Blood and urine samples from racehorses are routinely used in screening for illegal drugs. Typing DNA to identify the origin of a sample is currently not an integrated component of forensic testing in the horse racing industry. A microsatellite or STR is one of the polymorphic DNA markers that reflect great variation among individuals and is widely used for identification of a sample via PCR amplification.1 A horse can be identified by use of 12 dinucleotide STR loci recommended by the International Society for Animal Genetics.2 However, it is critical that amplifiable DNA be recovered from a sample to enable acquisition of a complete DNA profile. In Pennsylvania, it takes 2 to 3 days for samples to reach the laboratory and 3 to 7 days before drug screening is completed. Samples are refrigerated until drug testing is completed, and some samples could remain refrigerated for several weeks before genotypic testing. To our knowledge, successful PCR-based DNA typing of samples stored for a long time has not been reported. Studies3,4 have been conducted on a limited number of stored samples of human origin. After drug testing is completed, samples may be refrigerated, frozen, or subjected to cycles of freezing and thawing.5,6 Thus, the study reported here was conducted to determine the effects of repeated freeze-thaw cycles and long-term refrigeration on DNA quantity and quality and viability for genotyping.
Deoxyribonucleic acid content in urine varies, and insufficient quantities of DNA pose the greatest challenge to the use of urine in DNA testing.6–8 Amplifiable DNA in urine originates from a limited number of nucleated cells.4 Centrifugation has been used to recover cell sediment for DNA extraction, whereas urine supernatant is usually discarded.8 However, cell-free DNA has been detected in human plasma and urine.9,10 Cell death results in the release of naked DNA. A portion of DNA from dying cells escapes intracellular degradation and phagocytosis and circulates in the blood stream.9 Some DNA from dying and dead cells passes through the barriers in the kidneys and is excreted in the urine.10 Low–molecular-weight DNA in supernatant of human urine can be detected by use of PCR techniques.10 To our knowledge, high–molecular-weight DNA in supernatants of human or horse urine has not been reported.
In several studies,5,6,11 samples that had positive results when tested for illegal drugs were genotyped for verification of source. However, whether the drugs in the test samples had an adverse effect on PCR amplification of microsatellite loci was unclear. Contaminants in isolated DNA may contain PCR inhibitors and thus inhibit amplification of STRs.12,13 In the study reported here, our objective was to determine whether supernatant of urine obtained from racehorses contained amplifiable DNA. We also wanted to identify factors that influence DNA recovery and amplification of STR loci as well as determine whether drugs illegally used in racehorses would inhibit PCR amplification. Finally, we wanted to validate an STR method to test DNA quality in a novel genetic analysis system.
Materials and Methods
Sample collection, storage, and transportation—Samples were obtained from 261 racehorses at a racetrack or at New Bolton Center of the University of Pennsylvania School of Veterinary Medicine. Samples were obtained in accordance with institutional animal care and use committee guidelines. Blood samples were collected in 10-mL glass tubesa containing potassium oxalate (20 mg) and sodium fluoride (25 mg) to prevent coagulation and inhibit plasma cholinesterases, respectively, and then stored at 4°C. Urine samples were collected in a 480-mL wide-mouth polypropylene jar with screw capb and then frozen; the next day, they were transported at ambient temperature to our laboratory. Blood tubes, urine containers, and sample boxes were labeled, sealed, and locked prior to shipment to the laboratory for forensic analysis.
Genomic DNA isolation—Deoxyribonucleic acid from fresh whole blood or plasma was isolated by use of a commercially available kit.7,c Deoxyribonucleic acid from stored blood or plasma was isolated by modification of the kit, whereas DNA from urine, urine sediment, or urine supernatant was isolated by use of precipitation methods.14 Deoxyribonucleic acid was isolated in duplicate for each sample.
Isolation of DNA from stored blood or plasma samples—Each tube of whole blood was inverted 5 times to yield a homogenous sample. An aliquot (0.3 mL) of whole blood or plasma was obtained and lysed by the addition of 0.9 mL of lysis buffer.d Nucleated cells (eg, WBCs) were centrifuged at 16,000 X g for 20 seconds. The cell pellet was resuspended and treated by the addition of 0.3 mL of cell lysis solutione and 2 μL of proteinase K solution (20 mg/mL). Cell lysis was performed at 55°C for 1 to 15 hours. Cell lysate was incubated with 1.5 μL of RNasef at 37°C for 15 minutes. The cell lysate was then thoroughly vortexed, incubated with 0.1 mL of protein precipitation solutiong on crushed ice for 5 minutes, and centrifuged at 16,000 X g for 5 minutes. The resulting supernatant was mixed with 0.3 mL of 100% isopropanol and incubated for 10 minutes at 25°C. The resultant DNA pellet was washed with 0.3 mL of 70% ethanol and resuspended in 0.3 mL of DNA hydration solution.h The DNA extraction procedure was repeated, and multiple centrifugations were conducted during the protein precipitation to deplete the protein pellet. Final volume of the DNA isolate was 50 μL.
Isolation of DNA from urine samples—Urine was directly digested by incubation for 15 hours at 55°C with one-tenth volumes of 10X digestion buffer (100mM Tris [pH, 8.0], 250mM EDTA [pH, 8.0], and 5% SDS) and proteinase K solution (100 μg/mL). Digested urine samples were placed on ice for 5 minutes, and then 7.5M ammonium acetate or 5M sodium chloride was added at one-third volume of the digestion mixture. The precipitate was incubated on ice for 15 minutes and then centrifuged at 3,000 X g for 30 minutes to deplete protein contaminants. The supernatant was mixed with an equal volume of 100% isopropanol containing glycogen (40 μg/mL). The DNA precipitate was obtained by incubation at 25°C for 10 minutes to 15 hours. The DNA precipitate was centrifuged at 3,000 X g for 30 minutes and dried under a stream of air for 10 minutes. The resultant DNA pellet was dissolved in 0.5 mL of Tris-EDTA buffer (10mM Tris HCl [pH, 8.0] and 1mM EDTA [pH, 8.0]) and subjected to a second purification. Multiple centrifugations were conducted during the second salt precipitation to deplete the pellet. Final volume of the DNA isolate was 20 μL.
Isolation of DNA from urine supernatant and urine sediment—Urine was mixed by vortex. An aliquot was then obtained and centrifuged (2,000 X g) for 10 minutes. Deoxyribonucleic acid was then isolated from the resulting supernatant. Each urine pellet was digested by incubation for 15 hours at 55°C with a half volume of digestion buffer (10mM Tris [pH, 8.0], 25mM EDTA [pH, 8.0], 0.5% SDS, and proteinase K [100 μg/mL]).
Quantification of isolated genomic DNA—The DNA concentration was estimated by use of a photometeri on the basis of the value for the optical density measured at 260 nm subtracted from the optical density measured at 320 nm; it was verified by ethidium bromide staining of 0.8% agarose gels by use of λ DNA-HindIII fragmentsj as a DNA size standard. Deoxyribonucleic acid bands were developed and photographed under UV lightk by use of a gamma camera.l The band containing high–molecular-weight DNA was quantified against the intensity of the band at 23,130 bp in the DNA ladder, with background subtraction.m Deoxyribonucleic acid was quantified 2 times, and a mean value was calculated.
Equine microsatellite loci and PCR primers—Five equine nuclear microsatellite loci (VHL20,15 HTG4,16 AHT4,17 HMS6, and HMS718) were chosen for evaluation of DNA quality (Appendix). Primers were used to amplify the entire dinucleotide repeat sequence of each microsatellite locus, as described elsewhere,15–18 with minor modification of the reverse primer for AHT4. Primer sequences were tested against formations of primer-dimer and hairpin.19,n The 5 equine STR loci were coamplified in a single tube. Polymerase chain reaction components and cycling conditions were optimized for DNA template, magnesium concentration, primer concentration, annealing temperature, and cycling number. Primers were assessed to determine the minimal concentration for an optimized balance of amplicon yield and resolution of allelic peaks among microsatellite loci.
PCR coamplification of 5 equine STR loci—The DNA isolates were evaluated via PCR amplification of the 5 equine STRs. A Taq DNA polymerase mixtureo was used to amplify microsatellite loci in a single tube on a thermocycler.p Polymerase chain reaction conditions for amplification of the equine STR loci were optimized and validated. Five STR markers were coamplified in a reaction of 15 μL by use of 2% to 5% DNA isolates (approx 20 to 50 ng) and primers. Polymerase chain reaction amplification was performed with 2.4mM MgCl2. Cycling conditions were 95°C for 15 minutes; 30 cycles of 94°C for 30 seconds, 54.5°C for 30 seconds, and 72°C for 1 minute; and 72°C for 10 minutes.
Determination of equine STR fragment size—Products of PCR amplification (7-μL aliquots) were electrophoresed on a 1.2% agarose gel to allow evaluation of amplicons. The size of each amplified STR fragment was analyzed on an automated system.q Briefly, the amplified STR fragments were labeled with 1 of 3 fluorescent dyesr–t (black, green, or blue). Amplification products (4 μL) were mixed with 0.6 μL of DNA size standardu in 25 μL of deionized formamide, and the mixture was loaded onto 96-well microplates. The DNA size standard contained fragments of 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, and 420 nucleotides labeled with red fluorescent dye.v Short tandem repeat fragments were separated by use of capillary electrophoresis, and fragment size was automatically determined against a DNA size standard by commercially available software.w Capillary electrophoresis was conducted at a capillary temperature of 50°C; sample was denatured at 90°C for 120 seconds, electrokinetically injected at 2.0 kV for 30 seconds, and separated at 6.0 kV for 25 minutes.
To identify an allele, the highest peak was considered to be an allele when it was not plus A (ie, a peak that is 1 nucleotide larger than the allelic peak),20 and the second highest peak was also considered an allele when it was at least 15% of the height of the allelic peak and was neither a stutter (ie, a peak that is 2 nucleotides or a multiple of dinucleotides smaller than the allelic peak) nor a plus A peak. A peak was automatically identified and assigned as a true allele at a locus. The allele was designated as the number of dinucleotide repeat units plus 0.1 when there was a partial repeat (ie, 1 nucleotide).21
Influence of freeze-thaw cycles on DNA quantity and quality—Blood or urine samples were divided into 2 equal portions; 1 was refrigerated and the other was frozen at −70°C. One week later, frozen samples were thawed at 25°C, and DNA was immediately isolated. The thawed samples were refrozen for another week, after which they were thawed again for DNA isolation. The samples were subjected to a total of 6 repeated freeze-thaw cycles, and the period during which samples were frozen for each cycle was 7 days. Refrigerated samples served as control samples.
Effect of select drugs on detection of microsatellite loci—Ten drugs were detected in 1 urine sample, and 4 of them were detected in plasma from the same racehorse. Six drugs were detected in 1 urine sample, and 5 drugs were detected in another urine sample. To investigate whether failure of STR typing was attributable to the presence of these drugs, an aliquot of 0.3 mL of blood or 1 mL of urine was mixed with 10 drugs (acetaminophen, caffeine, chlorpheniramine, dextromethorphan, dextrophan, ephedrine, nicotine, phenylpropanolamine, pseudoephedrine, and sertraline), 6 drugs (caffeine, cotinine, dextromethorphan, dextrophan, guaifenesin, and sertraline), or 5 drugs (chlorpheniramine, cotinine, dextromethorphan, dextrophan, and sertraline); each drug was at a concentration of 0 or 1 μg/mL. In another experiment, blood or urine was mixed with doxorubicin hydrochloride alone at concentrations of 0, 0.1, 0.5, or 1 μg/mL. The drugs were incubated with blood or urine samples for 6 hours at 25°C and overnight at 4°C with continuous shaking. Drugs were prepared in methanol, and a negative control sample with methanol was similarly incubated as in the drug-challenged samples. Deoxyribonucleic acid was isolated after overnight incubation.
Centrifugation—To deplete contaminants, centrifugation was conducted during salt precipitation by use of 30 samples of blood or urine. Aliquots of 0.3 mL of blood or 1 mL of urine from each refrigerated sample (after refrigeration for 7 days) were placed in 1.5-mL microcentrifuge tubes. Each tube was labeled and assigned to 1 DNA isolate on the basis of the number of centrifugations. For example, DNA isolate No. 1 was subjected to 1 centrifugation, whereas DNA isolate No. 2 was subjected to 2 centrifugations. Total number of DNA isolates was 5 for blood and 6 for urine.
Statistical analysis—Data on DNA quantity were acquired from each sample in serial treatments or during a time course. All data were analyzed by use of a 2-way ANOVA.x Significance was defined as values of P < 0.05.
Results
Validation of STR coamplification—Maximum amplification of 5 STR loci was obtained with 20 to 50 ng of DNA template (Figure 1). Polymerase chain reaction amplification of 5 equine STR loci was optimized with 2.4mM MgCl2. Results from gradient PCR assays indicated that the annealing temperature of 54.5°C yielded optimal amplification with high resolution and specificity of all 5 STR loci. Thirty cycles yielded reliable results as determined on the basis of peak resolution, number of stutter peaks, signal intensity (peak height), and background signals. It was determined that a minimal primer concentration for each microsatellite locus yielded a reproducible STR profile (Appendix).
Electropherograms of PCR amplification products of 5 equine microsatellite loci (VHL20, HTG4, AHT4, HMS6, and HMS7) for 20 ng of DNA (A) and 2.4mM MgCl2 (B), with annealing temperature of 54.5°C (C) and 30 cycles (D). Electropherograms were obtained from PCR products (4 μL) of DNA amplified from blood or urine. The y-axis provides peak intensity as an indicator of amplicon quantity, whereas the x-axis provides length of each amplified STR locus. Each STR locus was labeled with 1 of 3 fluorescent dyes, and the allele peak is indicated. Notice that values on the x- and y-axes differ among portions of the figure. RFU = Relative fluorescent unit. nt = Nucleotide.
Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.648
Amplifiable DNA in equine urine supernatant—Deoxyribonucleic acid isolates (n = 1,120) from urine supernatants of 35 horses were evaluated and compared with DNA isolates from urine sediment. Similar to the results for urine sediment, DNA was recovered from urine supernatant and was amplifiable in the 5-STR PCR (Figure 2). Approximately 36% and 39% of total DNA was isolated from urine supernatant when subjected to 1 and 2 freeze-thaw cycles, respectively, which decreased to 28% in 4 freeze-thaw cycles. After 2 freeze-thaw cycles, the yield of high– molecular-weight DNA was significantly reduced in urine supernatant (decreased by 36% [P < 0.05]) and in urine sediment (decreased by 43% [P < 0.001]). Deoxyribonucleic acid in supernatant and sediment of urine after 2 freeze-thaw cycles yielded complete DNA profiles and had an identical genotype to that of the nonfrozen control urine. Following 4 freeze-thaw cycles, there was significant (P < 0.001) reduction of DNA content in urine supernatant and sediment (86% and 81%, respectively). An insufficient quantity of DNA was recovered from urine supernatant; thus, the success of STR typing was only 22% for urine supernatant and 41% for urine sediment. This result indicated that repeated freeze-thaw cycles induced cellular degeneration and DNA degradation.
Results for DNA isolated from urine supernatant and sediment. A—Isolated DNA and the corresponding PCR products of 5 microsatellite loci on 0.8% (DNA; top row) or 1.2% (PCR products; bottom row) agarose gels after samples were subjected to 2 freeze-thaw cycles. Ten percent of total DNA isolate or 7 μL of the corresponding PCR products with 200 ng of a L DNA-HindIII fragment ladder or 100 ng of a 100-bp DNA ladder was loaded on each gel. B—Mean and SD DNA yield from urine sediment and supernatant (n = 35 urine samples and 1,120 DNA isolates). The DNA was quantified in sediment (white bars) and supernatant (gray bars) after samples were subjected to 4 freeze-thaw cycles (1 to 4). Control urine samples (cold) were refrigerated for the same duration of time as the frozenthawed samples. C and D—Electropherograms of 5 microsatellite loci from DNA isolated from urine supernatant (C) and urine sediment (D) after samples were subjected to 2 freeze-thaw cycles. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.648
Results obtained from 9 horses indicated that fresh urine supernatant contained amplifiable DNA that accounted for approximately 43% of the total DNA. Urine refrigerated for 35 days also contained amplifiable DNA in urine supernatant of all samples examined. The DNA yield of urine supernatant decreased by 35%, compared with that of fresh urine supernatant.
Effect of sample volume on DNA recovery from equine urine—Detectable and amplifiable DNA was obtained from 1 mL of urine in 174 of 261 samples tested. Increasing the volume to 5 mL for urine samples in which there was no detectable DNA yielded an additional 57 samples with amplifiable DNA. Progressively increasing the volume of sample to 20 and 80 mL improved the yield of DNA for all samples (Figure 3). Genotyping was successful for 67% of samples by use of 1 mL of urine and increased to 100% when 80 mL was used.
Results for DNA and the corresponding PCR products of 5 microsatellite loci on 0.8% (DNA; top row) or 1.2% (PCR products; bottom row) agarose gels for 20% of total DNA for various volumes of 2 urine samples (A) and the electropherogram of 5 STRs isolated from 20 mL of urine sample No. 2 (B). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.648
Recovery of DNA from refrigerated blood, plasma, and urine samples—Quantity of DNA in 0.3 mL of fresh equine plasma was 0.71 to 1.83 μg. The DNA yield significantly (P < 0.001) decreased when blood and urine samples were refrigerated for 35 days (Figure 4). There was a significant reduction in DNA quantity in urine samples, compared with the DNA quantity in blood samples, during the first 7 days of storage. Despite this reduction, complete DNA profiles were acquired from blood and urine samples that had been refrigerated for 35 days.
Effect of duration of refrigeration of samples on DNA recovery. A—DNA isolates (top row) and the corresponding amplified STR fragments (bottom row) on 0.8% and 1.2% agarose gels, respectively, for blood and urine samples refrigerated for various times. B—Mean ± SD DNA content obtained from samples of whole blood (n = 133; left) and urine (139; right) refrigerated for 0 to 5 weeks after collection. There were 1,596 DNA isolates for blood samples and 1,946 DNA isolates for urine samples. C—Electropherograms of 5 STR loci obtained from DNA isolated from samples of blood (left) and urine (right) after samples were refrigerated for 5 weeks. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.648
Effect of repeated freeze-thaw cycles on DNA recovery and genotyping—Repeated freeze-thaw cycles significantly (P < 0.001) decreased DNA yield from equine blood (n = 66) and urine (56) samples and had more effects on DNA recovery from urine than blood (Figure 5). The DNA yield per milliliter of urine was reduced by 57% (from 0.70 to 0.30 μg) and 96% (from 0.70 to 0.03 μg) after 2 and 6 freeze-thaw cycles, respectively. In contrast, DNA yield significantly decreased by only 11% (P < 0.05) after urine was refrigerated for 2 weeks and by 64% (P < 0.001) after it was refrigerated for 6 weeks.
Effect of freeze-thaw cycles on DNA recovery and detection of STR loci. A—DNA isolates (top 2 rows) and the corresponding amplified STR fragments (bottom 2 rows) on 0.8% and 1.2% agarose gels, respectively, for blood and urine samples subjected to 6 freeze-thaw cycles (Freeze) or refrigerated for the same duration of time (Cold). B—Mean and SD DNA quantity recovered from samples of whole blood (n = 66; left) and urine (56; right) after samples were subjected to 6 freeze-thaw cycles (white bars) or were stored in a refrigerator for the same duration (gray bars). There were 1,584 DNA isolates for blood samples and 1,344 DNA isolates for urine samples. C—Electropherograms of 5 STR loci obtained from DNA isolated from samples of whole blood after samples were refrigerated for 5 weeks (left) or subjected to 5 freeze-thaw cycles (right). Notice the loss of peaks for 2 STR loci (arrows). D—Electropherograms of 5 STR loci obtained from DNA isolated from samples of urine after samples were refrigerated for 5 weeks (left) or subjected to 5 freeze-thaw cycles (right). Notice the loss of peaks for 3 STR loci (arrows). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.648
The DNA yield per 0.3 mL of whole blood was significantly (P = 0.01) reduced from 4.04 to 3.42 μg (15%) and to 2.18 μg (46%) after 2 and 6 freeze-thaw cycles, respectively (Figure 5). In comparison, DNA yield decreased significantly by 8% (P < 0.05) and 33% (P < 0.001) when blood samples were refrigerated for 2 and 6 weeks, respectively. Amplification of equine microsatellite loci and the peak height in both blood and urine samples decreased as the number of freezethaw cycles increased. Consequently, STR loci were poorly amplified and rarely detectable. In a blood sample, for instance, VHL20 (14/17) and HTG4 (30/30) were not detected, whereas AHT4 (28/33) and HMS6 (20/20) were detected with altered allele sizes after 5 freeze-thaw cycles. Freezethaw cycles had drastic effects on detection of microsatellite loci in urine samples. For example, in a urine sample, VHL20 (17/18) was inaccurately genotyped and 3 markers (AHT4, HMS6, and HMS7) were not detectable after 5 freezethaw cycles. Successful genotyping decreased to 92% in blood samples and 44% in urine samples after 3 freeze-thaw cycles, but after 6 freeze-thaw cycles, it decreased to 67% for blood samples and only 10% for urine samples.
Effect of selected drugs on STR typing—Drugs in urine or plasma samples (n = 20 each) did not affect DNA yield or genotype profiles (Figure 6). Doxorubicin, which intercalates DNA base pairs, was used to determine whether the drug affected DNA isolation and PCR amplification of STR loci. Various concentrations of doxorubicin (0.1, 0.5, and 1 μg/mL) in equine blood or urine samples (n = 20 each) did not induce any inhibitory effect.
Results for DNA and the corresponding PCR products of 5 microsatellite loci on 0.8% (DNA; top row) or 1.2% (PCR products; bottom row) agarose gels for urine samples to which various drugs were added (A) and the electropherogram of 5 STRs isolated from a urine sample without drugs (B) and with 10 drugs (C). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.648
Effects of impurities in DNA isolates on incomplete STR profiles—To determine whether failure of STR detection resulted from impurities in DNA isolates, multiple centrifugations to deplete protein contaminants were performed. The DNA isolated from blood samples after 4 centrifugations yielded complete DNA profiles, but DNA isolated after 3 centrifugations did not contain the AHT4 locus (Figure 7). Similarly, DNA isolated from urine samples after 5 centrifugations yielded complete DNA profiles, but DNA isolated after 4 centrifugations did not contain 2 loci (HMS6 and HMS7). These results indicated that for a centrifugation-based protein-depletion procedure, a single centrifugation was insufficient for depletion of contaminants (such as PCR inhibitors), especially when the samples were not fresh. Success for STR typing was 59.6% for blood samples and 52.8% for urine samples after a single centrifugation, but it increased to 100% after 5 centrifugations when the salt-precipitation method was used.
Effect of contaminants in DNA isolates on PCR-based detection of STRs. A—Results for DNA and the corresponding PCR products of 5 microsatellite loci on 0.8% (DNA; top row) or 1.2% (PCR products; bottom row) agarose gels for samples of whole blood and urine after samples were centrifuged a various number of times. Notice that urine samples were centrifuged 6 times, whereas whole blood samples were only centrifuged 5 times. B—Electropherograms of 5 STRs isolated from samples of whole blood after the samples were centrifuged 3 times (left) or 4 times (right). C—Electropherograms of 5 STRs isolated from samples of urine after the samples were centrifuged 4 times (left) or 5 times (right). See Figures 1 and 5 for remainder of key.
Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.648
Discussion
Optimal conditions of PCR amplification are critical because inappropriate PCR conditions may lead to misinterpretation of genotyping results.22,23 Optimal methods for the 5 equine STRs were used in 8,981 DNA isolates with high reproducibility and reliability. Excess free magnesium may increase nonspecific amplification24 and induce drop-out of an allele, which results in a homozygote of a locus.25 Insufficient DNA template may yield nonspecific PCR products with similar fragment sizes that lead to misinterpretation of the results26 and may also generate larger amplicons than expected.22 An insufficient quantity of DNA is often related to allele drop-out27 or to inconclusive results.28 Excessive amounts of primers or their polymer forms, such as primer-dimer,27 may affect migration of the target fragment during capillary electrophoresis. Thus, minimizing primer concentrations is critical in determining the size of the unknown DNA fragment by use of multiplex PCR assays. A number of primer pairs routinely used for parentage testing or individual identification were designed before improved tools were available. Consequently, old primers often have extremely different melting temperatures, which results in poor primertemplate binding and thus poor amplification of the target locus. Primers derived from repeat sequences, such as long interspersed nuclear elements or long-term repeats, are likely to increase nonspecific amplification, thus complicating the identification of true alleles.
In contrast to DNA content in human urine, DNA content in equine urine has not been extensively studied. Urine supernatant typically is discarded because DNA of human urine is found in the urinary sediment, which contains nucleated cells (such as epithelial cells).4 In this study, high–molecular-weight DNA was isolated from equine urine supernatant and successfully amplified in a multiplex PCR assay. Deoxyribonucleic acid in urinary supernatant was comparable to DNA in urinary sediment. To our knowledge, this is the first evidence that supernatant of equine urine contains amplifiable DNA that could improve genotyping of urine samples and would thus support the use of urine as an alternative source of sample for forensic identity testing. A small DNA fragment without other cellular components is excreted in urine during disease states in humans.10 However, it is not clear whether DNA is excreted in urine of a healthy subject or the duration that circulated or naked DNA exists in urine. Recovery of amplifiable nuclear DNA in ultrafiltered urine supernatant was not successful, possibly because large molecules such as DNA remain unfiltered.26 Recovery of DNA from urine sediment and supernatant was reduced after freeze-thaw cycles, but reduction of recovery from supernatant was relatively lower with 2 freeze-thaw cycles, which indicated that a substantial number of nucleated cells may have been lysed and released nuclear DNA into the matrix after it was thawed. Multiplex STR markers have been successfully genotyped from 0.1 mL of total fresh or frozen human urine,29 which agrees with our results in that total urine would be considered for DNA isolation when low DNA content is expected.
The DNA content in urine samples varies among subjects.8,23 The quantity of DNA extracted from 1 mL of fresh human urine was 1 to 100 ng,28 and that from 50 mL of frozen human urine was 0 to 20 μg.23 In equine urine, quantifiable DNA was also variable and ranged from 0 to 2.4 μg/mL. The difference in DNA content in humans is also related to gender; urine from females contains more DNA (14 to 200 ng/mL) than urine from males (4 to 60 ng/mL) because of additional squamous cells derived from the vaginal tract.8 An aliquot of 0.1 mL of fresh human urine was sufficient for amplification of a single locus or a few loci in a multiplex assay.3,29 However, allele drop-out or problems with allele sizing have been reported26 when 0.1 mL of urine was used. Although DNA can be recovered from most human urine samples of 1 mL, the quantity and quality of DNA are sometimes insufficient for multiplex PCR amplification.26 Stored urine samples lose some proportion of amplifiable DNA; thus, large volumes (20 to 100 mL) of stored urine may be required.23 In a study,30 6 of 10 samples yielded successful results for PCR typing when 15 mL of fresh human urine was genotyped, whereas successful typing was achieved for all samples when 50 mL of urine was used.30 The success of genotyping for equine urine was 175 of 261 (67%) for 1 mL of urine that was 3 days old, frozen once, and assessed by use of 5 STR loci. When the samples were obtained from a pool of males, females, and castrated males, most of the samples with unsuccessful results could be attributable to male horses.
Genotype success for urine samples in a study28 was only 40%. This low success rate notwithstanding, urine may yield dependable results when stored properly. Equine blood and urine samples can be refrigerated for at least 35 days without affecting amplification of STR loci. Refrigeration of human urine for up to 35 days caused DNA degradation but did not ruin PCR amplification when the amplified STR sequence was not too long.3 High–molecular-weight DNA can still be recovered from human urine after 180 days of refrigeration, although there is a 10-fold decrease in yield.4 Analysis of results of that study4 revealed that isolated DNA was suitable for PCR amplification of DNA markers and that intact epithelial cells were detectable after storage at 4°C for 7 years. However, a prolonged duration of refrigeration destroys amplification of DNA makers and yields inconclusive genotype results because of degeneration of epithelial cells.4,28
One or 2 freeze-thaw cycles did not disable equine STR genotyping, although DNA yield decreased; however, > 2 freeze-thaw cycles made samples susceptible to failure for STR detection in urine by use of PCR amplification. This observation was supported by the notion that freeze-thaw cycles cause degeneration of nucleated cells and degradation of DNA molecules by releasing hydrolytic enzymes, such as nuclease.8 To our knowledge, this is the first evidence of the adverse effect of repeated freeze-thaw cycles on DNA integrity and effects on DNA amplification. There is no difference in genotyping between fresh urine and urine frozen only once for up to 180 days.4,29 In a study,23 89.3% of the urine samples frozen for 15 to 25 years were successfully genotyped. These results are contrary to those from another study7 in which only 34 of 48 (71%) urine samples frozen for 120 to 180 days were successfully genotyped. A urine sample obtained from a racehorse yielded partial STR profiles after 3 freeze-thaw cycles,5 and it was unclear whether storage conditions caused the failure to detect the other 8 markers. However, in the present study, 2 or 3 freeze-thaw cycles did not prevent successful STR typing in blood samples, and repeated freeze-thaw cycles had fewer deleterious effects on amplifiable DNA in blood samples than in urine samples.
Genotyping of drug-positive urine samples has been described.5,6,11 Human urine samples that had positive results when tested for nandrolone6 or anabolic steroids11 were successfully genotyped by use of mitochondrial DNA markers, but genotyping failed for autosomal STR markers. It is unlikely that the drugs contributed to poor nuclear DNA profiles because the same sample yielded a complete mitochondrial DNA profile. Partial DNA profiles were obtained from a drug-positive urine sample obtained from a racehorse after the urine sample was subjected to 3 freeze-thaw cycles.5 It was not clear whether the partial DNA profile for that horse was caused by poor DNA quality or by the presence of the drug in the sample. Failure to identify the origin of the urine or plasma in the study reported here cannot be attributed to the presence of drugs. However, whether metabolites of the drugs had inhibitory effects is unknown. Doxorubicin as a DNA-intercalating agent did not interfere with DNA amplification, which suggested that DNA intercalation can be overcome by denaturation of DNA molecules.
Although many DNA isolation procedures use ≤ 1 mL of sample,29 some samples (such as urine with low DNA content) require a larger volume for genotyping.23 A salt precipitation method by use of alcohol satisfies such requirements and avoids the use of hazardous organic solvents, such as phenol and isochloroform. We used multiple centrifugations during salt precipitation to increase DNA purity. A single centrifugation was insufficient for depletion of protein contaminants before DNA precipitation, especially when urine or blood samples were refrigerated for > 7 days. A longer period of storage caused degradation of cellular components. A similar DNA isolation procedure without multiple centrifugations was used and yielded DNA with low purity and poor amplification in 34 of 48 (71%) frozen urine samples.7 Contamination of DNA isolates may be attributable to insufficient depletion of PCR inhibitors12,13 during salt precipitation, and use of multiple centrifugations improved genotyping in the present study.
Finally, samples of equine blood and urine can be successfully genotyped when properly stored, although blood has more integrated DNA and is more suitable for STR typing than is urine.28 As a noninvasive method of sample collection, urine has a tremendous advantage over blood. Total urine (sediment and supernatant) is recommended for complete DNA profiling when there is low DNA content.
Abbreviations
| STR | Short tandem repeat |
Kendall Healthcare, Mansfield, Mass.
Qorpak, Bridgeville, Pa.
Puregene genomic DNA purification kit, Gentra, Minneapolis, Minn.
Puregene red blood cell lysis buffer, Gentra, Minneapolis, Minn.
Puregene cell lysis solution, Gentra, Minneapolis, Minn.
Puregene RNase A solution, Gentra, Minneapolis, Minn.
Puregene protein precipitation solution, Gentra, Minneapolis, Minn.
Puregene DNA hydration solution, Gentra, Minneapolis, Minn.
BioPhotometer, Eppendorf, Hamburg, Germany.
TrackIt, Invitrogen, Carlsbad, Calif.
FOTO/Phoresis UV transilluminator, FOTODYNE, Hartland, Wis.
FCR-10 camera, FOTODYNE, Hartland, Wis.
TotalLab TL100, Nonlinear Dynamics, Durham, NC.
Autodimer, NIST, Gaithersburg, Md.
HotStar Taq DNA polymerase 2X master mix, Qiagen, Valencia, Calif.
TC-512 gradient thermocycler, Techne, Burlington, NJ.
CEQ8800 genetic analysis system, Beckman Coulter, Fullerton, Calif.
D2-black, Wellred, Beckman Coulter, Fullerton, Calif.
D3-green, Wellred, Beckman Coulter, Fullerton, Calif.
D4-blue, Wellred, Beckman Coulter, Fullerton, Calif.
DNA size standard-400, Beckman Coulter, Fullerton, Calif.
D1-red, Wellred, Beckman Coulter, Fullerton, Calif.
CEQ fragment analysis software, Beckman Coulter, Fullerton, Calif.
JMP 7, version 4.0, SAS Institute Inc, Cary, NC.
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Appendix
Repeat motif, dyes, sequences and concentrations (nM) of PCR primers, amplicon size, and GenBank accession numbers for detection of 5 equine STRs in blood and urine samples obtained from racehorses.
| STR | Repeat motif | Dye | Primer sequences | Primer (nM) | Expected size (bp) | GenBank accession No. |
|---|---|---|---|---|---|---|
| VHL20 | CA | D4 | 1 5′-CAAGTCCTCTTACTTGAAGACTAG-3′ | 70 | 90 to 109 | X75970 |
| 2 5′-AACTCAGGGAGAATCTTCCTAG-3′ | ||||||
| HTG4 | GT | D3 | 1 5′-CTATCTCAGTCTTGATTGCAGGAC-3′ | 40 | 128 to 140 | AF169165 |
| 2 5′-CTCCCTCCCTCCCTCTGTTCTC-3′ | ||||||
| AHT4* | CA | D2 | 1 5′-AACCGCCTGAGCAAGGAAGT-3′ | 90 | 146 to 164 | Y07733 |
| 2 5′-TCCCAGAGAGTTTACCCTGG-3′ | ||||||
| HMS6 | TG | D3 | 1 5′-GAAGCTGCCAGTATTCAACCATTG-3′ | 70 | 160 to 170 | X74635 |
| 2 5′-CTCCATCTTGTGAAGTGTAACTCA-3′ | ||||||
| HMS7 | CA | D4 | 1 5′-CAGGAAACTCATGTTGATACCATC-3′ | 70 | 175 to 185 | X74636 |
| 2 5′-TCTTGTTGAAACATACCTTGACTGT-3′ |
