REFERENCES

(1)

(2)

(3)

(4)

(5)

(6)

L.Z. Benet, J.R. Mitchell, L.B. Sheiner, Pharma-
cokinetics: The dynamics of drug absorption,
distribution, and elimination, in: A.G. Gillman,
T.W. Rall, A.S. Nies, P. Taylor (Eds.), Goodman
and Gilman’s The Pharmacological Basis of Thera-
peutics, 8" ed., Pergamon Press, New York, NY,
1990, pp. 3-32.

W. Grellner, B. Madea, M. Staak, Individualiza-
tion of fresh and stored human urine samples by
PGM,- and Ge-subtyping, Forensic Science In-
ternational 68 (1994) 181-93.

L. Kobilinsky, Recovery and stability of DNA in
samples of forensic science significance, Forensic
Science Review 4 (1992) 67-87.

P.M. Schneider, Basic issues in forensic DNA
typing, Forensic Science International 88 (1997)
17-22.

M.M. Holland, R. Roy, M.D. Fraser, R.H. Liu,
Application of serological and DNA methods for
the identification of urine specimen donors, Fo-
rensic Science Review 5 (1993) 1-14.

GJ. Tsongalis, D.E. Anamani, A.H.B. Wu, Iden-
tification of urine specimen donors by the PM +
DQAI amplification and typing kit, Journal of
Forensic Sciences 41 (1996) 1031-4.

(7)

(8)

(9)

(10)

(11)

(12)

(13)

I. Medintz, L. Chiriboga, L. McCurdy, L.

Kobilinsky, Restriction fragment length polymor-

phism and polymerase chain reaction-HLA DQa

analysis of case work urine specimens, Journal of
Forensic Sciences 39 (1994) 1372-80.

D.R. Linfert, A-H.B. Wu, G.J. Tsongalis, The
effect of pathologic substances and adulterants on
the DNA typing of urine, Journal of Forensic
Sciences 43 (1998) 1041-5.

P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex®
100 as a medium for simple extraction of DNA
for PCR-based typing from forensic material,
Biotechniques 10 (1991) 506-13.

B. Budowle, J.S. Waye, G.G. Shutler, F.S.
Baechtel, Hae III—A suitable restriction endonu-
clease for restriction fragment length polymor-
phism analysis of biological evidence samples,
Journal of Forensic Sciences 35 (1990) 530-6.

QuantiblotTM, Human DNA quantitation kit,
package insert, Roche Molecular Systems, Inc.,
Alameda, CA, 1993.

AmpliType®, PM+DQA] PCRamplification and
typing kit, package insert, Roche Molecular Sys-
tems, Inc., Alameda, CA., 1995.

AK. Chaturvedi, N.T. Vu, R.M. Ritter, D.V.
Canfield, DNA typing as a strategy for solving

issues relevant to forensic toxicology, Journal of
Forensic Sciences 44 (1999) 187-90.


URINARY GENOTYPING FOR DQAI AND PM Loci Ustnc PCR-Basep
AMPLIFICATION: EFFECTS OF SAMPLE VOLUME, STORAGE TEMPERATURE,
PRESERVATIVES, AND AGING ON DNA EXTRACTION AND TYPING

INTRODUCTION

Elimination of drugs from the body involves vari-
ous processes that occur in the kidneys, liver, lungs,
and other organs, Renal clearance is the most impor-
tant elimination mechanism, which results in the
appearance of drugs and their metabolites in the
urine (1). Therefore, human urine is frequently ana-
lyzed for forensic toxicological evaluations. This is
particularly true with workplace drug testing, wherein
urine specimens are often submitted because of their
easy accessibility (2). In some instances, the integrity
of the urine sample may be challenged, and the origin
of the sample may need to be verified.

Methods for individualization of the urine samples
have largely been based on the indirect interpretation
of the expressed genetic markers—that is, individuals
are differentiated at the phenotypic level using sero-
logical techniques, on the basis of blood cell typing,
secretor status, and polymorphic proteins (3). At
present, identity testing at the DNA level allows the
detection of genetic variation by direct identification
of the allelic gene sequences, and the polymerase
chain reaction (PCR) has proven to be a powerful
tool for this purpose (4). The PCR technique is
particularly more effective in analyzing those types of
biosamples, wherein the nuclear material is scanty.
Although human urinary DNA typing results had
been reported (5-8), the effectiveness of the PCR-
based DNA technology has not been fully established
to characterize the genetic constitution of urine
samples: Information about sample requirement, stor-
age condition, preservation, and expected frequency
for a successful DNA typing has not been well evalu-
ated. Therefore, the present study was conducted to
obtain such information using the PCR-based typing
technology.

In the study, different methods of sample concen-
tration and DNA extraction were explored for urine
samples collected from human subjects. The effects
of storage temperature, storage time, sample volume,
and preservatives on the typing efficiency were also

investigated. Semi-quantitation of the extracted DNA
and typing of the loci for human leukocyte antigen
(HLA) and polymarkers (PMs) allowed comparisons
to be made among the alternative methods.

MATERIALS AND METHODS

Materials

Reagents, solvents, and other necessary supplies
were obtained from commercial sources. All reagents
were of analytical grade, and the solvents were of
molecular biology grade. The DNA analysis kits
(QuantiblotTM Human DNA Quantitation Kit;
AmpliType® PM+DQAI PCR Amplification and
Typing Kit) were supplied by Perkin-Elmer Corpora-
tion (Foster City, CA).

Sample Collection

All volunteers were fully informed about the study
and instructed on the method of mid-stream, clean
catch urine collection. Urine specimens were col-
lected in sterile Nalgene® sample bottles and were
mixed by gentle inversion for at least 30 min prior to
processing, using a hematology mixer. To avoid the
effects of repeated sampling and to study the storage
effects, samples of each urine specimen were aliquoted
fresh into different portions in the appropriate con-
tainers prior to the application of different treatment
conditions.

Blood specimens were also obtained from the
urine donors by finger prick, using sterile lancets and
aseptic techniques. Blood samples were processed
fresh and served as the subjects’ typing reference.

Assessment of Sample Concentration Techniques
An initial assessment on the adequacy of 3 concentra-
tion procedures was conducted on urine samples from
8 male volunteers, according to the scheme described in
Fig. 1. Male urine samples were selected over female
samples as the former samples contain relatively scanty


(Procedure 2), which could probably be due to the larger
initial sample volume used (5 ml vs. 1 ml). The DNA
content of the samples varied among the subjects. The
amount of DNA recovered from 1 ml of fresh urine
ranged from 14 to > 200 ng/ml for females and from 4
to 60 ng/ml for males. The quantity of DNA decreased
sharply after the first 24 h at frozen storage regardless of
the gender: The greater the amount of starting DNA,
the greater the decrease. After 24 h, a slower reduction
in the quantifiable DNA was observed for up to 7 days.
At RT storage, the amount of isolated DNA decreased
with the elapsed time. However, the amount of quanti-
fiable DNA from RT samples was significantly higher
than that of frozen samples at any given time interval.

DQAI and PM Typing

The DQA1 and PM typing results (Tables 2 and 3)
correlated well with the DNA amounts (Table 1).
Results within the male population showed that
typeable frequency was sharply reduced after 24 h of
storage at—20°C, then remained essentially unchanged
at 25% for up to 7 days (Figs. 4 and 5). Samples
stored at RT produced significantly higher typing

rates than that of frozen samples, independent of
concentration methods (Cochran-Q, p = 0.0001).
For female samples, there was no significant differ-
ence in the typing rates among the conditions tested
(Cochran-Q, p = 0.7750). Although centrifugation
(Procedure 1) yielded slightly higher quantifiable
DNA than micro-centrifugation (Procedure 2), as
explained by the larger sample volume used (5 ml vs.
1 ml), such effect was not evident on the typing
results. Typeable blots were obtained from samples
with DNA content < 0.30 ng/ml, which were below
the detectable level by QuantiblotTM.

Effects of Preservatives

As depicted in Fig. 3, fresh urine samples with
sodium azide were 100% typeable, while all the
samples containing sodium fluoride produced nega-
tive results. In the presence of sodium azide, typeable
results were obtained for all samples stored at 4°C for
up to 20 days, while only 3/6 of the frozen samples
were typeable. After the 30-day storage in the pres-
ence of sodium azide, refrigerated and frozen samples
were typed with equal rate of success (1/6).

Table 2. The typeable rates for DQA1 using Chelex® extracts from urine samples subjected to

different storage conditions over time.

Sample* Male (n = 12) Female (n = 13)
5 ml Imi 5 ml 1ml
Fresh 100% 100% 100% 100%
RT (24h) 94% 83% 88% 100%
RT (48 h) 94% 67% 100% 100%
RT (7 days) 11% 50% 100% 92%
-20°C (24 h) 18% 25% 100% 92%
-20°C (48 h) 29% 25% 82% 92%
-20°C (7 days) 35% 25% 76% 92%

* Samples were concentrated by centrifugation (Procedure 1; 5 ml) and micro-centrifugation (Procedure

2; 1 ml); RT = room temperature.


amounts of cellular components. Therefore, DNA typ-
ing of male urine samples provides a greater challenge
for a procedure. The 3 concentration procedures evalu-
ated were centrifugation, micro-centrifugation, and
dialfiltration. The obtained urinary concentrates were
subsequently used for the DNA extraction.

Centrifugation (Procedure 1)—Each 5-ml sample
was concentrated by centrifugation in a centrifuge
(GPR Centrifuge, Beckman, Fullerton, CA) at 1,500
x g for 15 min in a conical glass centrifuge tube. The
supernatant was discarded to the volume of about 0.5
ml, and the pellet was resuspended by gentle vortexing.
The suspension was then transferred to a 1.5-ml
Eppendorf tube, with 2 volumes of saline rinse (0.9%
NaCl), each of 0.3 ml. The obtained suspension was
centrifuged at 13,600 x g for 5 min (Microcentrifuge
235C, Fisher Scientific Co., Pittsburgh, PA). The
supernatant was discarded, leaving approximately 50
pl of the residue.

Micro-centrifugation (Procedure 2)—One-ml urine
samples were centrifuged at 13,600 x g for 5 min in the
micro-centrifuge. After discarding the supernatant layer,
a final volume of about 50 il was retained.

Dialfiltration (Procedure 3)—This filtration was
performed according to the manufacturer instruction
for CentriplusTM or CentriconTM (Millipore Corp.,
Bedford, MA). These devices had a molecular weight
cut-off (MWCO) covering the range of 3 to 100 kd
(CentriplusTM-100; CentriconTM-3, -10, -30, and -100).
Starting sample volumes were dictated by the device
capacity, thus 5-ml samples were used with CentriplusTM,
whereas 1- or 2-ml samples were used with CentriconTM.,
Using a centrifuge with a fixed angle rotor (Ivan Sorvall,
Inc., Newtown, CT), ultrafiltering occurred upon the
application of the centrifugal force that drives the sample
solution through the membrane. The relative centrifu-
gal force was 1,000-5,000 x g for 30 to 60 min, depend-
ing on the device used. The retained urinary materials
larger than the MWCO were recovered by inverted
centrifugation. The obtained pellet was resuspended by
gentle vortexing. This mixture was transferred to an
Eppendorf tube and followed by volume reduction toa
final volume of approximately 50 tl, as previously
described.

DNA Extraction

Depending upon the nature of a particular experi-
ment, Chelex® 100 resin and/or organic extraction
methods were used.

Chelex® extraction—DNA from the urine con-
centrates was extracted in the presence of Chelex®
100 resin (Bio-Rad Laboratories, Richmond, CA)
following the procedure of Walsh et al. (9). To
approximately 50 il of each of the concentrates, 200
pl of the 5 % Chelex® resin suspension in water was
added, and the mixtures were incubated in a water
bath at 56°C for 30 min and then at 90 to 100°C for
8 min. DNA from whole blood samples was also
extracted by this resin method.

Organic extraction—Approximately 50 il of each
urinary concentrate was incubated with 15 yl of pro-
teinase-K (10 mg/ml in deionized water) in 0.5 ml of
digest buffer at 56°C for 6 h. DNA was extracted from
the mixture by using a chloroform:phenol:isoamy] alco-
hol system (10).

Comparison of DNA Extraction Methods
Initially, Chelex® 100 resin and organic extrac-
tions were separately performed on 1 ml of each of the
fresh and 7-day frozen urine samples from 6 male
volunteers following concentration by micro-
centrifugation (Procedure 2; Fig. 2). The obtained
extracts were stored at —20°C until analyzed.

Urine samples
(6 males)

——

Chelex extract | | Organic extract

Fresh: 6/6
Frozen: 4/6

Fresh: 6/6
Frozen: 4/6

Typeable results

Fig. 2. Comparison of Chelex® and organic extraction
methods using fresh and frozen urine samples of male
volunteers; the sample volume was 1 ml. Details of
the procedures are given in the Materials and Methods
section. The typeable results reflect the number of
samples successfully typed out of the total number of
samples tested under a particular category.


JEquUNU |&}0} ay} JO ]no padAy

“Kuobaye9 sejnoyed e Japun payse} sajdwues jo

|Nysseoons sajdwes Jo Jaquinu ay} Joayai S}Nseu e|qeedA} ay +}x9} a4) UI paquosep ale seinpacoid

@se4L (001-9 ‘0€-O ‘OL-D '€-9 40 snid-d) (€ eunpadoig) UOHeNIYIeEIP pue “(7 einpedcid) UOHEBNyLNUSd-OJO]W ‘(| eiNpsD01q)

uoneBnyjuac :sajdwes aun (9, 0Z-) UeZO4J Y-~Z pue Ysa) Bulsn seunpadoid uoNesjJUBDUOD € JO JUaUSSasse AJeUT

ld *b “Bia

s}INsel ajqeadAL
orc waosg | | 9yz :wazosg} | 9/1 :uazorg UI suazo1g UI “uaro1y
9/9 “User ore susosg| | oyp ysarg| | 970 :uozorg 9/0 :uaz01g 9/0 :uazory Ue :Usany UE Usa
my mz my wy my wy ms ms
oneBnyuIue0-OD IW, 001-9 06-9 (igs) fe) snd-D woneSnginuaD
[ I I J
STIVIN9 SaTVNZ

SATdWVS ANTEN



RESULTS

Assessment of Sample Concentration Techniques

Typing results for DQA1 and PM loci suggested
that sample purification by dialfiltration (Procedure
3), using Centriplus® or Centricon®, did not im-
prove the typeable frequency relative to other con-
centration methods (Fig.1). The typeable blots
obtained from the dialyzed concentrates showed less
intense color reaction than the others, which indi-
cated low amplification efficiency or low concentra-
tion of the DNA templates. Therefore, centrifugation
(Procedure 1) and micro-centrifugation (Procedure
2) concentration methods were employed in the
subsequent experiments.

Comparison of Extraction Methods

As exhibited in Fig.2, the Chelex® resin extracts
and organic extracts yielded comparable positive typ-
ing rates. However, signals from organic extracts
were less intense than that from the resin extracts.
Thus, the Chelex® extraction method was selected
for further studies. This method is also relatively less
cumbersome and less time-consuming.

Amount of Isolated DNA

As is evident from Table 1, the amount of isolated
DNA was significantly higher in female than male
samples. Also, the DNA amount was slightly higher in
the extracts from concentrates obtained by centrifuga-
tion (Procedure 1) than by micro-centrifugation

Table 1. The amount of quantifiable DNA in 10 ul of the extracts obtained from concentrates? of male and
female urine samples at room temperature (RT) and -20°C over a storage period of 7 days.

Male (n = 12) Female (n = 13)
Sample
5 ml ml 5 ml ml
Fresh 83 %? 67 % 100 % 100 %
Tes! (0.20-3.00 ng) —(0.25-3.00 ng) (2.00->10.00 ng) (0.70->10.00 ng)
RT (24h) 67% 33 % 92% 100 %
(0.20-3.00ng) —(0.20-2.00 ng) (0.60->10.00 ng) _(0.60->10.00 ng)
42% 33% 100 % 100 %
RT@Bh) —0.15-1.20ng) —(0.15-0.50 ng) (0.30->10.00 ng) (0.50->10.00 ng)
RE-(7 days) 33% 17% 85% 92%
YS) @.20-0.50 ng) (0.20-0.25 ng) (0.40->10.00 ng) _(0.15-10.00 ng)
17% 8% 1% 69 %
20°C 24h) 30-2.00ng) (0.15 ng) (0.15-9.00ng) —_(0.20-9.00 ng)
5 8% 8% 62 % 54%
-20° C (48 h) (0.25 ng) (0.15 ng) (0.15-2.40ng) —_ (0.18-4.00 ng)
5 25% 8% 62% 69 %
“20°C (7 days) 20 ng) (0.25 ng) (0.20-1.25ng) —_(0.15-2.00 ng)

* The 2 concentration procedures employed were centrifugation (Procedure 1; 5 ml) and micro-
centrifugation (Procedure 2;1 ml). Both procedures are described in the text.
Percentage of samples in which DNA was found to be present within the range given in the parentheses.


DOT/FAA/AM-99/14

Office of Aviation Medicine
Washington, D.C. 20591

Urinary Genotyping for DQA1 and PM
Loci Using PCR-Based Amplification:
Effects of Volume, Storage Temperature,

Preservatives, and Aging on DNA
Extraction and Typing

Nicole T. Vu
Arvind K. Chaturvedi
Dennis V. Canfield

Civil Aeromedical Institute
Federal Aviation Administration
Oklahoma City, Oklahoma 73125

April 1999

Final Report

This document is available to the public
through the National Technical Information
Service, Springfield, Virginia 22161.

Q

U.S. Department
of Transportation

Federal Aviation
Administration


1205 (a) (b)
10 4

80 4

FREQUENCY (%)

20 T T T T 1 T T

STORAGE TIME (DAY)

—2— -20°C (female %) —e— RT (male %)
—o— .20°C (male %) = RT (female %)
= -20°C (total %) + RT (total %)

Fig. 4. The typeable frequency for DQA1 (a) and PM (b) markers using female
and male urine samples that had been maintained at RT and -20°C for a period
of 7 days. (The volume of the urine samples was 1 mL).

120 (a) (b)
100
= wo
Ms
a
2 60
g
&
40
20
r 1 r r 1 r r r T 1
0 2 4 6 8 0 2 4 6 8
STORAGE TIME (DAY)
—o— .20°C (male %) —e— RT (male %)
—2— 20°C (female %) —a— RT (female %)
= .20°C (total %) —4— RT (total %)

Fig. 5. The typeable frequency for DQA1 (a) and PM (b) markers using
female and male urine samples that had been maintained at RT and -20°C
for a period of 7 days. (The volume of the urine samples was 5 mL).


Table 3. The typeable rates for PM using Chelex® extracts from urine samples subjected to different
storage conditions over time.

Sample* Male (n = 12) Female (n = 10)
Sml Im Sml 1 ml
Fresh 100% 100% 100% 100%
RT (24h) 100% 92% 100% 100%
RT (48 h) 83% 67% 100% 100%
RT (7 days) 58% 42% 100% 100%
-20°C (24 h) 25% 33% 100% 100%
-20°C (48 h) 25% 33% 100% 100%
-20°C (7 days) 25% 25% 100% 100%

* Samples were concentrated by centrifugation (Procedure 1; 5 ml) and micro-centrifugation
(Procedure 2; 1 ml); RT = room temperature.


Effects of Sample Volume, Storage Temperature,
and Aging

To study the effects of these variables on urinary
DNA extraction and typing efficiency, urine speci-
mens from 25 institute volunteers (12 males and 13
females) were used. All specimens were processed
immediately, and samples were aliquoted fresh prior
to the application of treatment conditions as previ-
ously described. Samples were exposed to either room
temperature (RT) or -20°C for different time inter-
vals (fresh, 1 day, 2 days, and 7 days). Based on the
initial findings, all samples were concentrated by
centrifugation (Procedure 1) or micro-centrifuga-
tion (Procedure 2) and DNA from the concentrates
was extracted by the Chelex® method.

Effects of Preservatives

To the 6 male urine samples, sodium azide (0.25%;
w/v) or sodium fluoride (1%; w/v) was added (Fig.
3). At these concentrations, both chemicals act as
preservatives. Urine specimens were stored at 4°C
and -20°C up to 30 days. After the sample concentra-
tion by micro-centrifugation (Procedure 2) and the
DNA isolation by Chelex®, DNA samples were
amplified and typed.

DNA Quantitation

The amount of DNA in the obtained extracts was
determined using QuantiblotTM Human DNA Quan-
titation Kit. Each 10-pl extract was processed

following the recommendations of the manufacturer
(11). Chemiluminescent signals were detected on
HyperfilmTM-ECL (Amersham Life Science Inc., Ar-
lington Heights, IL) after 30 min exposure and devel-
opment using a QX-70 medical film processor (Konica
Medical Corporation, Wayne, NJ). The intensity of
the sample signals was visually compared with that of
the DNA standards processed simultaneously. This
method selectively determines human DNA semi-
quantitatively in the range of 0.15 to 10 ng. Based on the
DNA amounts, appropriate volumes of the extracts
were utilized in the subsequent DNA amplification.

DNA Amplification and Typing

In vitro DNA replication by Taq-polymerase was
carried out according to the manufacturer’s direc-
tions for DQAI and PM amplification (12). Target
regions of 6 genetic loci were amplified from
approximately 10 ng of the extracted DNA in a
thermal cycler (GeneAmp PCR System 9600, Perkin
Elmer Corporation, Foster City, CA) in 32 cycles. OF
the 6 loci, 1 locus was human leukocyte antigen
(HLA) DQAI. The remaining loci were PM: low
density lipoprotein receptor (LDLR), glycophorin A
(GYPA), hemoglobin G gammaglobin (HBGG),
D7S8 (locus on chromosome 7 linked with cystic
fibrosis), and group specific component (GC). All
amplified samples were kept at -70°C until typed.
Hybridization and detection of the amplified DNA
were carried out as per manufacturer’s recommenda-
tions (12).

Urine samples

(6 males)
J
[ ]
1% NaF 0.25% NaN3
Fresh: 0/6 Frozen Fresh: 6/6 Frozen
Refrigerated 7 days: 0/6 Refri., 7 days: 6/6 7 days: 3/6
7 days: 0/6 20 days: 6/6 20 days: 3/6
30 days: 1/6 30 days: 1/6

Typeable results

Fig. 3. The typeable results of fresh, refrigerated, and frozen urine samples over a storage period
of 30 days in the presence of sodium fluoride (NaF) or sodium azide (NaN). Details are given in
the text. The typeable results reflect the number of samples successfully typed out of the total
number of samples tested under a particular category.




Technical Report Documentation Page

1. Report No. 2. Government Accession No, 3. Recipient's Catalog No.
DOT/FAA/AM-99/14.

4 Title and Subtitle 5. Report Date

Urinary Genotyping for DQA1 and PM Loci Using PCR-Based April 1999

Amplification: Effects of Sample Volume, Storage Temperature, 6. Performing Organization Code
Preservatives, and Aging on DNA Extraction and Typing

7. Author(s) 8, Performing Organization Report No.

Vu, N.T., Chaturvedi, A.K., and Canfield, D.V.

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS)

FAA Civil Aeromedical Institute

P.O. Box 25082 11, Contract or Grant No.
Oklahoma City, OK_73125

12, Sponsoring Agency name and Address 13. Type of Report and Period Covered
Office of Aviation Medicine

Federal Aviation Administration
800 Independence Ave., S.W.

Washington, D.C. 20591 14, Sponsoring Agency Code

15. Supplemental Notes
This work was accomplished under the approved tasks AM-B-97-TOX-202 and AM-B-98-TOX-202.

16. Abstract

Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug
testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and
socioeconomic consequences of a positive drug test. Methods for individualization of biosamples have reached a
new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful
characterization of the urine specimens depends on the quantity and quality of DNA present in the samples.
Therefore, the present study investigated the influence of storage conditions, sample volumes, concentration modes,
extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of
PCR-based urinary genotyping for DQA1 and PM loci. Urine specimens from male and female volunteers were
divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by
dialfiltration, using 3,000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate
compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin
method gave comparable typing results. Larger sample volume yielded higher amount of DNA, but the typing rates
were not affected for sample volumes between 1 to 5 ml. The quantifiable amounts of DNA present were found to
be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under
both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage
samples produced significantly higher success rates than that of frozen samples, while there was only marginal
difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of
DQA1+PM genotype was achieved for all sampling of fresh urine, independent of gender, starting sample volume,
or concentration method. Preservation by 0.25% sodium azide was acceptable for sample storage at 4°C during a
period of 30 days. For longer storage duration, freezing at -70°C may be more appropriate. Thus, the applicability
of the DQA1+PM typing was clearly demonstrated for individualization of the urine samples.

17. Key Words 18. Distribution Statement

Polymerase Chain Reaction, DNA Typing, Genotyping, Document is available to the public through the

Genetic Markers, DQA1 and PM Loci, Urinalysis, National Technical Information Service

Aviation Accident Investigation Springfield, Virginia 22161

Security Classif. (of this report) 20. Security Classif, (of this page) 21. No. of Pages 22. Price
Unclassified Unclassified 13

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized



NOTICE

This document is disseminated under the sponsorship of
the U.S. Department of Transportation in the interest of
information exchange. The United States Government
assumes no liability for the contents or use thereof.


DISCUSSION

Because the nuclear material in human urine is
scanty and PCR inhibitors are likely to be present
(5,7), it was hypothesized that the concentration of
samples, as well as elimination of the inhibitors,
would improve the DNA amplification efficiency.
However, both centrifugation (Procedure 1) and
micro-centrifugation (Procedure 2) concentration
methods were found to be more suitable for the DNA
recovery than the dialfiltration method (Procedure
3). Apparently, the poor DNA recovery by
dialfiltration could have resulted from the adsorp-
tion of cells and DNA to the filtration apparatus and
from the extensive degradation of DNA after freeze-
thaw that prevented DNA retention. Alternatively,
extractable components from the membrane could
have interfered with the amplification efficiency. Of
the 3 methods evaluated, micro-centrifugation (Pro-
cedure 2) was the method of choice since it produced
more consistent results and required a lesser amount
of sample. The suitability ofthe Chelex® resin method
over the organic method for the DNA extraction
from the concentrates was also reflected in a previous
report on the DQAI+PM typing of postmortem
forensic tissues and body fluids (13).

Since the epithelial cells from genito-urinary tract
and the leukocytes comprise the primary sources of
urinary DNA (6), the quantity of DNA varied con-
siderably depending on the physiological state of the
subjects—for example, larger amounts of DNA were
recovered from female than male urine samples. The
present study clearly demonstrated that the
DQAI1+PM genotype could be successfully assigned
for fresh urine, using as little as a 1-ml sample,
independent of gender and concentration method.
As freezing affected DNA recovery and typing nega-
tively, preservation by freezing is not beneficial for
sample storage. It appears that the thawing process
damaged DNA, probably through a cell lysis mecha-
nism that would expose naked DNA to environmen-
tal insults. Furthermore, the time-dependent effect

of freezing on DNA recovery could have resulted
from the routine freeze-thaw cycles of the freezer.
Thus, storage at -70°C would probably be more
appropriate if freezing is required. For frozen urine,
the sample may be processed by micro-centrifugation
(Procedure 2) in multiple aliquots to increase the
DNA yield. Alternatively, the DNA extraction proce-
dure described by Linfert et al. (8) may be employed,
as it has been shown to produce 71% typing success.
However, the procedure requires 10- to 30-ml urine
samples.

The storage duration for urine samples could be
further extended by using the chemical preservative
sodium azide (0.25%), as storage at 4°C was accept-
able for up to 30 days. For longer storage periods,
freezing at -70°C is recommended to avoid the del-
eterious effects of the freeze-thaw cycles of the freezer
at higher storage temperature (> -20°C). Because
sodium fluoride at 1% might have interfered with the
PCR, either washing of the urinary residues with
saline prior to the extraction or alcohol precipitation
of the DNA from the Chelex® extracts did not
improve the typeable frequency. Whereas high salt
concentration (1% sodium fluoride) in samples may
inhibit the PCR, excessive washing could cause addi-
tional loss of nucleated cells or further DNA damage.
At present, the mechanism of sodium fluoride inter-
ference is not clear, but sodium azide may be em-
ployed as a preservative for prolonged storage at 4°C.
Since sodium azide and sodium fluoride are com-
monly used as preservatives (5) in urine samples for
forensic toxicology/drug testing, the DNA typing
can be successfully achieved by knowing the chemical
nature of the preservative used in a urine sample.

Overall, this study demonstrated that DNA
typing could be effectively applied in resolving the
integrity of urine samples submitted for drug analy-
sis. It has applicability not only in postmortem
forensic toxicology but also in work place forensic
drug testing. However, if the sample is kept frozen
for along duration, then the technique may not be
as effective.