Comparative Medicine
Copyright 2000
by the American Association for Laboratory Animal Science
Vol 50, No 3
June 2000
Deoxyribonucleic Acid Preparation in Polymerase
Chain Reaction Genotyping of Transgenic Mice
Klaus Zimmermann, Hans Peter Schwarz, and Peter L. Turecek
Background and Purpose: In an attempt to find a rapid and reproducible method for routine polymerase chain
reaction genotyping of transgenic mice, two novel approaches were developed.
Methods: One approach allowed reproducible amplification from crude lysates of tail snips, using a thermally
activated polymerase. In a second approach, for situations in which non-invasive techniques are necessary, oral
swab specimens were amplified after DNA extraction by use of an isolation kit. Samples from 10 transgenic factor
VIII knockout mice were genotyped after processing by use of these and other methods.
Results: False-negative results were not obtained by use of the two novel approaches. Despite their relative simplicity, both approaches yielded results comparable to those obtained by use of procedures known to be reliable,
such as organic extraction and a DNA extraction kit.
Conclusion: Both approaches are useful for PCR-amplification of DNA from mammalian sources.
Transgenic rodents, including knockout mice, are frequently
used as animal models to study the function of single gene products in an organism. Integration of the transgene into the genomic DNA of mice is usually verified by use of molecular
methods requiring the preparation of DNA for subjection to
polymerase chain reaction (PCR) analysis. The source of the
DNA is often the tail (1–7) or other tissues, such as ears (8), toes
(9,10), or blood (11). Alternatively, non-invasive approaches to
DNA preparation include use of epithelial cells from the inner
surface of the rectum (12) or nasopharyngeal aspirates (13). The
optimal method for purifying DNA from these sources is, however, a matter of debate. Use of crude tissue lysates without further purification has been described as the most rapid method
of DNA preparation (4, 9, 10, 14). Although this is a simple protocol, it is difficult to achieve reproducible results. Therefore, a
large number of researchers purify the samples after lysis in a
time-consuming process (1–3, 5–7, 15).
Since we routinely determine the genotype of transgenic
mice, we have tried a variety of methods of DNA preparation in
an attempt to find a feasible method that is rapid and produces
reproducible results. Here we describe two novel approaches to
DNA extraction for PCR genotyping: amplification of crude tail
snip lysates, using a novel heat-activated enzyme for PCR
analysis; or amplification of oral swab specimens after DNA extraction, using an isolation kit.
Materials and Methods
Breeding of knockout mice: The methods described here
were developed for routine genotyping of the offspring of heterozygous (carrier) females in our factor VIII knockout mouse
colony. The targeted disruption of the mouse factor VIII gene,
which was achieved by insertion of a neocassette into exon 17 of
the factor VIII gene, has been described (1, 2). Our colony was
generated by crossbreeding exon 17-knockout mice with C57BL/
6J mice (16). Heterozygous and homozygous breeding animals
Baxter Hyland Immuno, Vienna, Austria.
Corresponding author: Hans Peter Schwarz, MD, Industriestrasse 67, A-1221
Vienna, Austria.
314
were kept in a laminar flow unit at a temperature of 20 to 22⬚C,
humidity of 50 to 55%, and a light/dark period of 12 hours each.
All animal studies were performed in compliance with Austrian
federal law (Act BG 501/1989) regulating animal experimentation. The mice used in this study were females, weighing 25 to
30 g and aged 130 to 160 days.
Crude lysates of tail snips: Pieces of tail, approximately 5 mm long and weighing 5 mg, were digested for 5 hours
at 55⬚C, with rotation in 600 ␮l of lysis buffer containing 10
mM Tris- HCl, pH 8.3, 2 mM MgCl2, 0.01% Nonidet P-40 (Sigma
Chemical Co., St. Louis, MO) and 200 ␮g of proteinase K/ml
(Boehringer Mannheim, Mannheim, Germany). The enzyme
was inactivated for 10 minutes at 94⬚C, and 5 ␮l of this lysate
was used directly for PCR analysis.
Organic extraction of DNA from tail snips: A 200-␮l aliquot of the crude tail lysate was treated with an equal volume
of phenol/chloroform followed by ethanol precipitation. The purified DNA was dissolved in 200 ␮l of TE buffer (10 mM TrisHCl, pH 8, 1 mM EDTA), and 5 ␮l of the solution was subjected
to PCR analysis.
Extraction of DNA from tail snips, using a DNA isolation kit: The DNA was extracted from tail snips by use of the
QIAamp tissue kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. Briefly, the tails were digested for
5 hours at 55⬚C, and the samples were subsequently applied to
a spin column supplied with the kit, centrifuged, washed and
eluted with 200 ␮l of elution buffer (supplied by the manufacturer).
Two microliters of the eluted solution were used for PCR analysis.
Extraction of DNA from oral swab specimens without further processing: Tissue specimens were taken from
the oral cavity of the mice, using conventional cotton swabs (Today, Köln, Germany). To obtain cells, the swab was inserted into
the oral cavity and the mouse was allowed to suck on the cotton. For lysis of the cells, the cotton swab was incubated for 5
minutes at 94⬚C in 200 ␮l of 0.004N NaOH. The liquid in the
swab was then pressed out by squeezing it with the pipette
tip, and the supernatant was transferred into a new reaction
tube. One-tenth volume of 10x PCR buffer was added for neu-
Genotyping of Transgenic Mice
tralization, and 5 ␮l was used for PCR analysis without further purification.
Extraction of DNA from oral swab specimens by use of
a DNA isolation kit: Tissue specimens were taken from the
oral cavity of the mouse, using conventional cotton swabs, as
described previously. Swab specimens were processed, using the
QIAamp tissue kit (Qiagen), adding 500 ␮l of the extraction
buffer supplied in the kit to the swabs and incubating them for
5 minutes at room temperature. The liquid was then pressed
out of the swabs, and the samples were further processed by applying the supernatant to the columns following the manufacturer’s
instructions (protocol for blood samples). Five microliters of the
DNA eluted with TE buffer were used for PCR analysis.
Polymerase chain reaction analysis: For PCR we used either a conventional Taq polymerase obtained from 1 of 3 manufacturers (DynaZyme, Fynnzymes Oy, Espoo, Finland; AmpliTaq®
DNA Polymerase, Perkin Elmer, Norwalk, CT; or Taq DNA Polymerase, Pharmacia, Uppsala, Sweden) or a heat-activated Taq
polymerase (HotStarTaq™, Qiagen). The heat-activated polymerase develops its full activity only after a pre-incubation period at 94⬚C. We carried out the heat-activation step for this
enzyme for 14 minutes at 94⬚C just before PCR amplification.
All samples were subjected to PCR in a total volume of 50 ␮l
containing 1 U of the conventional or heat-activated polymerase
in the buffer (including magnesium ion) supplied by the manufacturer, 200 ␮M each dNTP, and 50 pmol of primers neoR 5'
TGTGTCCCGC CCCTTCCTTT 3', MC-18 5' GAGCAAATTC
CTGTACTGAC 3', and MC-19 5' TGCAAGGCCT GGGCTTATTT
3'. Samples were overlaid with mineral oil and amplified for 45
cycles in a TRIO-Thermoblock (BioMetra, Göttingen, Germany),
using the following cycling profile: 30 seconds at 94⬚C, 30 seconds at 55⬚C, and 60 seconds at 72⬚C, with final elongation for 60
seconds at 72⬚C. Eight microliters of each PCR product were analyzed on a 3.5% TBE NuSieve® GTG low-melting agarose gel (FMC
BioProducts, Rockland, ME) and stained with ethidium bromide.
The genotypes were determined by use of a three-primer PCR
serving simultaneously as an internal control for false-negative
reactions (9). Primer pair MC18/MC19 yields a 680-bp control
fragment from a FVIII gene carrying no insertion, whereas the
combination of primer pair MC18/neoR amplifies a 180-bp fragment resulting from the inserted neo-sequence. It should be noted
that an equivalent fragment was described by Bi et al. (1) as having 150 bp, possibly due to miscalculation of the molecular weight.
Results
Improvement of PCR genotyping from crude lysates of
tail snip tissue specimens: Although other tissues could have
been used as well, we used tail snips as the source of DNA for
PCR genotyping because tail cuts were made for various studies
of bleeding and characterization of coagulation parameters in
the knockout mice. Because a large number of publications describe direct subjection of crude lysates to PCR analysis after
inactivation of proteinase K (4, 10, 12, 14), we used a similar
method that has yielded highly reproducible results for cells
(17). However, when we used this method with conventional Taq
polymerases (from the 3 manufacturers), we did not find it to be
reliable for tissue lysates. Amplifications by use of these enzymes often resulted in a high background of unspecific bands,
sometimes to such an extent that false-negative results of PCR
analysis were obtained. In contrast, when we used the heat-ac-
Figure 1. Amplification of samples from crude tail snip lysates with
various polymerases Aliquots of 5 l of crude lysates from 3 samples
were amplified with 3 common polymerases or a heat-activated polymerase and were fractionated on a 3.5% TBE low-melting agarose
gel, then were stained with ethidium bromide. Lanes: 1 and 18, molecular weight marker (MspI digest of pBR322); 2–5, polymerase A;
6–9, polymerase B; 10–13, polymerase C; 14–17, heat-activated polymerase. Lanes 2, 6, 10, and 14 are negative controls.
tivated polymerase instead of the conventional polymerases, the
background was reduced to nearly undetectable levels. The reproducibility of this method was comparable with more complicated protocols that use purified DNA samples. Figure 1 is an
example of 3 samples of crude tail snip lysates, each PCR-amplified with 3 common Taq polymerases (lanes: 2–5, polymerase
A; 6–9, polymerase B; 10–13, polymerase C) and the heat-activated polymerase (lanes 14–17).
Sampling from oral swab specimens as a non-invasive
DNA extraction method: If the design of a study did not require tissue or blood from the experimental animals, we sought
to use a non-invasive method for extraction of DNA for
genotyping. This was especially important for factor VIII knockout mice because of their bleeding disorder. Use of nasopharyngeal aspirates (13) represented an elegant approach for
obtaining DNA, which to our knowledge, had never been used
for genotyping of chimeric mice. Because nasopharyngeal aspirates are difficult to obtain from mice, we adapted this method
by using an oral swab. However, PCR analysis was not highly
reliable when samples from oral swab specimens were lysed
without further processing. Extraction of the DNA by use of an
isolation kit achieved better results. Using an isolation kit, we
currently achieve a PCR signal after amplification in more than
95% of all samples. Although the reaction may yield negative
results if the amount of DNA is very small, virtually all samples
can be amplified after taking a second swab specimen.
Direct comparison of different DNA preparation methods: To directly test the reliability of our two novel DNA extraction methods for PCR genotyping—amplification of crude
lysates, using the heat-activated polymerase, and amplification
of oral swab specimens after extraction of DNA by use of an isolation kit—we selected 10 samples known to contain the target
DNA (from homozygous female knockout mice) and processed
them by use of these 2 methods, as well as conventional methods. Thus, we tested four methods of DNA preparation from tail
snips: crude lysates amplified by use of a common Taq polymerase; crude lysates amplified by use of the heat-activated
polymerase; organic extraction of DNA by use of phenol/chloroform, amplified by use of a common Taq polymerase; extraction
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Vol 50, No 3
Comparative Medicine
June 2000
Table 1. Reliability of various DNA extraction methods
Method
No. of positive reactions
(in 10 samples tested)
Tail snips
Crude lysate/common polymerase
Crude lysate/heat-activated polymerase
Crude lysate/heat-activated polymerase
Organic extraction
DNA isolation kit
Oral swab specimen
Oral swab specimen without further processing
Oral swab specimen/DNA isolation kit
8/10
10/10
10/10
10/10
10/10
6/10
10/10
of DNA by use of a commercially available isolation kit, amplified using a common Taq polymerase. We also tested 2 methods
of DNA preparation from oral mucosa, both amplified by use of
common Taq polymerases: lysates of oral swab specimens without further processing; lysates of oral swab specimens using
DNA extraction with an isolation kit. The results are summarized in Table 1. Amplification products of samples obtained
from crude tail snip lysates with a common polymerase yielded
8 positive reactions and 2 false-negative reactions, from
unpurified oral swab specimens yielded 6 positive and 4 falsenegative reactions. False-negative reactions were not observed
when crude lysates of tail snips were amplified with the heatactivated polymerase or when oral swab specimens were amplified after DNA extraction, using an isolation kit. Thus, the
reliability of these two novel approaches was comparable to the
known reliability of the widely used organic extraction and DNA
isolation kit procedures.
Discussion
Our study indicates that, although a further purification step
for crude lysates (1–3, 5–7, 15) or use of a DNA isolation kit is
widely recommended for the preparation of DNA prior to PCR,
it is doubtful whether such steps are necessary for reliable PCR
genotyping. For crude lysates of tail snips, we recommend use of
heat-activated enzymes, which allow a simple “hot-start” procedure (18). Despite the higher price of such enzymes, this method
appears preferable to more laborious protocols because of its
simplicity and rapidity. If a non-invasive alternative is required,
oral swab specimens are an elegant approach. In this instance,
however, the samples should be further purified, using a DNA
isolation kit for optimal results. These methods can be applied
not only for genotyping knockout mice, but also for all types of
PCR amplification of DNA from mammalian sources.
Acknowledgments
We thank Kathryn Nelson and Christine Pavesicz for editorial assistance and preparation of the manuscript.
316
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