Designing Polymerase Chain Reaction (PCR ... - ACS Publications

LABORATORY EXPERIMENT pubs.acs.org/jchemeduc

Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory Kelly M. Elkins* Chemistry Department and Criminalistics Program, Metropolitan State College of Denver, Denver, Colorado 80217-3362, United States

bS Supporting Information ABSTRACT: The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for PCR and analyze the results. However, primers can also be designed in undergraduate laboratories with students at this level. In a course that focuses on forensic DNA molecular biology for forensic chemistry students, students have used the Applied Biosystems AmpFlSTR SGM Plus kit that amplifies DNA at eleven regions in a single test tube. It is important for forensic chemistry students to be able to design and analyze a single set of primers and, more importantly, create multiplexes of primers. This enables students to more fully understand how the primers and the kits that are routinely employed by the crime laboratories function. Creating a single set of primers does not demonstrate the extent of design and engineering inherent in creating multiplexes or adequately prepare students for research and careers in the field. The in silico method described herein uses free bioinformatics tools and results in student-designed multiplexes for Combined DNA Index System (CODIS) loci. Sample student data are shown. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Internet/WebBased Learning, Problem Solving/Decision Making, Biotechnology, Forensic Chemistry, Molecular Biology, Nucleic Acids/DNA/RNA

T

he polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry and molecular biology courses as reagents and thermocyclers have become more affordable for institutions. There are numerous reports of the use of PCR in undergraduate laboratories with a forensic application. For example, PCR can be used to amplify a 369801 base pair D1S80 locus of the human genome,1 to amplify hypervariable regions of DNA from dog hair and saliva,2 to amplify a 440 base pair hypervariable region of human mtDNA from a simulated crime scene,3 and to amplify a 192 base pair DNA segment present in genetically modified foods.4 In the undergraduate lab, PCR has also been demonstrated to differentiate bacterial species,5 to genotype the normal variation in human color vision,6 to evaluate a metabolic polymorphism,7 in diagnostics,8 and to test for genetically modified organisms in foodstuffs.9,10 In all of these examples, the instructors designed the experiments and created and secured the primers, and the students prepared their PCR samples using the procedure as written. In the second semester of a two-semester forensic science course that focuses on DNA molecular biology for forensic chemistry students, students employ the Applied Biosystems AmpFlSTR SGM Plus multiplex kit that amplifies DNA at 11 regions (D21S11, FGA, TH01, vWA, D8S1179, D18S51, D3S1358, D19S433, D16S539, D2S1338, and the gender marker, amelogenin) including nine of the thirteen short tandem repeat (STR) loci of the U.S. Combined DNA Index System (CODIS) (all of the above except D19S433 and D2S1338) for Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.

PCR and DNA typing (Table 1). Most U.S. crime laboratories utilize 16-plex kits for human casework for sample completion so that the data can be entered into CODIS, as necessary. It is important for forensic chemistry students to be able to employ a kit for PCR and mimic crime lab work and, more importantly, to design and analyze a single set of primers and create primer multiplexes. This enables students to more fully understand how the primers and the kits that are routinely employed by the crime laboratories function and extends their research skills. Primers can be designed in undergraduate laboratories with students at this level.1114 In a single three-hour laboratory session, students design PCR primers in silico using free bioinformatics tools available on the World Wide Web and collaborate with a peer to create a multiplex PCR primer set for two loci. Creating only a single set of primers does not demonstrate the extent of design and engineering inherent in creating multiplexes or adequately prepare students for jobs in forensics or even modern clinical testing in medicine.

’ MATERIALS AND METHODS This study was approved by Metro State’s Institutional Review Board (IRB)—Human Subjects Review Committee (HSRC), and the students consented to participate in the study and to the release of the anonymous data shown here. Published: August 10, 2011 1422

dx.doi.org/10.1021/ed100676b | J. Chem. Educ. 2011, 88, 1422–1427

Journal of Chemical Education

LABORATORY EXPERIMENT

Table 1. CODIS STR Loci by NCBI Accession Number and Repeata

a

Data from ref 20.

Table 2. Sample PCR Primer Results

Figure 1. Genome sequence for TPOX CODIS locus region on human chromosome 2 thyroid peroxidase (hTPO) reference sequence and published validated forward and reverse primers (arrows and bold) producing a 64 base pair product.

This in silico lab does not require specialized materials, expensive chemicals, or extensive preparation. A reliable Internet connection and student laptops or computers (via a computer or department lab) are required. The utilized Web sites include NCBI,15 IDT’s OligoAnalyzer,16 NCBI Primer-Blast,17 and NIST’s STRbase.18 An example of the type of data collected and evaluated in this lab is presented to the students during the prelab lecture. The primers shown as an example (TPOX locus) are used in the same course in a subsequent laboratory (real-time PCR amplification) (Figure 1 and Table 2),19 and amplify a nonvariable portion of the locus and student-designed primers from this lab. Students are shown how to navigate to the Web sites and locate a STR repeat. The differences between the forward and reverse primers (both 50 to 30 but located on the plus and minus strands, respectively) and the STR repeat region for a sample locus from NCBI (Table 1) are highlighted prior to inputting the primers in the analysis Web sites, collecting the output to be saved in a text file, and creating multiplexes. Working in alone or in pairs, the students obtain the DNA sequence for a CODIS STR locus (Table 1)20 from the National

Center for Biotechnology Initiative (NCBI)15 using mini-Mac computers in one of the department computer laboratories. After locating the STR repeat (Figure 2), they initially try to create the primers from the 50 region directly upstream from the repeats and the region directly downstream for the 30 complement using the given guidelines for optimal primers including the following: 4060% guaninecytosine (GC) content, melting temperatures (Tm) within 5 °C, less than four identical base repeats, only weak primer dimer and hairpin formation, a length between 1830 bases, and overall Tm values in the range of 5072 °C. The amplicon should not exceed 500 base pairs. The primers are evaluated in Integrated DNA Technologies’ (IDT) OligoAnalyzer 3.116 by pasting the primer sequence into the sequence box and clicking on the Analyze, Hairpin: Submit and Self-Dimer 1423

dx.doi.org/10.1021/ed100676b |J. Chem. Educ. 2011, 88, 1422–1427


Figure 2. Genome sequence for TH01 CODIS locus region on human chromosome 11 tyrosine hydroxylase sequence, AATG (named for 30 strand) STR repeat (underlined), and student designed forward and reverse primers (arrows and bold) producing an 87 base pair product given the 9 repeats observed in the NCBI reference sequence (or 90 base pair product using K562 with the 9.3 repeat allele).


boxes on the right. The defaults are used for all calculations. The Hetero-Dimer: Calculate calculation is used to evaluate the two primers together. Then the primer sequences are submitted to NCBI Primer-Blast (Homo sapiens)17 to evaluate specificity by entering the NCBI accession number in the PCR template box and the designed primers in the forward and reverse primer boxes under primer parameters. The students also indicated the primer region for the NCBI accession number by base number in the boxes to the right of the PCR template box and the expected product size below the primer sequence boxes. After the students completed a reasonable single primer set (Table 3), the students paired themselves or their groups to create a multiplex of the two sites by first locating other students with similar melting temperatures for their primers. The multiplex primers were checked against each other in OligoAnalyzer using the Hetero-Dimer function. The students made adjustments to the primers to create their best multiplex using the remaining lab time or out of class while recording all attempts in the text file (Table 3). In a follow-up laboratory experiment, the students tested the primers they designed experimentally using real-time PCR19 in a

Table 3. Sample Student PCR Primer Multiplex Results

1424




Table 4. Student Designed STR PCR Primers

a

Size based upon NCBI sequence. sequence (both alleles indicated).

b

Size based upon K562 DNA

25 μL reaction. The students amplified human K562 DNA (using 1 ng total in the 25 μL reaction) (Promega, Madison, WI) using the published and validated TPOX primers21 and their designed primers (Table 4) (purchased from IDT, Coralville, IA) (1 μL each of 5 μM primers) in a real-time PCR experiment conducted on a Bio-Rad (Hercules, CA) iQ5 instrument using the iQ5 software (v. 2.0) and 12.5 μL of 2 iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) and nuclease-free water to total 25 μL on a 96-well plate according to the following protocol for a gradient plate: an initial 3 min denaturation at 95 °C and 40 cycles of denaturation at 95 °C for 15 s using an annealingextension gradient at 5065 °C for 60 s. A melting curve was produced by conducting 91 cycles of 30 s of melting every 0.5 °C from 50 to 95 °C. The SYBR Green I excitation wavelength is 497 nm, and the emission wavelength is 522 nm. Three primer sets from classes of previous years were also tested. A total of ten primer sets were evaluated. The production of the single reaction and multiplex PCR products was evaluated by the shape of the melt peaks from real-time PCR and post-PCR agarose gel electrophoresis using a 2% gel run in 1 TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) (National Diagnostics, Atlanta, GA) using the DNA Logic (Lambda Biotech, St. Louis, MO) and Lonza (Basel, Switzerland) 20 bp (base pair) ladders. Upon completion, digital photographs were recorded of each gel upon illumination under UV light using a digital photography system equipped with a SYBR filter. Hazards

No chemicals are needed, and there are no intrinsic chemical hazards associated with this laboratory experiment as it is performed entirely on a computer. For the follow-up lab, SYBR Green I is a weak mutagen and may be an intercalating agent and should be handled with care. Gloves, goggles, long pants, and closed-toed shoes should be worn at all times in the laboratory. The digital camera should be set to UVvis safety at all times, and gel pictures should be recorded with the illuminator door in the closed position.

’ RESULTS Sample results for the example TPOX primers are shown in Table 2. The TPOX PCR primers amplify a nonvariable, 64 base

pair amplicon of the Homo sapiens thyroid peroxidase on chromosome 2 (NCBI Accession number: NG_011581) from bases numbered 8126281325 (inclusive) (Figure 1). An analysis of these primers yields that the primer length varies by one base pair, the GC content varies by 7.1%, the Tm vary by 0.2 °C, and the best self-dimers and hairpins were 23 base pairs. The 30 primer Tm had the lower GC content, but the overall Tm was almost identical to that of the 50 primer due to the one base increase in length. The hairpins and self-dimers exhibited sufficiently low melting temperatures as compared to the annealing temperature of the primer to the template. The best heterodimer formed is four base pairs, which is more than optimal (three or less). The primers were determined to be specific for only this region in the human genome. Students work individually or in pairs in the lab. Each student or group of students chooses a different locus from the thirteen CODIS loci (Table 1). After inputting the accession number and locating the STR repeats from the NCBI nucleotide file for a CODIS locus (Table 1), the students evaluate the 50 region directly upstream from the repeats as a potential 50 primer and the complementary region directly downstream for the 30 complement. This is advantageous for forensic use as the recovered DNA is often degraded by environmental exposure, arson, or mass disaster events and shorter DNA segments are more easily amplified (e.g., mini-STRs). Then the students use currently available Web-based bioinformatics programs for single and multiplex primer evaluation including OligoAnalyzer 3.1 and PrimerBlast. OligoAnalyzer outputs the melting temperature, primerdimer and hairpin formation, percent GC content, sequence length, and heterodimer formation. Primer-Blast indicates for which sequences the primer pairs are specific in a human genome database (or other selected database). As the goal of this laboratory is to teach the students the basics of primer and multiplex design, automated primer design servers are not employed (e.g., Primer 3) although these are well suited to the research and teaching setting and are reported for use in a related lab experiment.14 Sample student results from the Web-based analysis for their designed STR primers, TH01 and D16S539, and sample multiplex results are given in Table 3. The designed primers indicate that the students successfully completed the in silico assignment as most of the data falls within the set criteria. The primers were determined to be specific for only those specific regions in the human genome and were 24 base pairs or less in length. The heterodimer pairs for the TH01 forward primer had a higher than desirable level of six heterodimer pairs, and the 50 primer multiplex of the opposite loci had a higher than desirable level of five heterodimer pairs. The observed hairpins in the primers have sufficiently low melting temperatures as to be denatured at the annealing temperature (e.g., 40.8 °C or less). The melting temperatures for the multiplex primers were within 5.5 °C. Although all students had sufficient lab time to test a few iterations of their primers and experiment with shifts in frame and region, the students did not have sufficient lab time to evaluate all possible primer sequences or to test multiple multiplexes and make multiple improvements. However, they were able to consider the challenges in editing the primers to better the multiplex by comparing to the time already spent to solve the problem. All of the student-designed primers from the class are shown in Table 4. The expected amplicon is shown for each in base pairs for both the NCBI sequence and the K562 genotype. In a subsequent laboratory using real-time PCR, students tested the ability of their primers to amplify human K562 DNA. Five of the 1425


Journal of Chemical Education seven primer pairs produced the expected amplicon as assayed by a post-PCR 2% agarose gel. Additionally, three of the primer pairs designed by students in the classes of the previous years were tested, and all produced the expected amplicons. Of the ten primer sets evaluated, eight successfully amplified DNA (an 80% success rate). The students also tested a few potential mutliplexes based on having varied amplicon sizes that could be diffentiated on the gel. The three multiplexes tested in class (D5S818: D13S317, D7S820:D5S818, and D7S820:D13S317) provided only low-level amplification; two of the multiplexes used one of the primer sets that failed to work in a single PCR reaction (D13S317). Subsequently four more multiplexes (TH01 with D16S539, D7S820, D8S1179, or D5S818) were tested using only primers that amplified the DNA successfully in a single reaction and that could be differentiated based on size. Three of these multiplexes produced the predicted DNA amplicons for both primer sets (including TH01:D16S539, TH01:D7S820, and TH01:D5S818) although the TH01 amplicon was preferentially amplified in all cases at the concentrations used. The D8S1179 amplicon is at the upper limits for PCR and too long for real-time PCR with a two-step reaction. This follow-up lab is not inexpensive: primer pairs per student cost approximately $15 each without a dye label (SYBR Green I was used for detection of the amplicon) and, if desired, $6080 per primer with a dye label plus shipping for the lot. The primers were purchased from IDT, and the total cost was $132.01 for the seven unlabeled primer sets for the current years’ class. The realtime PCR cost was approximately $4.50 per reaction; numerous reactions were run including one replicate each for each of the temperatures in the annealing temperature gradient, negative controls, and multiplexes. (The total cost much exceeded the collected lab fees collected by the college from the seven students for the course).

’ ASSESSMENT In an anonymous survey, student feedback for the laboratory was positive: students were excited about designing the primers to better understand how they work and being able to order and experiment with them in the follow-up real-time PCR laboratory. Students noted that an understanding of the background through lecture and the prelab lecture was important. They appreciated the opportunity to learn how to create primers using the Web tools and gained an appreciation in multiplex primer design and felt the concepts would have been confusing without performing the lab. The majority of the students responded that creating the primers was more difficult than expected, but predominately that the data analysis was easier than expected or as easy or difficult as expected. Students noted that they learned that the process can be very time-consuming including finding the repeats and pairing the primers. ’ DISCUSSION The students follow the instructions contained in the syllabus for writing concise weekly lab reports; that is, students complete sections including the title, preparer, partners, date, objective, method, data, analysis and results, sample calculations, discussion and answers to lab questions, and a conclusion. Working individually or in pairs, the students obtained the sequence of a CODIS STR region and created primers for a single CODIS locus and analyzed the primers using two free bioinformatics tools. Subsequently, two students or student groups were merged


and the students compared the primers of the individual unique loci and evaluated if they could be used simultaneously in a multiplex, single tube PCR reaction or if changes would be necessary. Students then suggested and tried alterations to the first-generation primers to create a suitable multiplex. Multiplexing is an important tool in forensics. This lab is invaluable for students pursuing molecular biology and forensics as bachelorlevel chemists or biologists and those continuing with postgraduate education. This lab is compatible with our budgetary constraints as the fixed costs of the mini-Mac computers had already been paid by the department and the cost of Internet access is borne by the students of the college. This laboratory exercise requires no expensive molecular biology reagents for the in silico portion (in stark contrast to the commercial multiplex DNA typing kits) and is highly accessible to instructors at all institutions, regardless of funding. The PCR primer design multiplexing laboratory experiment could easily be extended to other species and non-CODIS loci. The follow-up laboratory in which students tested the designed primers using real-time PCR was expensive, but the students were enthusiastic about the opportunity to evaluate their work experimentally. Students were especially pleased to determine that most of their primers amplified the DNA and that some of their multiplexes were successful.

’ ASSOCIATED CONTENT

bS

Supporting Information A student handout consisting of background information and instructions and instructions for the instructor. This material is available via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT ACS Project SEED is gratefully acknowledged for a summer stipend for Ain Ealey who tested revisions of this lab in the summer of 2009 and provided useful suggestions. I wish to thank my students in the spring 2011 CHE 3710 Criminalistics II course for trying the improved version of the experiment including experimentally testing their primers: Benjamin Bender, Audrionna Kingsley, Hannah Leger, Megan Owens, Tiffany Stone, and Francesca Garcia Wheeler. I wish to thank my students in the spring 2009 CHE 3710 Criminalistics II course for their suggestions for improvement: Patrick Bevins, Sarah Bonsall, Lindsay Christopherson Powis, Kali Gipson, Natalie Hernandez, Raelynn Kadunc, Jeffrey Minutillo, Carrie Melanson, Tanya Mokelki, Michelle Montoya, and Zachary Morin. I wish to thank my students in the spring 2008 CHE 3710 Criminalistics II course for their patience, cooperation and input in the inaugural running of this lab: Katelin Arnold, Jennifer Auger, Lydia Benyam, Megan Jones McDowell, Jacqueline Keller, Erin Knopka, Gina Mann, Susan McLaughlin, Andrea Moore, Melanie Newman, Stephanie Sauter, Kurt Smith, Nia Travers, and Nikole Whitsitt. ’ REFERENCES (1) Jackson, D. D.; Abbey, C. S.; Nugent, D. J. Chem. Educ. 2006, 83, 774–776. 1426




(2) Carson, T. M.; Bradley, S. Q.; Fekete, B. L.; Millard, J. T. J. Chem. Educ. 2009, 86, 376–378. (3) Millard, J. T.; Pilon, A. M. J. Chem. Educ. 2003, 80, 444–446. (4) Taylor, A.; Sajan, S. J. Chem. Educ. 2005, 82, 597–598. (5) Suwanjinda, D.; Eames, C.; Panbangred, W. Biochem. Mol. Biol. Educ. 2007, 35, 364–369. (6) Lubin, I. M.; Wilson, B.; Grant, K. B. J. Chem. Educ. 2003, 80, 1289–1291. (7) Childs-Disney, J. L.; Kauffmann, A. D.; Poplawski, S. G.; Lysiak, D. R.; Stewart, R. J.; Arcadi, J. K.; Dinan, F. J. J. Chem. Educ. 2010, 87, 1110–1112. (8) Claros, M. G.; Quesada, A. R. Biochem. Mol. Biol. Educ. 2000, 28, 223–226. (9) Thion, L.; Vossen, C.; Couderc, B.; Erard, M.; Clemenc-on, B. Biochem. Mol. Biol. Educ. 2002, 30, 51–55. (10) Brinegar, C.; Levee, D. Biochem. Mol. Biol. Educ. 2004, 32, 35–38. (11) Kim, T. D. Biochem. Mol. Biol. Educ. 2000, 28, 274–276. (12) Lima, A. O. S.; Garcês, S. P. S. Biochem. Mol. Biol. Educ. 2006, 34, 332–337. (13) Bornhorst, J. A.; Deibel, M. A.; Mulnix, A. B. Biochem. Mol. Biol. Educ. 2004, 32, 173–182. (14) Thornton, B.; Basu, C. Biochem. Mol. Biol. Educ. 2011, 39, 145–154. (15) National Center for Biotechnology Initiative. http://www.ncbi. nlm.nih.gov/ (accessed June 2011). (16) Integrated DNA Technologies’ OligoAnalyzer 3.1. http:// www.idtdna.com/analyzer/Applications/OligoAnalyzer/ (accessed June 2011). (17) NCBI Primer-Blast. http://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed June 2011). (18) Masibay, A.; Mozer, T. J.; Sprecher, C. J. Forensic Sci. 2000, 45, 1360-1362, http://www.cstl.nist.gov/strbase/promega_primers.htm (accessed June 2011). (19) Elkins, K. M.; Kadunc, R. E. J. Chem. Educ. 2011, in press. (20) Butler, J. M. Forensic DNA Typing, 2nd ed.; Elsevier: Burlington, MA, 2005; pp 1660. (21) Horsman, K. M.; Hickey, J. A.; Cotton, R. W.; Landers, J. P.; Maddox, L. O. J. Forensic Sci. 2006, 51, 758–765.

1427


Designing Polymerase Chain Reaction (PCR ... - ACS Publications

Recommend Documents