Electrophoresis 2001, 22, 755±762 755

Timothy Nock1 Temperature and pH studies of short tandem

Jim Dove1
Bruce McCord1 repeat systems using capillary electrophoresis
David Mao2 at elevated pH
1
Department of Chemistry and
The DNA secondary structure can affect the migration time and precision of DNA sepa-
Biochemistry,
136 Clippinger Laboratories, rations in the physical gels used in capillary electrophoresis (CE). To counteract these
Athens, OH, USA effects, DNA typing is performed using elevated temperatures (60oC) and high concen-
2
Agilent Technologies/ trations (7 M) of urea. These conditions affect the precision and lifetime of the analysis.
J&W Scientific, To better understand the effects of these conditions on the reproducibility of DNA
Folsom, CA, USA migration, we examined the effects of temperature and pH on short tandem repeat
(STR) analysis using the PE/ABI 310 Genetic Analyzer. Separations were performed
using the Profiler + multiplex system, a set of coamplified STRs with a 4-base repeat
motif, labeled at the 5©-end using fluorescent dyes. The analytical separations were ob-
tained using a commercial buffer at pH 8 and an experimental buffer consisting of 3%
hydroxyethylcellulose at pH settings ranging from 8±12. Multichannel laser-induced flu-
orescence detection was used. Temperatures were examined from 30±70oC. The
results demonstrate the fact that highly efficient separations can be carried out at alka-
line pH. In addition, improvements in temperature stability were seen when compared
to results at lower pH. However, high concentrations of urea were found to be neces-
sary to achieve optimal resolution.

Keywords: Short tandem repeat analysis / DNA separation EL 4294

1 Introduction
In the past few years there has been an expansion in the
application of capillary gel electrophoresis (CE) to DNA
analysis. Methods have been developed and validated for
DNA quantitation [1, 2], genotyping [3, 4], mutation analy-
sis [5, 6], and sequencing [7]. The reliability and stability
of the technique has progressed to the point that the use
of many of these applications is becoming routine in
assays for product analysis and quality control. CE sys-
tems using multicapillary, multichannel fluorescence
detection are playing an increasing role in sequencing the
human genome due to their high speed and easy automa-
tion [8]. CE is also playing an increasingly important role
in forensic human identification [9±11]. In this application,
CE using entangled polymer buffers is used to analyze
sets of highly polymorphic microsatellite DNA known as
short tandem repeats (STRs). The STRs used in human
identity testing consist of four base motifs such as AATG
that repeat a certain number of times at particular loca-
tions on a given individual©s DNA. The STRs are targeted
Correspondence: Dr. Bruce McCord, Department of Chemistry
and Biochemistry, 136 Clippinger Laboratories, Athens,
OH 45701, USA
E-mail: mccord@helios.phy.ohiou.edu
Fax: +740-593-0148
Abbreviation: HEC, hydroxyethylcellulose
CE and CEC
using fluorescently labeled primers and a multiplex ampli-
fication with 3±16 different sets of primers. This process
conserves sample and reduces analysis time. The loca-
tion at which each primer anneals to the template DNA is
selected to ensure that there is no overlap between differ-
ent coamplified loci. As there is a linear relationship be-
tween DNA size and migration time from approximately
75±500 bases in size [12], up to four different STR loci
can be analyzed with a single dye label by simply adjust-
ing the location at which the primers anneal to the sam-
ple. Additional loci can be analyzed simply by changing
the dye label.
In DNA typing, identity is determined by calculating the
probability that a given individual will have a specific set
of alleles at an STR locus. In the United States, a set of
13 STR loci have been selected for use in a database
known as CODIS (combined DNA identification system)
[13]. Convicted sex offenders and certain other classes of
felons are compelled to submit samples for DNA analysis,
the results of which are stored in this database. By com-
bining the frequencies of all thirteen loci, extremely high
probabilities of identity are reached, ensuring that repeat
offenders can be quickly detained through comparison of
crime scene samples with results in the CODIS database.
In our research, we are interested in the application of the
multiplex STR systems as a mechanism to explore elec-
trophoretic principles. The repetitive nature and regular

 WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 0173-0835/01/0404-755 $17.50+.50/0

756 T. Nock et al.
spacing of the STR systems provides a useful tool for the
characterization of the effect of different parameters on
the separation. Another issue in STR analysis is the pres-
ence of variant alleles that contain one and two base
deletions from the four base repeat motif. To detect these
variants, methods must be developed with optimal resolu-
tion and precision. In addition, the judicial aspects of the
forensic application require a high degree of confidence in
the reliability of the results. Thus, the development of sys-
tems for DNA typing by CE represents a unique challenge
for the analyst.
To meet these requirements, analytical systems have
been developed with multiple controls to maintain preci-
sion [10]. For each set of analyses, both internal and
external standards are utilized. To each amplified sample
an internal standard is added which consists of a mixture
of DNA fragments labeled with a red (ROX) fluorescent
dye. The internal standard is then used to estimate the
size of each allele in the amplified samples. An external
standard is also part of the analytical protocol. It consists
of a mixture of the most common alleles present in each
STR locus in the multiplex combined with the ROX-
labeled internal standard. To perform a determination, the
size of an unknown allele is determined with reference to
the internal standard and the identity of the allele is deter-
mined by comparing the calculated size of the unknown
with the known allele sizes determined in the external
standard.
The multiple comparisons between internal and external
standards are necessary in order to maintain analytical
precision, however, additional measures are also
required. Secondary structure differences can occur be-
tween the amplified sample and the internal standard and
will result in a loss of precision. These effects are caused
by conformational and sequence differences and will
result in poor sizing accuracy. To minimize DNA secon-
dary structure effects, analyses are performed using ele-
vated temperatures (60oC) and high concentrations of
urea (7 M).
The use of high temperature and urea in these systems
necessitates careful control of instrumental parameters.
High urea concentrations limit the shelf life of the buffers
and can result in sublimation and crystal formation. The
elevated temperatures required can be difficult to main-
tain in conditions where high humidity or rapid room tem-
perature shifts occur. Although these issues can be easily
mitigated by proper validation of the analytical systems, a
less cumbersome approach would be to perform separa-
tions at room temperature and with denaturants other
than urea.
Electrophoresis 2001, 22, 755±762
In order to investigate the plausibility of reducing the tem-
perature and concentration of urea in these assays, we
have characterized the effect of temperature and urea
concentration on the analysis of a particular multiplexed
set of 9 STRs, the Profiler+ system. These 9 STRs are an
important subset of the 13 STRs recommended for use in
the CODIS database of convicted sexual offenders and
other felons. The analyses in this study are performed
using the PE/ABI 310, the CE system utilized in the vast
majority of DNA profiling assays.
To perform this work, we have utilized an experimental
sieving buffer consisting of 3% hydroxyethylcellulose
(HEC) as well as a commercial buffer POP4 consisting of
4% poly(dimethylacrylamide), at varying temperatures
and/or concentrations of urea. We have also examined
the application of elevated pH as an alternative to high
temperatures and urea in an effort to minimize DNA sec-
ondary structure. The elevated pH work was performed
using fluorocarbon-coated capillaries. Our results illus-
trate the effect of urea, pH and temperature on the preci-
sion and accuracy of DNA genotyping.
2 Materials and methods
2.1 Materials
Fluorescently labeled allelic ladders and GS500 ROX
standards were acquired from Perkin Elmer (Foster City,
CA, USA). Trizma base, boric acid, CAPS, EDTA, HEC
(#30,863-3; Aldrich Chemical, Milwaukee, WI, USA), and
Amberlite MB-150 Resins were obtained by Sigma Chem-
ical (St. Louis, MO, USA). Urea was acquired from Spec-
trum (Gardena, CA, USA). High-purity formamide was ob-
tained from Amresco (Solon, OH, USA).
2.2 Sample preparation
Allelic ladder samples (AmpFISTR Profiler+; Perkin
Elmer) were prepared by adding 11 mL of formamide to
0.5 mL of allelic ladder and 0.5 mL of the ROX standard.
To denature the samples, the sample tubes were placed
in a PE/ABI 9700 Thermal cycler, heated to 95oC for
2 min, and snap-cooled to 4oC in an ice bath. Blood stains
on FTA paper were extracted according to the manufac-
turer©s protocols (part #10786-010; Gibco BRL, Grand
Island, NY, USA), and amplified using the protocols de-
scribed in the AmpFISTR Profiler+ Users Manual (part
#4303812; Perkin-Elmer).
2.3 Capillary electrophoresis
An ABI Prism 310 Genetic Analyzer (Perkin Elmer) was
used for all experiments and calibrated using matrix dye
standards labeled with ROX, JOE, NED, and FAM fluo-
Electrophoresis 2001, 22, 755±762 Temperature and pH studies of STR systems 757

rescent dyes. J&W Scientific (Folsom, CA, USA) mSil-FC
capillary columns with 50 mm inner diameter were used.
The sieving medium was 3% w/v HEC and contained
20 mM CAPS, pH 11, and varying concentrations of urea;
7, 5, 3.5, 2, and 0 M. The HEC sieving polymer solution
was prepared by adding various amount of urea to 25 mL
of CAPS buffer and shaking until dissolved, to obtain the
proper concentration of urea. 1.5 g of HEC was stirred
into the solution, the solution was allowed to mix overnight
and approximately 0.5 g of Amberlite mixed-bed ion-
exchange resin were added and stirred for about 1 h
using procedures similar to those described by Baskin et
al. [14]. This solution was centrifuged at 3000 rpm for
10 min and the polymer solution removed from the ion-
exchange resin. The solution was then filtered through a
5 mm syringe filter (Millipore, Bedford, MA, USA). So-
lutions were stored in the refrigerator. Buffers containing
HEC in Tris-borate/EDTA were prepared as described
previously [9], and buffers containing POP4 (Perkin
Elmer) were prepared using the manufacturer©s sug-
gested protocols. Samples were electrophoretically sep-
arated using 50 mm ID capillaries with 41 cm length
(30 cm to the detector) and a field strength of 350 V/cm.
The capillaries were prepared by rinsing with buffer for
5 min prior to running the samples. Injections were made
at 15 kV for 5 s. Electrophoresis was conducted at 30±
70oC and data were analyzed using GeneScan software
(Version 2.0.1; Perkin Elmer). Regression analysis on the
slope and standard deviation of the slope was performed
using the LINEST function present in Microsoft Excel
(Version 5.0). The resolution between two peaks was cal-
culated using the standard chromatographic definition,
modified to permit measurement using peak width at half
height:
Resolution = 2(T1 - T2)/(W1 + W2) = (2ln2)1/2(T1 - T2)/
[(W(1/2)1 + W(1/2)2)]
where T is the peak retention time, W is the baseline peak
width and W(1/2) is the peak width at half height [15].
Dividing the resolution into the DNA fragment size differ-
ence between any two peaks produces an estimate of the
resolution in units of bases.
3 Results and discussion
In a paper by Rosenblum and co-workers in 1997 [16], a
technique was described for the analysis and mitigation of
anomalously migrating DNA fragments. In their paper,
they identified specific fragments in a TAMRA dye labeled
Genescan 500 standard whose migration times were tem-
perature sensitive. The effect of this temperature sensitiv-
ity was to produce size estimates for these fragments that
varied with temperature. The size estimates were ob-
tained through comparison with DNA fragments which
were insensitive to temperature. Separations performed
under stronger denaturation conditions produced size
estimates closer to the true (sequenced) size.
In a previous work in our laboratory we have detected a
concomitant improvement in precision with increasing
temperature which we believe is also associated with the
DNA secondary structure. It is possible that elevated tem-
peratures and strong denaturing conditions limit the varia-

Table 1. Slope of the relationship between temperature (45±70oC) and estimated size for individual alleles in the Profiler+
multiplex
STR Allele# Sizea) Run 1 SDb) Run 2 SD Run 3 SD Run 4 SD Average SDc)
Slope slope slope slope slope
D3S1358 12 111.2 ±0.12 0.01 ±0.091 0.003 ±0.098 0.006 ±0.097 0.005 ±0.10 0.01
VWA 21 194.9 ±0.083 0.009 ±0.085 0.006 ±0.058 0.005 ±0.055 0.008 ±0.07 0.02
FGA 30 264.7 ±0.144 0.005 ±0.163 0.01 ±0.129 0.006 ±0.131 0.003 ±0.14 0.02
Amel. x 103.5 ±0.147 0.01 ±0.12 0.004 ±0.118 0.007 ±0.117 0.004 ±0.13 0.01
D8S1179 19 170.4 ±0.16 0.016 ±0.193 0.014 ±0.148 0.004 ±0.15 0.006 ±0.16 0.02
D21S11 36 232.4 ±0.037 0.009 ±0.042 0.01 ±0.019 0.004 ±0.02 0.004 ±0.03 0.01
D18S51 26 341.9 ±0.186 0.006 ±0.191 0.009 ±0.167 0.011 ±0.161 0.011 ±0.18 0.01
D5S818 7 131.2 ±0.107 0.009 ±0.082 0.003 ±0.084 0.002 ±0.078 0.003 ±0.09 0.01
D13S317 8 205.0 ±0.121 0.005 ±0.127 0.003 ±0.109 0.006 ±0.108 0.005 ±0.12 0.01
D7S820 15 292.8 ±0.094 0.008 ±0.095 0.005 ±0.082 0.003 ±0.078 0.004 ±0.09 0.01
Results are given in units of bases/C
a) Estimated size at 61oC
b) Standard deviation of the calculated slope
c) Standard deviation of the average of the four estimated slopes
Slope and standard deviations are calculated using Microsoft Excel. POP4 buffer; 43 cm, 50 mm uncoated capillary;
350 V/cm; 5 s 15 kV injection. See also Fig. 1 for an example dataset.
758 T. Nock et al.
Figure 1. Temperature vs. estimated size for POP4, and
CAPs buffer (pH 11) at 0 and 7 M urea. Other conditions
as stated in Section 2.3. (^) POP4, (~) 0 M urea; (&) 7 M
urea. Each plot represents an individual STR locus.
bility in migration times by melting out different DNA con-
formers. Certainly, analyses are more stable in regions
where minor variations in temperature do not affect size
estimates. As mentioned above, current instrumentation
in forensic DNA analysis utilizes a two-step calibration
process to produce precise estimates of DNA size based
upon migration time. Allele calls are produced through a
binning process that fits the data into narrow migration
time windows, typically  0.5 bases.
We have been interested in characterizing the environ-
mental and experimental factors that affect this precision.
Previous reports demonstrate that control of temperature
and the addition of denaturants is an important parameter
in the production of precise and accurate DNA size estim-
ates [16±18]. Thus, we undertook a study to examine the
effect of temperature on size estimates of the Profiler +
Electrophoresis 2001, 22, 755±762
STR multiplex, a commonly used set of STR markers. In
this work, we examined the effect of the capillary oven
temperature on the size estimates of STR fragments. A
series of temperatures from 45±70oC were utilized to
examine their effect on the size estimates for ten different
alleles in the Profiler+ multiplex.
3.1 Allele size estimates
For these experiments, we utilized the manufacturer©s
standard protocols with the exception of the temperature,
which is normally set at 60oC. To make the measure-
ments, allelic ladders were denatured in formamide,
snap-cooled in an ice bath, and analyzed on a PE/ABI
310 using the POP4 polymer. POP4 polymer consists of
4% poly(dimethylacrylamide) in 100 mM TAPS at a pH of
8.0 [16]. 8 M urea and 5% 2-pyrrolidinone are added as
denaturants. The capillary used was a 43 cm, 50 mm ID
uncoated capillary from Polymicro Technologies (Phoe-
nix, AZ, USA) at a field strength of 350 V/cm and all sam-
ples were injected electrokinetically at 15 kV. The size in
bases of each STR allele was estimated using the PE
ROX 500 internal size standard fit to a global southern
sizing algorithm. DNA fragments from 75±400 bases in
length are included in the calibration estimate.
The selected alleles incorporate a size range from 100±
350 bases in length and allowed us to probe the entire
range of the calibration curve produced by the ROX
standard. The results of this work are reproduced in Table
1, which illustrates the differences in temperature re-
sponse from 45 to 70oC for each allele. While the
sequenced size of these alleles has not been published,
the response of the size estimate with respect to tempera-
ture can be used to examine the overall stability of the
system. In Table 1 the results of four different analyses
are compared. With the exception of the last two data
sets, which were run in the same 2-day period, the runs
were taken at different times over a period of several
months and on different capillaries. In spite of this fact,
the estimates of the slope of the temperature response
are fairly precise with standard deviations of 0.01±0.02
bases/oC.
Of particular interest is the differential response in slope
from one set of markers to the next. For example, a tem-
perature change of 2oC in the capillary changes the size
estimate of allele 26 in D18S51 0.36 bases while allele 36
in D21S11 changes only 0.06 bases. Another interesting
pair of alleles is vWA and D13S317. These alleles occur
very close to the 200 bp fragment in the ROX size stand-
ard, however, the slope for these alleles is markedly dif-
ferent, indicating that differences in temperature response
for this pair of alleles is more likely due to the amplified
Electrophoresis 2001, 22, 755±762 Temperature and pH studies of STR systems 759

Table 2A. Slope of the relationship between temperature Table 2B. Slope of the relationship between temperature
(45±70oC) and estimated size for individual (45±70oC) and estimated size for individual
alleles in the Profiler+ multiplex, 0 M urea alleles in the Profiler+ multiplex, 7 M urea
STR Allele Size 61 Run 1 SD Run 2 SD Average STR Allele Size 61 Run 1 SD Run 2 SD Average
(oC) slope slope slope (oC) slope slope slope
D3S1358 12 114.10 ±0.016 0.004 ±0.014 0.003 ±0.015 D3S1358 12 115.11 ±0.024 0.003 ±0.017 0.005 ±0.021
vWA 21 196.96 ±0.032 0.004 ±0.031 0.003 ±0.032 VWA 21 197.75 ±0.040 0.006 ±0.041 0.006 ±0.039
FGA 30 254.34 0.022 0.003 0.026 0.004 0.024 FGA 30 258.97 0.039 0.005 0.042 0.006 0.040
Amel. X 106.12 ±0.032 0.003 ±0.028 0.003 ±0.030 Amel. X 108.59 ±0.063 0.009 ±0.067 0.008 ±0.062
D8S1179 19 167.17 ±0.039 0.005 ±0.033 0.004 ±0.039 D8S1179 19 171.12 ±0.066 0.012 ±0.057 0.009 ±0.060
D21S11 36 238.86 ±0.036 0.009 ±0.030 0.003 ±0.033 D21S11 36 239.25 0.027 0.004 0.017 0.008 0.021
D18S51 26 328.60 0.046 0.004 0.052 0.008 0.048 D18S51 26 333.42 0.041 0.007 0.015 0.006 0.026
D5S818 7 136.76 ±0.030 0.005 ±0.026 0.002 ±0.028 D5S818 7 137.31 ±0.021 0.004 ±0.023 0.005 ±0.022
D13S317 8 203.14 ±0.033 0.003 ±0.027 0.003 ±0.030 D13S317 8 207.80 ±0.050 0.005 ±0.030 0.007 ±0.039
D7S820 15 293.74 ±0.011 0.005 ±0.020 0.005 ±0.016 D7S820 15 294.60 0.008 0.004 0.023 0.005 0.015
20 mM CAPS buffer, pH 11; 3% HEC; 43 cm, 50 mm J&W 20 mM CAPS buffer, pH 11; 3% HEC; 43 cm, 50 mm J&W
msil FC capillary msil FC capillary

fragments and not to the size standard. The overall
results clearly illustrate the importance of temperature
control on the size estimates of alleles in the multiplex.
In controlled studies from these and other laboratories,
the precision (standard deviation) of allele size estimates
at 60oC ranges from 0.24±0.07 bases, with 0.17±0.12
bases being fairly typical [19±23]. An issue in forensic
DNA typing of STRs is the existence of variant alleles.
These alleles contain deletions of one to two bases from
the standard 4-base motif. The level of precision, required
for reproducibly determining the difference between two
alleles differing in size by one base, is 0.15 bp [21]. Thus,
present CE technology is just adequate to perform this
task. The appearance of these variant alleles can some-
times lead to an inconclusive result at one locus in a multi-
plex. While the absence of one allele from a total of 13
sets of STR loci has minimal effect on the statistics of the
analysis (in fact the rarity of these types often results in a
unique profile), improved precision would be useful in the
identification of these variant alleles. Our findings on the
temperature dependence of sizing indicate that improved
control of temperature should result in increased preci-
sion.
3.2 Electrophoresis at alkaline pH
An alternate method for affecting the conformational
effects of ssDNA is to adjust buffer pH. Recent reports
have demonstrated the applicability of CE for DNA analy-
sis at elevated pH [24, 25]. The denaturing effect of alka-
line pH is believed to be due to the development of neg-
ative charges on guanine and thymine bases. In both
reports, CE with UV detection was used to analyze DNA
at pH 11 and above. The authors found the separation
mechanism in alkaline buffers to be very similar to that at
lower pH using urea as a denaturant. However, DNA res-
olution of only 10±11 bases was illustrated and little infor-
mation was given on the reproducibility of the results. In
one study, separation efficiency was maximized at pH 11
although complete denaturation of DNA was expected at
pH 11.5 [24].
A goal of the present work was to determine if it was
possible to produce high-resolution separations under
alkaline conditions using fluorescently labeled DNA. Pre-
vious studies in the literature utilized UV detection tech-
niques and peak resolution was limited. Our expectation
was that resolution would improve using higher concen-
trations of polymer and that the fluorescent dye linkers
would remain stable at high pH. We were also interested
in how buffers at elevated pH would affect the tempera-
ture response, precision and resolution of the separation.
We expected that the use of high pH buffers could obviate
the need for elevated temperatures and high concentra-
tions of urea. Initially, we examined buffers at pH 11 and
12 using a 50 mm fluorocarbon-coated capillary (msil FC)
filled with 3% HEC. This specially coated capillary was
necessary because of the solubility of silica at pH above
8.5. However, the bulk of this work was performed in a
20 mM CAPS buffer at pH 11, as the fluorocarbon coating
was found to have limited stability at pH 12. The HEC
polymer was chosen as a result of our previous work
demonstrating that high precision DNA typing can be per-
formed with this system [9] and due to its demonstrated
stability at high pH. It should be noted that these results
should be fairly independent of the type of polymer sys-
tem as long as the polymer remains chemically stable. In
order to characterize the effect of alkaline pH, the temper-
ature dependence of the DNA size estimates using the
HEC/CAPS buffer was compared to the results previously
obtained with the POP4 system (Fig. 1).
760 T. Nock et al. Electrophoresis 2001, 22, 755±762

dependence of allele size, peak resolution suffered. Dur-

Figure 2. The effect of urea concentration on the resolu-
tion of the blue allelic ladder from the Profiler+ multiplex
kit. The ladder consists of sets of alleles amplified from
the D3S1358, vWA, and FGA STR loci. The X-axis is nor-
malized to the DNA fragment size using the ROX 500
(Perkin-Elmer) internal standard. Conditions: 20 mM
CAPS buffer, pH 11; 30oC; 43 cm J&W msil FC capillary;
350 V/cm; 15 kV, 5 s injections. (A) 7 M urea; (B) 3 M urea;
(C) 0 M urea.
Experiments were performed by analyzing the profiler+
allelic ladder at pH 11 using three different concentrations
of urea: 0, 3, and 7 M. The results at 0 and 7 M urea are
illustrated in Table 2. The slopes of the regression line be-
tween allele size and temperature in the table show a
nearly flat response at all concentrations of urea. Average
slopes range from 0.015 to 0.06 and show both positive
and negative slopes. These results are different from the
data in Table 1 in which most of the measured alleles had
slopes of ±0.09oC/base or greater. The results indicate
that a stabilizing effect on DNA temperature response is
occurring at elevated pH that is not present under stand-
ard conditions using POP4. We believe this effect to be
the result of improved denaturing of the DNA fragments
under alkaline conditions.
3.3 Resolution at elevated pH
While the results at pH 11 exhibited superior characteris-
tics to the POP4 polymer with respect to the temperature
ing additional experiments to test the effect of urea con-
centration on the estimation of size, an interesting effect
was shown. Experiments were performed at 30oC to
examine the separation system at near ambient tempera-
tures. The results showed that peak resolution improved
with increasing quantities of urea. The improvement of
resolution is likely to be the result of hydrogen bonding
between the DNA and urea, increasing the mass of the
DNA fragments. Increased urea concentrations can affect
the solution viscosity and may alter the sample focusing
effects during injection. These results are interesting in
that they illustrate that urea has a dual role in ssDNA sep-
arations. In addition to its denaturation effects, it also pro-
vides improved resolution.
The effect of the increased urea concentration is also
illustrated in the migration times of the DNA fragments.
For example, the migration time of allele 15 from D7S820
increased from 14.54  0.07 min to 22.08  0.07 min as
the concentration of urea was increased from 0 to 7 M. In
situations requiring increased sample throughput, the
shorter migration times in the non-urea buffer could be
considered an advantage. Figure 2 illustrates the effect of
increasing urea concentration on the separation of the
blue allelic ladder from the profiler + multiplex. Urea con-
centration also affected the size estimates, but the tem-
perature response as measured by the slope of the rela-
tionship between estimated size and temperature did not
markedly change (Fig. 1). Thus, the observed differences
in size estimates at different pH and urea content are the
result of changes in DNA mobility resulting from hydrogen
bonding. The effect of elevated pH on the guanine and
thymine bases and the fluorescent dyes used in labeling
the DNA may also affect size estimates.
Overall results from three different concentrations of urea
at pH 11 and 30oC are shown in Table 3. The table gives
resolution values for allele sizes from 100±316 bp. Reso-
lution is slightly degraded for late eluting peaks. Other-

Table 3. Resolution and size estimates for the analysis of selected alleles in the profiler+
multiplex at three different concentrations of urea
0 M Urea 3.5 M Urea 7 M Urea
Locus Allele# Resolution Allele size Resolution Allele size Resolution Allele size
Amel. X 2.04 +/± 0.12 111.09 +/± 0.24 1.09 +/± 0.02 108.61 +/± 0.04 0.63 +/± 0.01 107.14 +/± 0.06
D21S11 26 2.71 +/± 0.09 199.10 +/± 0.19 1.94 +/± 0.06 199.24 +/± 0.03 1.26 +/± 0.01 197.99 +/± 0.05
D18S51 20 3.26 +/± 0.09 316.62 +/± 0.05 2.88 +/± 0.17 314.66 +/± 0.08 2.26 +/± 0.27 312.54 +/± 0.10
D13S317 14 2.64 +/± 0.17 237.47 +/± 0.11 2.07 +/± 0.07 230.17 +/± 0.09 1.38 +/± 0.12 229.47 +/± 0.08
VWA 20 2.91 +/± 0.16 194.87 +/± 0.10 1.82 +/± 0.13 194.53 +/± 0.05 1.26 +/± 0.07 193.88 +/± 0.06
Experimental error is measured as the SD of ten replicates. Conditions: 20 mM CAPS buff-
er, pH 11; 3% HEC; 43 cm, 50 mm J&W msil FC capillary; 5 s, 15 kV injection, 30oC. Results
are the average of ten runs.
Electrophoresis 2001, 22, 755±762
wise, the results are roughly equivalent to those reported
for the 3% HEC and POP4 polymers at 60oC with urea
but at far lower temperatures [26]. The reproducibility of
the size estimates are also shown in Table 3 and range of
standard deviations from 0.06 to 0.012 for a set of ten
analyses using 7 M urea. In a final series of experiments,
a set of 10 samples was extracted from FTA blood stain
cards and the results were amplified and analyzed using
POP4 at 60oC, 3% HEC in 100 mM Tris-borate-EDTA
(TBE) buffer with 7 M urea at 60oC [9] and in 20 nM CAPS.
The results were typed using Genotyper software and
shown to be in complete concordance with each other.
Figure 3 illustrates the analysis of an amplified sample at
30oC in a pH 11 CAPS buffer at 7 M urea.
4 Concluding remarks
This paper demonstrates a varying response to tempera-
ture in the different alleles among the forensic 10 loci
studied at pH 8 and underscores the importance of tem-
perature control when performing these analyses. The
results obtained in this study also demonstrate that highly
efficient separations with good resolution can be per-
formed using elevated pH. The effect of temperature on
the size estimates obtained using the internal/external
standard calibrations is shown to be reduced when
compared to standard procedures at lower pH. Separa-
tions near room temperature still required urea to achieve
maximal resolution. This effect was believed to be the
result of hydrogen bonding between the urea and DNA
molecules and not the result of improvements in the dena-
turing ability of the buffer. Our results indicate the poten-
tial for the use of high pH with coated capillaries in DNA
Temperature and pH studies of STR systems 761
Figure 3. Separation of a blood
stain amplified from an FTA
stain card using the Profiler+
STR kit and multichannel fluo-
rescence detection. 3% HEC in
20 mM CAPS at 30oC. Other
conditions as in Section 3. Panel
(1) alleles labeled with the FAM
dye from D3S1358, vWA, and
FGA; (2) alleles labeled with the
JOE dye from Amelogenin,
D8S1179, D21S11, D18S51;
(3) alleles labeled with NED dye
from D5S818, D13S317, and
D7S820; (4) ROX-labeled inter-
nal standard. The ROX labeled
peaks shown are 139, 150, 160,
200, 250, 300, and 340 bases in
size.
analysis. Reduction of the temperature dependence of
allele-size estimates will improve the long-term precision
of the analysis. Future work will explore the effects of buff-
er additives other than urea to improve DNA resolution at
elevated pH.
The authors would like to thank John Pedersen for con-
tributing samples utilized in this study, and J&W Scientific
for the donation of certain capillaries used in this study.
Federica Crivellente, Sharon Williams, and Alice Isenberg
are acknowledged for helpful discussions. Major parts of
this work were funded through grant # 1999-IJ-XC-K014
from the National Institute of Justice and through start-up
funds provided by Ohio University.
Received July 27, 2000
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