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Rapid and easy apolipoprotein E genotyping using an improved PCR-RFLP

technique

Article · January 2000

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Customer application article QIAGEN

Rapid and easy apolipoprotein E

genotyping using an improved
PCR-RFLP technique
M. Ossendorf and W. Prellwitz
Institut für Klinische Chemie und Laboratoriumsmedizin, Johannes Gutenberg-Universität, Mainz,
Germany

Here we describe a new genotyping technique for distinguishing ApoE alleles utilizing PCR-RFLP
that is designed to provide more easily interpretable results than previous methods. The
QIAamp® DNA Blood Mini Kit is used to isolate genomic DNA from whole blood, which is
PCR-amplified and then restriction-enzyme digested to produce large, easily identifiable
fragments. Internal controls are included to reduce the risk of misclassification of fragments.

Apolipoprotein E (apoE) occurs in all types resolve. In addition, misclassification due to

of lipoproteins and is thought to be involved partially digested fragments is avoided
in the conversion of very low-density lipo- through the use of internal controls.
proteins to intermediate-density lipoproteins.
Two of the three common apoE isoforms Materials and methods
are associated with disease: apoE4 with Genomic DNA was purified from 200 µl
Alzheimer’s disease, and apoE4 and apoE2 whole blood with the QIAamp® DNA Blood
with coronary heart disease. Mini Kit according to the Blood and Body
Fluid Spin Protocol in the accompanying
ApoE isoforms can be distinguished by handbook. DNA was eluted in 200 µl elution
isoelectric focusing using the very low-density buffer and stored at –20°C. A 318 bp
lipoprotein fraction of blood. However, some fragment from the apoE gene was PCR-
individuals (e.g., diabetic subjects) are amplified in a 50 µl reaction containing 10 µl
difficult to classify using this method, presum- (0.1–0.4 ng) purified genomic DNA,
ably due to posttranslational modification of
the protein (1). The apoE alleles (ε2, ε3, and
ε4) that encode the different isoforms (2) can
Fragments Amplified from ApoE Alleles using
be distinguished by PCR-RFLP (polymerase Improved PCR-RFLP
chain reaction – restriction fragment length
ApoE allele
polymorphism) analysis. A region of the apoE
gene that contains variable HhaI recognition
A A H
sites is amplified from genomic DNA, and the
PCR fragment is subsequently digested with ∑2 Primer E2mut
the restriction endonuclease HhaI. Although
Primer E3
each apoE allele can be identified from its
A: AflIII recognition site
unique RFLP pattern, the fragments are small A A H H H: HaeII recognition site
(19–91 bp) and difficult to resolve on an
∑3
agarose gel. Poor resolution and other
technical difficulties associated with this
technique are responsible for a high rate of
A H H Figure 1 Primer E2 introduces an AflIII site in the
misclassification (2). amplified product. Variable AflIII and HaeII sites
∑4 result in a unique RFLP pattern for each allele.
The new genotyping procedure described Invariant sites are used to detect partial digestion.
here involves PCR-RFLP but allows more
reliable classification than the old technique
because the resulting fragments are easier to

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QIAGEN
References
1. Black, S.C., Hewett, S.,
Kotubl, Y., Brunt, R.V., and
Reckless, J.P.D. (1990)
Isoform patterns of
apolipoprotein E in
diabetes mellitus. Diabet.
Med. 7, 532.
2. Paik, Y.K., et al. (1985)
Nucleotide sequence and
structure of the human
apolipoprotein E gene.
Proc. Natl. Acad. Sci.
USA 82, 3445.
3. Lahoz, C., et al. (1996)
Frequency of phenotype-
genotype discrepancies at
the apolipoprotein E locus
in a large population
study. Clin. Chem.
42, 1817.
Issue No. 1, 2000
Apolipoprotein E Genotyping
E2/E2 E2/E3 E2/E4
M U A H U A H U A H
Figure 2 A 318 bp fragment from the apoE gene was amplified from individuals with the indicated apoE
genotype using primers E2mut and E3. PCR fragments were digested with AflIII (A) or HaeII (H) and elec-
trophoresed with undigested PCR fragment (U) on a 4% agarose gel. M: DNA Molecular Weight Marker V
(Boehringer Mannheim, Mannheim, Germany).
1x QIAGEN® PCR Buffer, 0.25 µM each
primer, 200 µM each dNTP, 1x Q-Solution,
and 1.5 U QIAGEN Taq DNA Polymerase.
Two primers were used in the amplification:
upstream primer E2mut (5’ ACT GAC CCC
GGT GGC GGA GGA GAC GCG TGC)
and downstream primer E3 (5’ TGT TCC
ACC AGG GGC CCC AGG CGC TCG
CGG). Primer E2mut differs from the genomic
sequence at one position (underlined) which
creates an additional AflIII recognition site in
the amplified fragment.
Reaction mixtures were incubated at 94°C for
3 min, subjected to 40 cycles of amplification
(94°C, 10 sec; 65°C, 30 sec; 72°C, 30 sec),
and incubated at 72°C for 7 sec. Restriction
digests containing 10 µl amplification reaction
and either 2.5 U AflIII or 1.5 U HaeII were
incubated at 37°C overnight. The products of
digestion were analyzed on a 4% agarose gel.
Results and discussion
The new procedure for apoE genotyping uses
primers E2mut and E3 to amplify a region of
the apoE gene that contains variable numbers
of restriction sites, depending on the allele
(Figure 1, page 11). AflIII and HaeII digestion
12 www.qiagen.com/clinical/
ApoE genotyping using PCR-RFLP
E3/E3 E3/E4 E4/E4
U A H U A H U A H M
bp
– 318
– 295
– 267
– 231/232
of the fragment amplified from the e3 allele
results in fragments of 231 and 232 bp,
respectively. The e2-derived fragment lacks
the HaeII variable site, and HaeII digestion
results in a 267 bp fragment. The fragment
amplified from e4 lacks the variable AflIII site,
and digestion with this enzyme results in a
295 bp fragment. In addition, all three alleles
contain an invariant HaeII site which is used
to monitor the completeness of digestion. AflIII
digestion is evaluated with the AflIII site
introduced by the E2mut primer.
As shown in Figure 2, each apoE allele can
be identified by its unique RFLP pattern. All of
the allele-specific fragments are smaller than
the uncut (318 bp) PCR fragment, and
incomplete digestion cannot result in misclassi-
fication.
This new technique — PCR amplification with
primers E2mut and E3 followed by digestion
with AflIII and HaeII — allows rapid, simple,
and reliable apoE genotyping. The QIAamp
Blood DNA Mini Kit, QIAGEN Taq DNA
Polymerase, and PCR buffer contributed to
the reproducibility and ease of this procedure,
by providing both reliable DNA preparation
and specific amplification. ■
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ApoE genotyping using PCR-RFLP QIAGEN

Further reading
General methods
Dixon, S.C., Horti, J., Guo, Y., Reed, E., and Figg, W.D. (1998) Methods for extracting and
amplifying genomic DNA isolated from frozen serum. Nat. Biotechnol. 16, 91.
12 different DNA extraction methods (both home-made and commercial) were evaluated for DNA
yield, DNA quality, and suitability in downstream PCR. The QIAamp DNA Blood procedure was
found to be the most efficient and reproducible, and was best overall.
Plaschke, J., Voss, H., Hahn, M., Ansorge, W., and Schackert, H.K. (1998) Doublex sequencing in
molecular diagnosis of hereditary diseases. BioTechniques 24, 838.
DNA was isolated from peripheral blood using the QIAamp DNA Blood Mini Kits and from
mucosa samples of the colon using QIAamp DNA Mini Kits. Doublex sequencing was performed
with two different labeled primers in a single reaction, to analyze mutations.

Prenatal diagnosis
Denomme, G.A. et al. (1995) The prenatal identification of fetal compatibility in neonatal
alloimmune thrombocytopenia using amniotic fluid and variable number of tandem repeat (VNTR)
analysis. Br. J. Haematol. 91, 742.
DNA was isolated from peripheral white blood cells, from amniotic fluid, or from cultured
cells obtained from amniotic fluid samples using QIAamp DNA Blood Mini Kits for demanding
PCR-RFLP–based HPA-1 genotyping or for direct Southern-blotting–based VNTR analysis.
Lo, D.Y.M. et al. (1998) Quantitative analysis of fetal DNA in material plasma and serum:
implications for noninvasive prenatal diagnosis. Ann. J. Genet. 62, 768.
Fetal DNA was isolated from maternal plasma using the QIAamp DNA Blood Mini Kit, and
analyzed by TaqMan® PCR. Results show that concentration of fetal DNA increases during pregnancy
from 25 to 292 genome equivalents per milliliter, which corresponds to 3.4% and 6.2% of total
plasma DNA.

Factor V analysis
Zehnder, J.L. and Benson, R.C. (1996) Sensitivity and specificity of the APC resistance assay in
detection of individuals with Factor V Leiden. Am. J. Clin. Pathol. 106, 107.
Genomic DNA from leukocytes was purified with the QIAamp DNA Blood Mini Kit and used in a
PCR assay for factor V genotyping (of the G1691A mutation). Compared to classical tests for
activated protein C (APC), genotyping is more sensitive and the authors recommend it for analysis
of the factor V status of patients.
Voelkerding, K.V. et al. (1996) Factor V R506Q gene mutation analysis by PCR-RFLP: optimization,
Literature

comparison with functional testing for resistance to activated protein C, and establishment of cell
Literature

line controls. Am. J. Clin. Pathol. 106, 100.

The authors present a method to genotype factor V mutations using PCR and analysis by restriction-
fragment–length polymorphism. Genomic DNA is purified from leukocytes using the QIAamp DNA
Blood Mini Kit. Genotyping is recommended by the authors for increased sensitivity.

www.qiagen.com/clinical/ 13 Issue No. 1, 2000

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