 10 pmol/lL primer exon 6-1, 12.5 lL of 2) Aliquot 3.5 lL of premix into three labeled micro-
10 pmol/lL primer exon 6-2, 5 lL of 10 mM centrifuge tubes.
dNTPs, and 1.25 lL of Taq DNA polymerase (USB, 3) Add 11.5 lL of the appropriate PCR product. Exon 6
5 U/lL). Prepare the exon 7 premix; this premix is products are added into the tubes containing Kpn I
identical to the exon 6 premix, except primers and BstE II premixes, whereas exon 7 products are
exon 7-3 and exon 7-4 are used in place of exon added to the tube containing Hpa II premix.
6-1 and exon 6-2. Gently mix both premixes. 4) Briefly centrifuge the reaction mixtures.
143
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.
TABLE I
In contrast to the exon 6 SNP, most SNPs do not Fragments produced by Hpa II digestion of exon 7
cause a specific phenotype, but they can be associated
Exon 7 fragment (bp) A1 A2 B O1 O2
with a phenotype-causing SNP at a high frequency and
would be considered in linkage disequilibrium in this sce- 309 þ þ þ þ þ
223 þ
nario. Over many generations, DNA recombination that 204 þ þ þ þ
occurs between the two SNPs can eventually erode this 145 þ
uneven distribution. Two SNPs in exon 7 alter the amino 137 þ þ
acids that are encoded at positions 266 and 268 in the 119 þ þ þ þ
96 þ þ þ
amino acid sequence and are sufficient to produce the
altered substrate-specificity [8]. The A alleles possess Each sample will have a combination of these alleles present
unless the individual is homozygous for a particular allele. Also, the
C796 (Leu) and G803 (Gly), whereas the B allele has A and O1 alleles cannot be distinguished by this method alone.
1
A796 (Met) and C803 (Ala). In this exercise, students do ‘‘þ’’ indicates that the fragment will be present.
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.
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
FIG. 5. Results of exon 7 digestion by Hpa II. Exon 7 PCR introduction, with this section receiving multiple drafts
products were digested by Hpa II for 2 hours, followed by
electrophoresis on a 4% high-resolution agarose/13 TBE gel. and reviews; previous reports have undergone scrutiny
Sample genotypes are shown above each lane. Fragment sizes of other sections. The most common mistake is that
(in bps) are given on the right. students do not use technical terms to describe the
ABO phenotype; for example, ‘‘A sugar’’ is used in
place of N-acetylgalactosamine.
Interpretation of the exon 7 data is critical to deter- This exercise is a useful tool to teach the principles
mine all genotypes, except the O1O1 genotype. Stu- of DNA isolation, gel electrophoresis, PCR, and restric-
dents can have difficulties if they have not given suffi- tion digests. In addition, introduction to SNP analysis
cient thought to the possible fragment patterns that in a laboratory setting is a valuable experience for stu-
can be observed, especially when the genotype is dents, as SNPs are important in the field of pharmaco-
heterozygous. They are required to construct a table in genomics in which cancer diagnosis and prognosis,
which each heterozygous genotype (10 possible com- determination of drug efficacy, and genetic predisposi-
binations) is present, and a plus symbol is used tion to certain diseases are correlated with SNPs [10].
whenever a particular fragment is expected (Table II). For example, polymorphisms affect the ability of pro-
Subsequently, students are easily able to eliminate teins to catabolize medications, such as the leukemia
genotypes when the pattern of fragments does not drug 6-mercaptopurine [11] and the chemotherapeutic
match their predictions. agent 5-fluorouracil [12]. Currently, there are tests to
Occasionally, students will get an insufficient amount determine the genotype of individuals who are candi-
of exon 7 PCR product to observe fragments smaller dates to take these drugs. Physicians can more accu-
than 204 bp following digestion. In this case, gels can be rately choose the appropriate dosage or consider alter-
stained in 13 TBE supplemented with 0.5 lg/mL ethi- native drugs if an incompatibility exists. The ABO
dium bromide for 30 minutes. If this additional staining is blood group is able to be tested with minimal concerns
unsuccessful, students are asked to discuss the likeli- about student privacy. If these methods are applied to
hood of each allele being present based on their exon 6 other genes, careful selection of genetic markers must
data and by determining the allelic frequencies for all al- be done in order to maintain student privacy. However,
leles that have not been ruled out. These calculations are these techniques can be applied to other organisms in
TABLE II
Exon 7 fragment pattern for heterozygous samples
Exon 7 fragment (bp) A1, A2 A1, B A1, O1 A1, O2 A2, B A2, O1 A2, O2 B, O1 B, O2 O1, O2
309 þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ
223 þ þ þ þ
204 þþ þ þþ þþ þ þþ þþ þ þ þþ
145 þ þ þ þ
137 þ þ þ þ þ þþ þ
119 þ þþ þþ þþ þ þ þ þþ þþ þþ
96 þþ þ þþ þ þ þþ þ þ þ
A single ‘‘þ’’ indicates that one allele will produce the fragment, whereas ‘‘þþ’’ indicates that both alleles will produce the fragment.
146 BAMBED, Vol. 36, No. 2, pp. 139–146, 2008
which SNP information is available without the ethical Acknowledgments— The authors thank DNA Genotek for pro-
concerns. 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.
STUDY QUESTIONS