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Simple & rapid generation of complex DNA profiles for the undergraduate laboratory.

DNA profiles can be generated by a variety of techniques incorporating different types of DNA markers. Simple methods are commonly utilized in the undergraduate laboratory, but with certain drawbacks. Here, I present an advancement of the Alu dimorphism technique involving two tetraplex PCR analyses that yield 6,561 possible genotypes. This technique is simple to perform in an undergraduate laboratory.

DNA markers are segments of nucleotides with known locations in the genome that can be informative in DNA profiling and genetic mapping studies if variations exist. Mini-satellites, a.k.a. variable number tandem repeats (VNTRs), consist of variants that are the result of 15-100 base pair (bp) repeated DNA fragments, and microsatellites are variants resulting from 2-7 bp repeated DNA fragments. VNTRs are commonly analyzed using the Southern blot procedure in order to detect numerous loci simultaneously, thereby generating a highly informative DNA profile. This is accomplished using a radioactive probe that consists of a short stretch of DNA nucleotides that are shared among the VNTR loci. Microsatellites require the use of high-resolution techniques such as standard acrylamide gel electrophoresis or the use of an automated DNA sequencer. Several microsatellite loci can be analyzed simultaneously by the multiplex polymerase chain reaction (PCR) technique. This methodology involves using primer pairs from several loci in a single reaction. Although there are commercial suppliers that offer kits for this technique, including the primers and markers for various alleles, cost becomes a limiting factor in undergraduate teaching laboratories. Based on cost, practicality, equipment availability, and isotope use, undergraduate laboratories are restricted in the types of analyses that can be performed.

The two techniques typically used in the undergraduate laboratory are either the isolation of the VNTR locus referred to as D1S80, or an Alu dimorphism such as TPA-25 (Bloom et al., 1996), and are available as kits through commercial suppliers. Both simply involve PCR and analysis by electrophoresis on an agarose gel. D1S80 is a choice marker since it contains 29 different alleles yielding a possible 435 different genotypes [n (n+1)/2]. The primary drawback is the resolution limitation of agarose gels, creating difficulty in distinguishing individual alleles. Alu dimorphisms are variants that differ by the presence or absence of an Alu element. Alu elements are 300 bp retroposons (RNA-mediated transposable elements) that are amplified in the primate genome by the process of retrotransposition (retroposition) (Figure 1). The more recent integrations are not fixed in the human genome and therefore yield this type of variation (Roy-Engel et al., 2001), which has been useful in human population studies (Batzer et al., 1994; Roy-Engel et al., 2001) supporting the African origins of humans. The advantages of using the Alu dimorphism include its ease of use and simplicity of identification of the allelic variants. The drawback is the limited information attainable with only three possible genotypes (Figure 2). I previously developed a system to assay four Alu-containing loci using multiplex PCR (Kass, 2003). This is a simple, rapid technique for analyzing 81 ([3.sup.4]) possible genotypes. However, when using this for a simple forensic-type study in an undergraduate genomics laboratory, two individuals had the same genotype; and when incorporating this for a paternity test illustration, it was difficult to rule out one potential father. Therefore, I developed a second tetraplex reaction presented here for the first time. The two tetraplex reactions yield 6,561 combinations, dramatically increasing forensic and paternity capabilities of this tool. Additionally, the individual Alu variants that were chosen are referred to as "intermediate frequency (IF) polymorphisms", i.e., both the presence and absence forms are commonly found among populations (Roy-Engel et al., 2001; Carroll et al., 2001). The selected markers (Table 1) represent neutral variants. Although the Alu dimorphism within the angiotensin converting enzyme (ACE) gene has garnered attention in linkage studies to various cardiovascular phenotypes (reviewed in Niu et al., 2002; Pilate et al., 2004), the results have been conflicting (Lindpaintner et al., 1995; Pilate et al., 2004) and there is no direct evidence of influence on the expression or structure of the ACE gene. Therefore, this Alu-based variant can also be depicted as simply an inert intronic marker (Niu et al., 2002; Katzov et al., 2004).

[FIGURES 1-2 OMITTED]

The Alu tetraplex technique is simple, involving PCR and agarose gel electrophoresis; it is robust and reproducible; and it therefore warrants promotion as an ideal tool to study DNA profiling in the undergraduate laboratory. Although the most expensive reagent is the Epicentre Failsafe polymerase (which includes PCR buffer and deoxynucleotide triphosphates [dTNPs]), if other reagents are readily available (e.g., agarose, electrophoresis buffer, nuclease-free water), the cost is relatively comparable to the kits. One hundred reactions can be performed with one tube of the 100 unit enzyme mix. Also, primers are very inexpensive through commercial suppliers whereas kits merely provide enough for the set of reactions associated with the kit. The following is a simple protocol for utilizing this technique.

Methods

Latex gloves must be worn throughout the experiment to avoid DNA damage by nucleases from the hands as well as for protection from the potential mutagen ethidium bromide.

Materials

Major equipment includes a thermal cycler, agarose gel electrophoresis apparatus with power supply, micropipettors, UV transilluminator, and documentation system (Polaroid or digital camera). Additionally, a hot plate, heat block or water bath, and plastic trays are needed. Consumables include gloves, pipet tips (preferably aerosol barrier tips when performing PCR to avoid DNA contamination), and microcentrifuge tubes. Reagents include nuclease-free water, oligonucleotide primers, DNA polymerase enzyme mix and buffer, DNA isolation reagents (kit or Chelex), agarose, electrophoresis buffer, and ethidium bromide.

DNA Isolation

DNA can be obtained from the students or supplied to them (from various sources including potential volunteers unknown to them). If already supplied, the DNA extraction step can be bypassed. However, using DNA from students appears to elevate their enthusiasm, as they are interested in viewing their own profiles. Approval was obtained through the institutional human subjects review panel, and a consent form was developed with the information that there is no penalty (e.g., grades) for not volunteering. To insure confidentiality, students draw numbers "out of a hat" and each student knows only his/her number, unless they reveal it to other students. An alternative, whereby no one knows any of the numbers, is to mix up the samples after DNA isolation and then label them. Either technique will work for this laboratory. The source for DNA for the paternity analysis could be from the instructor and his/her family, or a family of a friend or colleague (unknown to the students), with two or more random samples serving as "potential fathers."

I would recommend the use of a commercial kit for extraction of DNA from the students. The Epicentre BuccalAmp[TM] DNA Extraction Kit (www.epibio.com) consists of a swab to obtain buccal cells and a single reaction tube to isolate the DNA. The protocol is provided by the manufacturer and is very simple to use. The procedure takes just five to ten minutes for the class. I have had 100% success with student samples. An alternate lower cost technique involves the use of Chelex (Bloom et al., 1996). However, it consists of a few more steps than the Epicentre kit, and there was a lower rate of success in generating PCR products. A 50% success rate was not atypical and consistent with the findings of others (Phelps et al., 1996).

Polymerase Chain Reaction

Two tetraplex reactions are provided. Oligonucleotide primers (purchased from Integrated DNA Technologies, Inc.) for the various loci are shown in Table 1. The reactions were developed using the Epicentre Fail Safe kit. This system is designed for difficult PCR procedures such as multiplex reactions. This is an important consideration, since the several Alu-containing loci are subject to heteroduplex formation and hence increase the difficulty of generating a quality profile. When using standard Taq enzyme to save on cost, the outcomes diminished considerably (Kass, 2003), and therefore this is not recommended. DNA polymerases from other suppliers may be tried, but there may be a trial and error process to generate the robust results obtained here.

Tetraplex 1: 51, 182, ACE, TPA

1. It is recommended that stocks of 2.5 [micro]M of each primer (51FA, 51RA, ACEF, ACER, 182F, 182R) and 5 [micro]M of TPAF and TPAR be prepared and stored at -20[degrees] C.

2. Due to the small volume, a 0.2 ml tube is recommended for PCR. A master mix (all reagents excluding the DNA) should be made based on the number of students plus a negative control (i.e., for nine students, multiply the amount of each reagent by 10). The fewer the number of individuals who handle the stocks, the less chance there is for DNA contamination. Each reaction consists of:

* 12.5 [micro]l buffer E (Epicentre)

* 1.4 [micro]l 5.0 [micro]M TPAF primer

* 1.4 [micro]l 5.0 [micro]M TPAR primer

* 1.8 [micro]l 2.5 [micro]M ACEF primer

* 1.8 [micro]l 2.5 [micro]M ACER primer

* 0.9 [micro]l 2.5 [micro]M 51FA primer

* 0.9. [micro]l 2.5 [micro]M 51RA primer

* 1.45 [micro]l 2.5 [micro]M 182F primer

* 1.45 [micro]l 2.5 [micro]M 182R primer

* 0.4 [micro]l Epicentre Fail-Safe DNA polymerase

3. Aliquot 24.0 microliters of the master mix into each tube.

4. Have students place 1.0 [micro]l of their DNA preps into the tube that corresponds with their number.

5. Place samples in a thermal cycler and run under the following conditions:
 94[degrees] C for two minutes; followed by 32 cycles of 94[degrees]
 C for 30 seconds, 58[degrees] C for 30 seconds, 72[degrees] C for
 one minute, and then, one final extension cycle of
 72[degrees] C for five minutes, then maintained at 4[degrees] C.
 Since the cycling conditions are the same for both
 tetraplex reactions, these can be run simultaneously
 on the same thermal cycler (we use the MJ Research
 PTC-150).


Tetraplex 2: 9, 60, 225, 50

1. It is recommended that stocks of 5.0 [micro]M of each primer be made.

2. Due to the small volume, a 0.2 ml tube is recommended for PCR. A master mix should be made based on the number of students plus a negative control (i.e., for nine students, multiply the amount of each reagent by 10). The fewer the number of individuals who handle the stocks, the less chance there is for DNA contamination. Each reaction consists of:

* 12.5 [micro]l buffer E (Epicentre)

* 1.1 [micro]l 5.0 [micro]M 50F primer

* 1.1 [micro]l 5.0 [micro]M 50R primer

* 1.2 [micro]l 5.0 [micro]M 60F primer

* 1.2 [micro]l 5.0 [micro]M 60R primer

* 1.2 [micro]l 5.0 [micro]M 225F primer

* 1.2.[micro]l 5.0 [micro]M 225R primer

* 1.2 [micro]l 5.0 [micro]M 9F primer

* 1.2 [micro]l 5.0 [micro]M 9R primer

* 0.4 [micro]l Epicentre Fail-Safe DNA polymerase

* 2.2 [micro]l autoclaved (sterile, nuclease-free) water

3. Aliquot 24.5 microliters of master mix into each tube.

4. Have students place 0.5 [micro]l of their DNA into one tube that corresponds with their selected number.

5. PCR cycling conditions are identical to Tetraplex 1.

Analysis by Agarose Gel Electrophoresis.

1. Prepare a 2% agarose gel in 1 X TAE buffer (0.04 M Trisacetate, 1.0 mM EDTA). I have been using a 50 ml gel in a 7 cm x 10 cm casting tray (Fisher Scientific) with a 10 well (1.5 mm) comb.

2. Place 1 X TAE running buffer in gel chambers covering the gel.

3. Add 2.7 [micro]l of 10X loading buffer to each sample; then load 25 [micro]l of sample per well. Markers providing good resolution for 100-700 bp should be used (I use either a 100 bp ladder or the 1kb+ markers from InVitrogen).

4. Run the electrophoresis gel at about 12 V/cm of length of gel (we use the high voltage, which is 135V, on the BioWorld power supply). Allow the samples to run until the bromophenol blue band is about 1 cm near the end of the gel (the 100 bp fragments run just below it, so do not run the band off the gel).

5. Place the gel in a small container adding about half the buffer. Stain with 25 [micro]l of 10 mg/ml ethidium bromide, shaking for three minutes. Place the ethidium solution in a hazardous liquid waste container. Rinse the gel in deionized distilled water and destain the gel in deionized distilled water for at least 30 minutes (a two to three hour destain yields a nicer picture). Take a picture, preferably with a UV gel documentation system (e.g., Fotodyne) to get the best possible snapshot. Photocopies or pictures on thermal paper can be provided to each member of the class (film would be costly). Alternatively, electronic images may be provided for the students and subsequently printed. If a documentation system is not available, then a Polaroid camera or digital camera system (e.g., Fisher Scientific) may be used with a UV transilluminator.

Safety Considerations

The risk of spreading any infectious agent is probably far less than natural processes such as coughing or sneezing (Bloom et al., 1996). To further minimize risks, each student extracts his/her own DNA. For extra precaution, swabs may be placed in a biohazard bag after placing in isolation solution. Samples are boiled in the extraction process, aiding sterilization. Care is also required using ethidium bromide. Students must wear gloves. Solutions containing ethidium bromide should be collected in a jar as opposed to tossing in the sink, and the gels discarded in a separate solid waste container. Any surface that comes in contact with the gel (e.g., transilluminator) should be rinsed with water. The paper towels and gloves can be placed in the ethidium bromide solid waste container. Additionally, if gels are viewed directly on a UV transilluminator, eye protection that filters out UV light should be worn.

Data Analysis

Two grids have been provided for students to incorporate their data (Figures 3A and 3B), one for each tetraplex reaction. Each student could place his/her number (assigned anonymously) in the empty box under his/her profile. For example, if the genotype for Student Number One for Markers 51, 182, ACE, and TPA was ++, ++, ++, and --, respectively, then Number

One would be placed in the box directly under the corresponding profile, as shown below:
 51 ++ ++ ++
 182 ++ ++ ++
 ACE ++ ++ ++
 TPA ++ +- --

 1

 51 ++ ++ ++
 182 +- +- +-
 ACE ++ ++ ++
 TPA ++ +- --

 51 ++ ++ ++
 182 ++ ++ ++
 ACE +- +- +-
 TPA ++ +- --

 51 ++ ++ ++
 182 +- +- +-
 ACE +- +- +-
 TPA ++ +- --

 51 ++ ++ ++
 182 ++ ++ ++
 ACE -- -- --
 TPA ++ +- --

 51 ++ ++ ++
 182 +- +- +-
 ACE -- -- --
 TPA ++ +- --


Students then have the opportunity to determine the unknown sample, as well as to analyze a sample paternity test and determine which individual is the father (see below).

Results

Generation of DNA profiles

Using crude DNA preps from cheek cells, clearly identifiable multiplex PCR products were attainable from all the individuals analyzed. The process is simple and utilizes standard equipment generally found in colleges and universities. On occasion, slight modifications may be made. Depending on results obtained, it may sometimes be helpful to optimize primer concentrations depending on the intensities of the bands, or altering the amount of DNA used (compensating with water). The conditions as presented yielded reproducible results. A sample profile for Tetraplex 1 can be observed in Figure 4 and in Kass (2003). The profile with band size for all eight alleles would be as follows:
51 (+ Alu) 652 bp

182 (+ Alu) 563 bp

ACE (+ Alu) 490 bp

TPA (+ Alu) 400 bp

51 (- Alu) 355 bp

182 (- Alu) 287 bp

ACE (- Alu) 190 bp

TPA (- Alu) 100 bp


[FIGURE 4 OMITTED]

A sample profile for Tetraplex 2 is presented in Figure 5. The profile with band size for all eight alleles would be as follows:
9 (+ Alu) 655 bp

60 (+ Alu) 522 bp

225 (+Alu) 447 bp

50 (+ Alu) 406 bp

9 (- Alu) 322 bp

60 (- Alu) 205 bp

225 (- Alu) 135 bp

50 (-Alu) 101 bp


[FIGURE 5 OMITTED]

Of interest was the finding that there are two variants of the 225 (+ Alu) allele. These differ slightly in size and are indicated by a bracket in Figure 5. Sequencing of these variants is underway to determine the nature of the size difference. Although this further increases the number of potential profiles (three variants instead of two for the 225 locus), for purposes of using the grids and maintaining simplicity of this analysis, both 225 + Alu forms were simply considered presence (+) variants.

Use in Forensics

For the class, one student sample was chosen by the instructor to represent the unknown. A small aliquot of this sample was placed in a tube and labeled "u." The goal was to identify the individual and rule out all other "suspects." Various "stories" can be devised to make the lab more interesting. In this case, we said that this sample was from a hair follicle left near the location of the stolen balance, and all students in the class were suspects. Fifteen student samples were then analyzed and two individuals had the same genotype using Tetraplex 1, but were then differentiated by Tetraplex 2; also two individuals had the same genotype using Tetraplex 2 but were differentiated by Tetraplex 1. Therefore, upon using the two tetraplex reactions, each student had a unique profile, and the unknown sample was clearly identified with all others ruled out. Hence this demonstrates the applicability of this technique as a forensic science tool.

Use in Paternity Testing

For this analysis I used DNA from a family (husband, wife, and two children) and DNA from two additional individuals suggesting them as potential fathers of the children (Figure 5). Based simply on Tetraplex 2, both Prospective Father #1 and Prospective Father #2 were ruled out, leaving Prospective Father #3 as the only viable candidate. Although this test does not provide the over 99% accuracy that commercial tests do, it allows students to assess how paternity tests are performed. Basically, if the child has alleles that were not present in the mother, then he/she must have received it from the father. An analysis of Child #1 finds a 9+ band which is absent in the mother and Prospective Father #1; hence this ruled him out. Child #1 also has the 60- and 50- alleles which are absent in the mother and Prospective Father #2 which then ruled him out, leaving Father #3 as the only viable candidate. Child #2 also has 9+, which rules out Father #1, and has the 50- allele, which ruled out Father #2 (who lacks the allele), again leaving Father #3.

Discussion

I have presented a simple rapid method of generating complex DNA profiles that can easily be performed in an undergraduate laboratory. Students have an opportunity to analyze their own DNA and incorporate it into a forensic analysis. Additionally, with the same technique, students have the opportunity to assess paternity. Furthermore, students can use the data to determine allele frequencies and perform Hardy-Weinberg analysis. This laboratory exercise was originally developed for an undergraduate genome analysis course, which has been incorporated as part of a bioinformatics program. We plan on developing a computer-based program to incorporate allele frequencies in a manner prepared for the PV92 Alu locus (http://www.bioservers.org/bioserver). Students would then have the opportunity to incorporate their (anonymous) profiles into a database. Students also have the opportunity to learn about retrotransposons, which account for roughly one-third of the human genome (Lander et al., 2001). For more adventurous laboratories, additional profiles can be generated using intermediate frequency Alu dimorphisms found in Roy-Engel et al. (2001) and Carroll et al. (2001) with the approach presented by Kass (2003). Also, profiles can be modified by substituting alternative dimorphic Alu loci in the multiplex reactions.

Acknowledgments

This work was supported by National Science Foundation Grant DUE-0126640, and a Faculty Research and Creative Activity Fellowship award from Eastern Michigan University. I thank Dr. Daniel Clemans for helpful comments on the manuscript.

References

Batzer, M.A., Stoneking, M., Alegria-Hartman, M., Bazan, H., Kass, D.H., Shaikh, T.H., Novick, G.E., Ioannou, P.A., Scheer, W.D., Herrera, R.J. & Deininger, P.L. (1994). African origin of human-specific polymorphic Alu insertions. Proceedings of the National Academy of Science U. S. A., 91,12288-12292.

Bloom, M.V., Freyer, G.A. & Micklos, D.A. (1996). Laboratory DNA Science. Menlo Park, CA: The Benjamin/ Cummings Publishing Company, Inc.

Carroll, M.L., Roy-Engel, A.M., Nguyen, S.V., Salem, A.H., Vogel, E., Vincent, B., Myers, J., Ahmad, Z., Nguyen, L., Sammarco, M., Watkins, W.S., Henke, J., Makalowski, W., Jorde, L.B., Deininger, P.L. & Batzer, M.A. (2001). Large-scale analysis of the Alu Ya5 and Yb8 subfamilies and their contribution to human genomic diversity. Journal of Molecular Biology, 311,17-40.

Kass, D.H. (2003). Generation of human DNA profiles by Alu-based multiplex polymerase chain reaction. Analytical Biochemistry, 321,146-149.

Katzov, H., Bennet, A.M., Kehoe, P., Wiman, B., Gatz, M., Blennow, K., Lenhard, B., Pedersen, N.L., de Faire, U. & Prince, J.A. (2004). A cladistic model of ACE sequence variation with implications for myocardial infarction, Alzheimer disease and obesity. Human Molecular Genetics, 13,2647-2657.

Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., et al. (2001). Initial sequencing and analysis of the human genome. Nature, 409,860-921.

Lindpaintner, K., Pfeffer, M.A., Kreutz, R., Stampfer, M.J., Grodstein, F., LaMotte, F., Buring, J. & Hennekens, C.H. (1995). A prospective evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. New England Journal of Medicine, 332,706-711.

Niu, T., Chen, X. & Xu, X. (2002) Angiotensin converting enzyme gene insertion/deletion polymorphism and cardiovascular disease: therapeutic implications. Drugs, 62,977-993.

Phelps, T.L., Deering, D.G. & Buckner, B. (1996). Using the polymerase chain reaction in an undergraduate laboratory to produce "DNA fingerprints". The American Biology Teacher, 58,106-110

Pilate M., Cicoira, M., Zanolla, L., Nicoletti, I., Muraglia, S. & Zardini, P. (2004). The role of angiotensin-converting enzyme polymorphism in congestive heart failure. Congestive Heart Failure, 10,87-95.

Roy-Engel, A.M., Carroll, M.L., Vogel, E., Garber, R.K., Nguyen, S.V., Salem, A.H., Batzer, M.A. & Deininger, P.L. (2001). Alu insertion polymorphisms for the study of human genomic diversity. Genetics, 159,279-290.

David H. Kass is Professor of Biology, Eastern Michigan University, Department of Biology, Ypsilanti, MI 48197; e-mail: dkass@emich.edu.
Table 1. Loci and oligonucleotide primers utilized in multiplex PCR
in this study. ACE and TPA 25 are described in Batzer
et al. (1994); Yb8NBC9, Yb8NBC225, Ya5NBC182, and YaNBC51
are described in Carroll et al. (2001); and Yc1NBC50 and
YC1MNC60 are described in Roy-Engel et al. (2001).

Alu I.D. Primer F

ACE 5'-ctggagaccactcccatcctttct-3'
TPA 25 5'-gtaagagttccgtaacaggacagct-3'
Yb8NBC9 5'-gtccccaccaatccctatct-3'
Yc1NBC50 5'-ggtatggggccaaatttaatcca-3'
Yb8NBC225 5'-gagtccagcccattttagca-3'
Yc1NBC60 5'-gaaaccgccaagattctcacc-3'
Ya5NBC51A * 5'-tttccttacatctagtgcccc-3'
Ya5NBC182 5'-gaaggactatgtagttgcagaagc-3'

Alu I.D. Primer R + Alu

ACE 5'-gatgtggccatcacattcgtcagat-3' 490
TPA 25 5'-ccccaccctaggagaacttctcttt-3' 400
Yb8NBC9 5'-tgctcaaagtcccacagcta-3' 655
Yc1NBC50 5'-tccaagagaagccaaacctacaga-3' 406
Yb8NBC225 5'-cccagcacaaacatgtcatt-3' 447
Yc1NBC60 5'-tctccatcatgattcccaactga-3' 522
Ya5NBC51A * 5'-cctccaagtaaagctacaccct-3' 652
Ya5NBC182 5'-aacccagtggaaacagaagatg-3' 563

Alu I.D. - Alu Chr. #

ACE 190 17
TPA 25 100 8
Yb8NBC9 322 14
Yc1NBC50 101 5
Yb8NBC225 135 12
Yc1NBC60 205 10
Ya5NBC51A * 355 3
Ya5NBC182 287 7

* Primer set redesigned in Kass (2003).

Figure 3. Grids to incorporate genotype data. A. Tetraplex 1. B.
Tetraplex 2. Empty boxes below the genotype allow for the
incorporation of profiles for each individual.

 51 + + + + + +
 182 + + + + + +
 ACE + + + + + +
 TPA + + + - - -

 51 + + + + + +
 182 + - + - + -
 ACE + + + + + +
 TPA + + + - - -

 51 + + + + + +
 182 - - - - - -
 ACE + + + + + +
 TPA + + + - - -

 51 + - + - + -
 182 + + + + + +
 ACE + + + + + +
 TPA + + + - - -

 51 + - + - + -
 182 + - + - + -
 ACE + + + + + +
 TPA + + + - - -

 51 + - + - + -
 182 - - - - - -
 ACE + + + + + +
 TPA + + + - - -

 51 - - - - - -
 182 + + + + + +
 ACE + + + + + +
 TPA + + + - - -

 51 - - - - - -
 182 + - + - + -
 ACE + + + + + +
 TPA + + + - - -

 51 - - - - - -
 182 - - - - - -
 ACE + + + + + +
 TPA + + + - - -

 9 + + + + + +
 60 + + + + + +
 225 + + + + + +
 50 + + + - - -

 9 + + + + + +
 60 + - + - + -
 225 + + + + + +
 50 + + + - - -

 9 + + + + + +
 60 - - - - - -
 225 + + + + + +
 50 + + + - - -

 9 + - + - + -
 60 + + + + + +
 225 + + + + + +
 50 + + + - - -

 9 + - + - + -
 60 + - + - + -
 225 + + + + + +
 50 + + + - - -

 9 + - + - + -
 60 - - - - - -
 225 + + + + + +
 50 + + + - - -

 9 - - - - - -
 60 + + + + + +
 225 + + + + + +
 50 + + + - - -

 9 - - - - - -
 60 + - + - + -
 225 + + + + + +
 50 + + + - - -

 9 - - - - - -
 60 - - - - - -
 225 + + + + + +
 50 + + + - - -

 51 + + + + + +
 182 + + + + + +
 ACE + - + - + -
 TPA + + + - - -

 51 + + + + + +
 182 + - + - + -
 ACE + - + - + -
 TPA + + + - - -

 51 + + + + + +
 182 - - - - - -
 ACE + - + - + -
 TPA + + + - - -

 51 + - + - + -
 182 + + + + + +
 ACE + - + - + -
 TPA + + + - - -

 51 + - + - + -
 182 + - + - + -
 ACE + - + - + -
 TPA + + + - - -

 51 + - + - + -
 182 - - - - - -
 ACE + - + - + -
 TPA + + + - - -

 51 - - - - - -
 182 + + + + + +
 ACE + - + - + -
 TPA + + + - - -

 51 - - - - - -
 182 + - + - + -
 ACE + - + - + -
 TPA + + + - - -

 51 - - - - - -
 182 - - - - - -
 ACE + - + - + -
 TPA + + + - - -

 9 + + + + + +
 60 + + + + + +
 225 + - + - + -
 50 + + + - - -

 9 + + + + + +
 60 + - + - + -
 225 + - + - + -
 50 + + + - - -

 9 + + + + + +
 60 - - - - - -
 225 + - + - + -
 50 + + + - - -

 9 + - + - + -
 60 + + + + + +
 225 + - + - + -
 50 + + + - - -

 9 + - + - + -
 60 + - + - + -
 225 + - + - + -
 50 + + + - - -

 9 + - + - + -
 60 - - - - - -
 225 + - + - + -
 50 + + + - - -

 9 - - - - - -
 60 + + + + + +
 225 + - + - + -
 50 + + + - - -

 9 - - - - - -
 60 + - + - + -
 225 + - + - + -
 50 + + + - - -

 9 - - - - - -
 60 - - - - - -
 225 + - + - + -
 50 + + + - - -

 51 + + + + + +
 182 + + + + + +
 ACE - - - - - -
 TPA + + + - - -

 51 + + + + + +
 182 + - + - + -
 ACE - - - - - -
 TPA + + + - - -

 51 + + + + + +
 182 - - - - - -
 ACE + - + - + -
 TPA + + + - - -

 51 + - + - + -
 182 + + + + + +
 ACE - - - - - -
 TPA + + + - - -

 51 + - + - + -
 182 + - + - + -
 ACE - - - - - -
 TPA + + + - - -

 51 + - + - + -
 182 - - - - - -
 ACE - - - - - -
 TPA + + + - - -

 51 - - - - - -
 182 + + + + + +
 ACE - - - - - -
 TPA + + + - - -

 51 - - - - - -
 182 + - + - + -
 ACE - - - - - -
 TPA + + + - - -

 51 - - - - - -
 182 - - - - - -
 ACE - - - - - -
 TPA + + + - - -

 9 + + + + + +
 60 + + + + + +
 225 - - - - - -
 50 + + + - - -

 9 + + + + + +
 60 + - + - + -
 225 - - - - - -
 50 + + + - - -

 9 + + + + + +
 60 - - - - - -
 225 + - + - + -
 50 + + + - - -

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Title Annotation:Inquiry & Investigation
Author:Kass, David H.
Publication:The American Biology Teacher
Date:Mar 1, 2007
Words:4164
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