Response of short tandem repeat systems to temperature and sizing methods

Transcription

1 Forensic Science International 133 (2003) Response of short tandem repeat systems to temperature and sizing methods Brittany Hartzell, Kylie Graham, Bruce McCord * Department of Chemistry and Biochemistry, 136 Clippinger Laboratories, Athens, OH 45701, USA Received 10 September 2002; received in revised form 5 February 2003; accepted 11 February 2003 Abstract In capillary gel electrophoresis (CE), changing run conditions such as temperature can result in minor variations in the size determination of an allele. These effects are caused by secondary structure differences that can occur between the amplified sample and the internal standard. The type of method chosen to generate the sizing curve in STR analysis can influence the relationship between estimated allele size and temperature. To better understand the effects of temperature and sizing method on the reproducibility of DNA migration, two fluorescently labeled allelic ladders, CTTv and Y-PLEX 6 were analyzed using the ABI Prism 310 Genetic Analyzer. The default method on the Genetic Analyzer utilizes an electrophoretic temperature of 60 8C and a Local Southern method to generate a sizing curve from the fragment migration times of the internal lane standard. In this work, electrophoresis was conducted at C using the commercially available POP 4 buffer at ph 8 and two sizing methods, Global Southern and Local Southern, were compared. The slopes of the regression line between estimated allele size and temperature, using either sizing method, were measured in order to demonstrate the temperature sensitivity of migration time and the importance of the operator-chosen method. Our results indicate that the Global Southern method is a better choice in situations where temperature fluctuations can occur. In addition, the temperature dependence of the DNA size estimates using the POP 4 system were compared to results obtained using an experimental buffer consisting of 3% hydroxyethylcellulose at ph 11. These results demonstrate that secondary structure effects are minimized at an elevated ph, increasing the precision of size estimates obtained. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DNA; STR; Temperature; Sizing 1. Introduction The analysis of short tandem repeats (STRs) by capillary gel electrophoresis (CE) has become widely accepted as a rapid and precise method for DNA typing. Many labs are now using dedicated instruments, such as the ABI Prism 310 Genetic Analyzer (Perkin-Elmer) that allow for the automated injection, separation, and detection of STR loci for forensic human identification [1 6]. The judicial aspects of STR analysis require a high degree of confidence in the accuracy of the sizing results. Therefore, systems for genotyping by CE have been developed with multiple controls to ensure precision. Both internal and * Corresponding author. Tel.: þ ; fax: þ address: mccord@ohiou.edu (B. McCord). external standards are utilized. The internal standard is added to each amplified sample and consists of a commercial mixture of fragments of known size that are labeled with a particular fluorescent dye such as ROX. The external standard is an allelic ladder comprised of the most common alleles present in the STR loci to be investigated. The fragment size of an unknown allele is determined by a comparison with the internal standard and the allele s identity is then determined by a comparison between the unknown s calculated size and the known allele sizes in the external standard. This protocol for DNA sizing using CE requires that analyses be performed at a constant, elevated temperature (60 8C) in order to prevent secondary structure differences between the amplified sample and the internal standard [7,8]. These secondary structure differences are a result of sequence differences between the sample and the standard /03/$ see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi: /s (03)

2 B. Hartzell et al. / Forensic Science International 133 (2003) and cause the fragments to migrate through the gel-filled capillary at different rates. Unfortunately, the elevated temperature required to minimize these effects can be difficult to maintain where there are shifting room temperatures. The lack of strict control over this instrumental parameter can affect the precision of the STR analysis results by causing changes in the size estimate for DNA fragments at different temperatures [9]. This becomes particularly important when variant alleles are present, especially those that contain sizes differing by one base from the four base pair repeat unit. The research described herein was completed to determine the magnitude of the effect that temperature has on DNA typing. Two fluorescently labeled allelic ladders, CTTv from Promega and Y-PLEX 6 from ReliaGene TM Technologies Inc. were analyzed using the ABI Prism 310 Genetic Analyzer. CTTv includes the forensic loci vwa, TH01, TPOX, and CSF1PO while Y-PLEX 6 contains Y-chromosomal specific STR loci, DYS 393, DYS 19, DYS 389, DYS 390, DYS 391, and DYS 385. While completing this study it was discovered that the type of method chosen to generate the sizing curve in STR analysis plays an important role in the extent of the temperature effect. From the fragment migration times of the internal standard, the Genetic Analyzer s Genescan software generates a sizing curve using an operator-chosen sizing method. Two sizing methods were compared in our investigation, Global Southern and the default method in the software, Local Southern. The Global method generates a best-fit curve through all of the points of the selected size standard and then by using values found on that line, calculates the size of the unknown fragment lengths. The Local Southern method instead uses the four fragments of the internal standard that are nearest in size to the unknown fragment to determine the best-fit curve [10]. Therefore, unlike the Global Southern method, only the region of the internal size standard that is close to the unknown fragment s length is used to calculate size. The slopes of the regression line between estimated allele size and temperature, using either Global or Local sizing, Table 1 Slope of the relationship between temperature ( C) and estimated size for individual alleles in the CTTv and Y-PLEX 6 ladder, using the Local Southern method of sizing STR Allele # Run 1 slope S.D. a Run 2 slope S.D. Run 3 slope S.D. Average slope S.D. b (A) CTTv CSF1PO CSF1PO CSF1PO CSF1PO TPOX TPOX TPOX TH TH TH vwa vwa vwa vwa (B) Y-PLEX 6 DYS DYS DYS DYS DYS DYS DYS DYS Non-linear N/A Non-linear N/A Non-linear N/A Non-linear N/A DYS DYS DYS Results are given in units of bases/8c. Runs 1, 2, and 3 were completed on different capillaries and at different time periods. Slope and standard deviation were calculated using Microsoft Excel. Experimental conditions: POP 4 buffer; 41 cm, 50 mm uncoated capillary; 366 V/ cm; 5 s 15 kv injection. See also Fig. 1 for examples of data plots. a Standard deviation of the calculated slope (five runs completed at each temperature). b Standard deviation of the three estimated slopes.

3 Fig. 1. Temperature vs. estimated size for selected alleles from the DYS-385 locus in the Y-PLEX 6 ladder, using Local Southern sizing. Conditions as stated in Table 1(B). 230 B. Hartzell et al. / Forensic Science International 133 (2003)

4 B. Hartzell et al. / Forensic Science International 133 (2003) were measured in order to demonstrate the temperature sensitivity of migration time and the importance of these operator-chosen sizing methods. In previous work using the Profiler Plus system, the temperature dependence of the DNA size estimates was eliminated by utilizing a buffer system with an alkaline ph [8]. This type of system makes the capillary environment very denaturing and prevents secondary structure in the DNA strands, resulting in more precise sizing. Therefore, an experiment was performed using the CTTv ladder to demonstrate the stabilizing effect of an elevated ph on DNA temperature response. 2. Materials and methods 2.1. Materials The fluorescently labeled allelic ladders, CTTv and Y-PLEX 6 were obtained from Promega (Madison, WI) and ReliaGene TM Technologies Inc. (New Orleans, LA), respectively. GeneScan-500 ROX size standard was purchased from Perkin-Elmer (Foster City, CA). Amberlite MB-150, 3-[cyclohexylamino]-1-propane-sulfonic acid (CAPS) and hydroxyethyl cellulose (HEC, # 30,863-3; Aldrich Chemical, Milwaukee, WI) were obtained from Sigma Chemical (St. Louis, MO). Urea was acquired from Spectrum Quality Products Inc. (Gardena, CA) and high-purity formamide was purchased from Amresco (Solon, OH). Uncoated fused silica capillaries with an internal diameter of 50 mm wereobtained from Polymicro Technologies (Phoenix, AZ) and FC-coated capillary columns with an internal diameter of 50 mm were acquired from J&W Scientific (Folsom,CA) Sample preparation A 0.5ml volume of allelic ladder and 0.5 ml of ROX standard were added to 11 ml formamide. For denaturation, sampleswereheatedat95 8Cfor 2 min and snap-cooled onice. Table 2 Slope of the relationship between temperature ( C) and estimated size for individual alleles in the CTTv and Y-PLEX 6 ladder, using the Global Southern method of sizing STR Allele # Run 1 slope S.D. a Run 2 slope S.D. Run 3 slope S.D. Average slope S.D. b (A) CTTv CSF1PO CSF1PO CSF1PO CSF1PO TPOX TPOX TPOX TH TH TH VWA VWA VWA VWA (B) Y-PLEX 6 DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS Results are given in units of bases/8c. Runs 1, 2, and 3 were completed on different capillaries and at different time periods. Slope and standard deviation were calculated using Microsoft Excel. Experimental conditions: POP 4 buffer; 41 cm, 50 mm uncoated capillary; 366 V/cm; 5 s 15 kv injection. a Standard deviation of the calculated slope (five runs completed at each temperature). b Standard deviation of the three estimated slopes.

5 232 B. Hartzell et al. / Forensic Science International 133 (2003) Separation and detection Capillary electrophoresis and multichannel laser-induced fluorescence detection were carried out using an ABI Prism 310 Genetic Analyzer (Perkin-Elmer) and data was analyzed with GeneScan software. The ABI Prism 310 was calibrated using matrix dye standards labeled with ROX, JOE, FAM, and NED fluorescent dyes for CTTv analysis and ROX, JOE, FAM, and TAMRA dyes for Y-PLEX 6 analysis. Capillaries with a length of 41 cm (30 cm between the injection inlet and detection zone) were used. The capillary was filled with Performance Optimized Polymer 4 (POP-4) from Perkin- Elmer, consisting of 4% poly(dimethylacrylamide), 8 M urea, 5% 2-pyrrolidinone, and 100 mm N-tris(hydroxymethyl)- methyl-3-aminopropanesulfonic acid (TAPS), at ph 8.0 [9]. An additional study involved the use of an alkaline separation system: 3% (w/v) HEC, 7 M urea, 20 mm CAPS at ph 11.0 following a procedure described by Nock et al. [8]. This sieving medium was prepared by adding 17.8 g of urea to 25 ml of deionized water. Once the urea had dissolved, 1.25 g of HEC was slowly added to the solution. The solution was stirred overnight to ensure that all of the HEC dissolved. Then approximately 0.5 g of Amberlite mixed-bed ion exchange resin was added and stirred for 1 h. This solution was centrifuged at 3000 rpm for 15 min and 30 ml of the supernatant liquid was removed. The 3.33 ml of a 10 1 CAPS buffer (200 mm, ph 11.0) was added to the HEC/urea solution and stirred for about 15 min. Finally, the solution was then filtered through a 5 mm syringe filter (Millipore, Bedford, MA). Fluorocarbon-coated capillaries were used for the elevated ph work. Samples were electrokinetically injected using 15 kv for 5 s. The fragments were separated for 24 min at a field strength of 366 V/cm. Capillary temperatures were varied by adjusting the ABI 310 Genetic Analyzer s heat plate between 35 and 70 8C, at 5 8C intervals. Fig. 2. Temperature vs. estimated size for vwa allele 20 in the CTTv ladder using Global Southern sizing. Conditions: (A) POP4, ph 8; (B) 3% HEC in 20 mm CAPS, ph 11.

6 B. Hartzell et al. / Forensic Science International 133 (2003) Data analysis Data was analyzed using the ABI Prism 310 GeneScan v2.1 software using both Local and Global Southern sizing calculations. The 250-base fragment from GeneScan-500 was not included in the generation of size standard curves, due to its tendency to migrate anomalously under non-ideal conditions on the ABI 310. The LINEST function in Microsoft Excel 98 was used to determine the slopes of the regression lines for plots of estimated allele size versus temperature. 3. Results and discussion 3.1. Allele size estimates Experiments with this CE system were performed using the manufacturer s standard protocol with the exception that temperature was varied. The alleles present in CTTv and Y-PLEX 6 include fragments ranging in length from 116 to 385 bases, allowing investigation of the entire calibration curve produced by the ROX 500 internal size standard. Three different analyses of the response of the size estimate with respect to temperature were completed for both of the ladders examined. The estimates of the slope of the temperature response were fairly precise with standard deviations of bases/8c even though the runs were taken at different times over a period of a few months and on different capillaries. The results of this work for the CTTv and Y-PLEX 6 ladders, sized using the default, Local Southern method are reproduced in Table 1(A) and (B), respectively. Local Southern sizing involves using only the four fragment sizes of the internal standard that are nearest to the unknown fragment to make a calculated DNA size estimate [10]. Using this method the slopes varied both between and within loci in both ladders. For example, while the slope for allele 7 in CSF1PO was 0.027, the slope for allele 14 within the same locus was and similarly, in the Y-PLEX 6 ladder the slopes for the DYS 389 alleles 28, 30, and 33 were 0.230, 0.300, and bases/8c, respectively. The slopes of the temperature response for all of the alleles in the CTTv ladder were negative, i.e. allele size estimates decreased with increasing temperature. However, the Y-PLEX 6 ladder had one locus, DYS 385, which gave positive slopes with increasing temperature. All of the alleles in DYS 385 gave linear, positive slopes except for allele 8 which produced a curving response each time that it was measured. Fig. 1 shows representative plots of the alleles within this locus. The reason behind this unique temperature behavior is unknown but it may be the result of high AT content in the locus [11,12]. Further study of this locus with conformational computer modeling of the sequence may be helpful in elucidating the cause of this non-linear response. Table 2(A) and (B) include the results of the analyses using the Global Southern sizing method. This method involves using all of the fragment sizes of the internal standard to estimate the size of an unknown DNA fragment. Using this method of sizing the slopes varied consistently for the different alleles within each of the loci. For example, the slopes for TPOX alleles 6, 10, and 12 were 0.094, 0.100, and bases/8c, respectively. However, there was a differential response in slope between loci, as had been seen using the Local Southern method of sizing. For example, the slope for allele 7 in CSF1PO was while allele 10 in TPOX had a slope of and allele 14 in vwa had a response of bases/8c. The slopes determined for the various alleles within the Y-PLEX 6 ladder followed a similar trend when Global Southern sizing was used. All of the alleles within a locus produced slopes with only minor variability while the slopes among the six loci varied over a wide range, with both negative and positive responses. Also of interest was the completely different response of allele 8 in the DYS 385 locus when sized using the Global Southern method. Instead of a curved response, the slope was positive and linear Electrophoresis under alkaline conditions Previous research has demonstrated that the secondary structure differences, which are thought to cause these slopes, can be reduced by utilizing an elevated ph [8]. To characterize the effect of alkaline ph, the temperature dependence of the size estimates of the CTTv ladder using 3% HEC in 20 mm CAPS at ph 11 was compared to the results previously obtained with the POP 4 system at ph 8 Table 3 Slope of the relationship between temperature ( C) and estimated size for individual alleles in the CTTv ladder, using the Local Southern method of sizing, under alkaline conditions STR Allele # Slope S.D. a CSF1PO CSF1PO CSF1PO CSF1PO TPOX TPOX TPOX TH TH TH vwa vwa vwa vwa Results are given in units of bases/8c. Slope and standard deviation were calculated using Microsoft Excel. Experimental conditions: 20 mm CAPS buffer, ph 11, 3% HEC, 7 M urea; 50 mm J&W msil FC capillary; 366 V/cm; 5 s 15 kv injection. a Standard deviation of the calculated slope (two runs completed at each temperature).

7 234 B. Hartzell et al. / Forensic Science International 133 (2003) (Table 3). As expected, at ph 11 the slopes of the regression line between allele size and temperature were nearly zero. Fig. 2 shows representative plots of this result for an allele at both a normal and an elevated ph. TPOX was the only locus that still had significant slopes, the other slopes ranged between and bases/8c. These results were much different than Table 1 in which most of the measured alleles had slopes of 0.06 bases/8c or greater. Therefore, our research shows that using an elevated ph minimizes the effect of temperature on the size estimates obtained using the standard internal and external controls. This result supports the proposed cause of these slopes, secondary structure differences between the internal standard and the DNA fragments to be typed. 4. Conclusions The presence of these slopes and their variation in magnitude demonstrates that temperature is an important factor in maintaining a high level of precision in DNA separation. Our results indicate that fluctuation of this variable can lead to band shifts, which in genotyping can sometimes occur at one locus and not at another. This is in agreement with our plots, which had significantly different slopes among the loci, regardless of the sizing method used. For example, using these results, a temperature change of 18C in the capillary changes the Local Southern size estimate of allele 31 in DYS bases while allele 10 in DY385 changes only 0.09 bases. Thus, while one fragment might be sensitive to a temperature fluctuation in the laboratory, resulting in a loss of calibration, another fragment with a different sequence might remain unaffected. However, the Global Southern method did provide minimal variability among the alleles within each locus, for both of the ladders tested. Calculation of an unknown fragment s length using the Local Southern method, where only the sizes of the nearest internal standard fragments are used, is more likely to be altered by effects local to one or more nearby sizing ladder fragments. Therefore, while the default method in GeneScan analysis software is the Local Southern sizing, our results indicate that the Global Southern method is a better choice in situations where temperature fluctuations can occur. This conclusion is further supported by a recent study completed by Klein et al. in which the Local and Global Southern methods were compared for the sizing of Profiler Plus alleles with ambient temperature variation [13]. Acknowledgements The authors would like to thank ReliaGene TM Technologies Inc. for contributing the Y-PLEX 6 ladder used in this study and the National Institute of Justice (Grant # 1999-IJ- CX-K014), which provided the primary funding for this research. References [1] J.M. Butler, Forensic DNA Typing: Biology and Technology Behind STR Markers, Academic Press, San Diego, CA, USA, [2] A. Tagliabracci, L. Buscemi, C. Sassaroli, M. Paoli, D. Rodriguez, Allele typing of short tandem repeats by capillary electrophoresis, Int. J. Leg. Med. 113 (1999) [3] L. Buscemi, A. Tagliabracci, C. Sassaroli, F. Bianchi, S. Canestrari, D. Rodriguez, Polymerase chain reaction typing of D21S11 short tandem repeat polymorphism by capillary electrophoresis: allele frequencies and sequencing data in a population sample from central Italy, Forensic Sci. Int. 92 (1998) [4] R. Pawlowski, W. Branicki, T. 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