instructor's lab manual - promega

Solving a Crime Using DNAAnalysis and Chemistry(basic forensics): Instructor’s Manual

I. Purpose and Concepts Covered .........................................................................1

II. Protocol for Crime Scene Investigation Using RFLP Analysis.........................2

A. Preparation for the Laboratory.........................................................................2

B. Teaching Suggestions .....................................................................................3

C. The Scenario: The Last Bag of Chocolate Chips.............................................4

D. Chromatography of Marker Inks ......................................................................5

E. RFLP Analysis of DNA Samples .....................................................................6

III. STR-Based Analysis of DNA Using Silver-Stained Gel .....................................9

A. Before You Begin .............................................................................................9

B. Amplification ..................................................................................................10

C. Polyacrylamide Gel Preparation ....................................................................12

D. Polyacrylamide Gel Electrophoresis ..............................................................15

E. Silver Staining................................................................................................17

F. Generating Film Images ................................................................................19

G. Analyzing Data ..............................................................................................20

H. Obtaining Single-Source Human Genomic DNA...........................................23

IV. STR Analysis of DNA Using the PowerPlex® 16 System ................................24

V. Supplier and Ordering Information ...................................................................25

VI. Resources ...........................................................................................................26

I. Purpose and Concepts Covered

This introductory forensics laboratory is for use in courses that cover basic topics in molecular biology and genetics. While modern DNA-based forensics uses short tandemrepeat (STR) analysis, the first lab in this manual uses restriction fragment length poly-morphism (RFLP) analysis. RFLP analysis is the conceptual predecessor for modernSTR analyses conducted by crime scene investigators and medical laboratories.

We have included simple chromatography analyses of evidence in addition to the DNAanalysis to remind students that crime scene evidence includes more than DNA.

This teaching unit has a second laboratory for instructors of more advanced students.The second laboratory is an STR-based laboratory and will require access to sophisti-cated laboratory equipment including a polyacrylamide gel apparatus. Instructors maychoose to run all or part of this lab as a demonstration for students.

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II. Protocol

II.A. Preparation for the Laboratory

Note to the Instructor

The identification of the "perpetrator" of the crime is based on RFLP analysis, a con-cept that is similar to STR analysis, which is used to identify individuals in modernforensics laboratories. A completely separate laboratory protocol for institutions thathave the ability to conduct STR-based analyses is also included in this unit.

The main exercise in this laboratory is the RFLP analysis of DNA. However, addi-tional evidence may be analyzed, including the note found at the crime scene. Thesimple chromatography experiment is included to encourage students to view DNAevidence as one piece of the puzzle of a crime scene. You can work with a chemistryfaculty member to "dress up" the chemical analysis of the crime scene, if you wouldlike to cover more complex chemical topics along with the DNA analysis.

Materials Required

For Chromatography Experiments

• crime scene tape• empty glass (one for each team of students)• two black markers of different brands (one each for each team of students)• 500 ml beakers (two for each team of students)• acetone• gloves• 3 mm Whatman filter paper• hole punch• glass stiring rod

For RFLP Analysis

• XmnI restriction enzyme (5 units or 0.5 µl at 12 u/µl concentration)• HincII restriction enzyme (6 units or 1.5 µl at 10 u/µl concentration)• nuclease-free water• Buffer B (restriction enzyme buffer)• DNA sample from crime scene (pGL4.11[luc2P] Vector, Cat.# E6661; 1 µg for

each team of students)• DNA sample from suspect one (pGL4.11[luc2P] Vector, Cat.# E6661; 1 µg for

each team of students)• DNA sample from suspect two (pGL4.12[luc2CP] Vector, Cat.# E6671; 1 µg for

each team of students)• Agarose LE, Analytical Grade (Cat.# V3121)• TBE Buffer, 10X (Cat.# V4251)• ethidium bromide stock solution, 10 mg/ml• DNA gel electrophoresis apparatus and power supply• UV light box and camera or scanner• pipettors and pipet tips• 1.5 ml sterile tubes• DNA markers (BenchTop pGEM® DNA Markers Cat.# G7521)• gloves• acetylated BSA (provided with restirction enzymes)• 37°C water bath• 65°C water bath• Blue/Orange Loading Dye, 6X (Cat.# G1881)



II.B. Teaching Suggestions

1. Personalize this scenario with names and locations from your institution, orif you have another idea for a crime scenario that will work with this labora-tory, write your own case to solve.

2. Create a learning community around this laboratory. Have genetics andchemistry students perform the scientific analysis. Ask students in prelaw,criminal justice or other appropriate majors to "prosecute" the case. Thecase can go to trial before a jury of peers from the college campus. Sciencestudents can act as "expert" witnesses to explain the evidence and testingto the rest of the community. Mass communications students can "cover" thecase.

3. This laboratory builds on several other laboratories.

For instance, DNA has to be isolated from a crime scene before it can beanalyzed. Consider leading your students through Unit 4: Genomic DNAPurification before performing this laboratory to give them background inDNA isolation.

Additionally, STR analysis requires amplification of DNA using PCR. PCR isintroduced in Unit 2: The Chemistry of Inheritance.

These units are available at www.promega.com/education

Each of these other units has laboratories, lectures and other teachingmaterials like animations that can be used in concert with this laboratory.

4. Have the students make all of their stock and working solutions to reinforcemoles-to-gram concepts taught in basic chemistry.



II.C. The Scenario: The Last Bag of Chocolate Chips

The manager of the college food services promised the president of the college aplate of his favorite chocolate chip cookies for the upcoming meeting with the execu-tive council of the Board of Directors. The chocolate chip cookies are a specialty ofChef Lombardo's and are famous across the college campus. Almost like Pavlov'sdogs, students, professors, faculty and staff emerge from their offices and dormrooms in a grand migration toward the Dining Hall when the aroma of these cookiesbaking drifts across campus. The appeal of these cookies can be traced, in part, tothe chocolate chips that are imported from Europe.

The day before the board meeting, Chef Lombardo checked the pantries in the mainkitchen and discovered that he only had one bag of these special chocolate chipsleft. He placed all of the ingredients onto the lower shelf of the pantry so that hewould be ready to bake the cookies first thing the next morning. Chef Lombardolocked the pantry, turned off the kitchen lights and left for the night.

When Chef Lombardo returned the next morning, he was greeted with a horrificsight. The door to the kitchen had been pried open, and the metal door on the pantrywas bent and torn where someone had pried the lock of the pantry door. The ingre-dients for the cookies were scattered around the kitchen. The bags of sugar andflour were busted on the floor. The open and empty bag of chocolate chips lay on thekitchen counter along with a glass that had lipstick marks on it and a note printed inblack marker that read:

This is just to sayI have eatenthe chocolatethat was inthe pantry

and whichyou were probablysavingfor tomorrow.

Forgive meit was deliciousso sweetand so warm.

Apparently the thief had a literary bent, having adapted text from William CarlosWilliams for the thank you note. The thief had cut his or her hand on the pantry doorand left blood stains.

Chef Lombardo immediately called the college president to report the tragedy. Thecriminal justice, genetics and general chemistry professors were all then called tothe crime scene.

The investigators interviewed a group of students who had returned home late froman outing and said that they had seen two professors walking back to campus at2:00 am. The professors were walking toward the dining hall. One of the professors,Dr. Johnson in history, sat on the executive council as the faculty representative tothe board and would have known about the chocolate chip cookies for the meeting.



Interestingly, both Dr. Johnson and the other professor, Dr. Lundquist in EnglishLanguage Arts, each had a hand bandaged when they were interviewed. The chem-istry professor confiscated the black markers that were on each professor's desk forfurther analysis. Each professor also gave buccal swab sample for DNA analysis.

II.D. Chromatography of Marker Inks

Before the Lab

1. Create a mock crime scene with evidence for your students to analyze. On apiece of 3 mm Whatman filter paper use one of the markers to write thethank you note found at the crime scene. Be sure to leave enough roombetween the bottom of the filter paper and the writing (about 2.5 cm) forchromatography. Make sure that the writing covers a sufficient area that thenote can be cut into strips and distributed to each team of students.

2. Each team of students should receive two markers, representative of thekind collected from the suspects.

Lab Protocol

Note: Be sure to wear gloves when you handle the thank you note and yourown filter paper.

1. Obtain a cut strip from the thank you note found at the crime scene.

2. Cut a clean piece of filter paper into two strips. Draw a line across the widthof the first strip using one of your sample markers. Do the same thing withthe other strip and the other marker. Your mark should be about 2.5 cmabove the bottom of the strip.

3. Make a hole using a hole punch at the top of your evidence and samplestrips.

4. Space all three strips along a glass stirring rod.

5. Carefully add acetone to the beaker so that it will justcover the bottom 0.5cm of the filter paper strips. Theexact volume will depend on how far down the stripsextend into the beaker when they are suspended bythe glass stirring rod.

6. Allow the acetone to wick up the filter paper strips. Once the acetone hasreached the top of the strips, remove them from the beaker and allow themto dry. (Place them on a nonabsorbent surface or hang them to dry).

Analysis

1. Can you see a difference between the inks of the two sample markers?

2. Does either one of them clearly give the same chromatography pattern asthe ink on the note?

3. Can you determine which type of marker was used to write the note? Whyor why not? If you think additional experiments are needed, what would youdo?



II.E. RFLP Analysis of DNA Samples

Before the Lab

1. Distribute the crime scene DNA, suspect 1 DNA and suspect 2 DNA to thestudents.

2. Label 9 tubes for the following restriction enzyme digests:

Reaction Tube #Crime scene sample Hinc II digest 1Crime scene sample Xmn I digest 2Crime scene sample HincII/XmnI digest 3Suspect 1 sample Hinc II digest 4Suspect 1 sample XmnI digest 5Suspect 1 sample Hinc II/XmnI digest 6Suspect 2 sample Hinc II digest 7Suspect 2 sample XmnI digest 8Suspect 2 sample Hinc II/XmnI digest 9

Restriction Enzyme Digestion of DNA Samples

1. Prepare the following master mixes for your single-enzyme digests asdirected in the table below. Prepare enough master mix for 8 digests tocompensate for pipetting errors.

Master Mix for Single Digests

2. To the three HincII digests (Tubes 1, 4 and 7), add the following:

Single-Digest Master Mix 18.5 µlDNA (1µg/ml) 1.0 µlHinc II (12 U/µl) 0.5 µl

3. To the three XmnI digests (Tubes 2, 5 and 8), add the following:

Single-Digest Master Mix 18.5 µlDNA (1 µg/ml) 1.0 µlXmn I (10 U/µl) 0.5 µl

4. Prepare the master mix for your double-enzyme digests according to thetable below. Prepare enough Master Mix for 5 digests to compensate forpipetting errors.

Master Mix for Double Digests

ComponentVolume Needed for Each Reaction

Volume Needed for 8 Reactions

Nuclease-free water 16.3 µl 130.4 µl

10X Buffer B 2.0 µl 16.0 µl

Acetylated BSA 0.2 µl 1.6 µl

ComponentVolume Neededfor Each Reaction

Volume Needed for 5 Reactions

Nuclease-free water 15.8 µl 79.0 µl

10X Buffer B 2.0 µl 10.0 µl

Acetylated BSA 0.2 µl 1.0 µl



5. To the three double-enzyme digests (Tubes 3, 6 and 9), add the following:

Double-Digest Master Mix 18.0 µlDNA (1µg/ml) 1.0 µlHinc II (12 u/µl) 0.5 µlXmn I (10 u/µl) 0.5 µl

6. Incubate the digests for 2 hours at 37°C.

7. Heat inactivate the reactions by placing the tubes at 65 °C for 15 minutes.

Notes:

The double digest will result in 5 fragments for pGL4.12 and 4 fragments forpGL4.11. This is because Xmn I does not cut pGL4.11.

Fragments from double digest of pGL4.12: 2215bp, 1306bp, 463bp, 328bp,and 110bp.

Fragments from double digest of pGL4.11: 1306bp, 740bp, 110bp and2214bp.

Agarose Gel Electrophoresis of Restricted DNA

1. Prepare 1X TBE for your Gel Running Buffer.

2. Weigh out the required amount of agarose, and add it to the appropriateamount of 1X TBE buffer in a flask or bottle. For example to prepare a 2%agarose gel, add 2.0 g of agarose to 100 ml of buffer.

3. Heat the mixture in a microwave oven or on a hot plate for the minimum timerequired to allow all of the agarose to dissolve. Interrupt the heating at regu-lar intervals, and swirl the contents. Do not allow the solution to boil over.

3. Cool the solution to 50–60 °C and pour the gel. Be sure to insert a gel combto create sample wells. Allow the gel to cool completely. Remove the combfrom the gel and place the gel in the electrophoresis apparatus.

4. To analyze samples on the gel, prepare the following:

Enzyme digest 5 µlBlue/Orange Loading Dye, 6X 1 µl

5. Add enough 1XTBE gel running buffer to cover the gel.

6. Load the samples onto the gel, and run at the voltage recommended by thethe gel box manufacturer.

7. Run the gel until the orange dye front (runs at approximately the same rateas a 50 bp piece of DNA) is near the bottom of the gel.

8. Remove the gel, and stain it by soaking it in a solution of 0.5 µg/ml ethidiumbromide (this is diluted from the 10 mg/ml) for 30 minutes at room tempera-ture.

Note: Ethidium bromide is a carcinogen. Wear gloves and, be sure to dis-pose of it in accordance with your institution’s guidelines.

9. Place the gel on a UV light box, and photograph the gel. Wear protectiveeyewear when using the UV light box. If the gel is too orange you candestain the gel in water for a few minutes at room temperature.



Analysis

1. Do either of your suspect DNA samples have the same RFLP pattern as thecrime scene sample?

2. The two DNA samples probably have different restriction fragments. Canyou figure out why (what is the polymorphism that you were able to detect)?

3. What is the purpose of the heat inactivation step at the end of the reaction ?

4. How does agarose gel electrophoresis separate DNA fragments?

5. TBE buffer is composed of tris, borate and EDTA. What function does eachof these ingredients serve in the buffer?

6. What is the function of the running buffer during agarose gel electrophore-sis? Can you use deionized water instead?

7. Many chemicals stain DNA. Compare the way the following DNA stainswork: ethidium bromide, propidium iodide, methylene blue and DAPI.

RFLP analysis of crime scene and suspect DNA.

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III. STR-Based Analysis of DNA and Silver Stain Gel

III.A. Before You Begin

This protocol will require running a polyacrylamide gel. You will need glass platesand the accompanying gel electrophoresis apparatus. You can purchase precast 6%polyacrylamide gels from GE Life Sciences and Invitrogen, but you will also need theappropriate gel electrophoresis apparatus for the precast gels.

The quality of the purified DNA sample and choice of thermal cycler, as well as smallchanges in buffers, ionic strength, primer concentrations, and thermal cycling condi-tions, can affect amplification. We suggest strict adherence to recommended proce-dures for amplification, denaturing gel electrophoresis, silver stain analysis andrecording data on film.

PCR-based STR analysis is subject to contamination by very small amounts ofhuman DNA. Extreme care should be taken to avoid cross-contamination whenpreparing sample DNA, handling primer pairs, setting up amplification reactions andanalyzing amplification products. Reagents and materials used prior to amplification(STR 10X Buffer, K562 Control DNA and 10X Primer Pairs) are provided in a sepa-rate box and should be stored separately from those used following amplification(allelic ladders, STR 2X Loading Solution and pGEM® DNA Markers).

Always include a negative control reaction (i.e., no template) to detect reagent cont-amination. We highly recommend the use of gloves and aerosol-resistant pipettetips.

Some of the reagents used in the analysis of STR products are potentially haz-ardous and should be handled accordingly. Table 1 describes the potential hazardsassociated with such reagents.

Table 1. Hazardous Reagents

Reagent Hazard

acetic acid (fix/stop solution) acetic acid (fix/stop solution)

acrylamide suspected carcinogen, neurotoxin

ammonium persulfate oxidizer, corrosive

bisacrylamide toxic, irritant

formaldehyde (staining solution anddeveloper solution) highly toxic, suspected carcinogen

formamide (STR 2X Loading Solution) irritant, teratogen

methacryloxypropyltrimethoxysilane(bind silane) toxic, moisture sensitive

silver nitrate (staining solution) highly toxic, oxidizer

sodium thiosulfate (developer solution) irritant, hygroscopic

TEMED corrosive, flammable

urea irritantxylene cyanol FF (STR 2X LoadingSolution) irritant

Note: Be sure to followyour institution’s safetyguidelines and proceduresfor using and disposing ofhazardous materials.

Note: To avoid workingwith unpolymerized acry-lamide and several otherchemicals in Table 1, useprecast acrylamide gels.Remember that you willneed a gel electrophoresisapparatus that can acco-modate whatever precastgel you chose.



III.B. Amplification

The GenePrint® STR Systems were developed for amplification without artifactsusingTaq DNA polymerase. Use the buffer provided in the kit for your amplificationswith Taq DNA polymerase.

This protocol is for the amplification of CTT and amelogenin, allowing your students toprofile three STR loci and determine whether the DNA is of a male or female origin.

Materials to Be Supplied by the User• GenePrint® System (CSF1PO, TPOX, TH01; Cat.# DC6001• GenePrint® Sex Identification, Amelogenin (Silver Detection; Cat.# DC4081)• bind silane• silver nitrate• formaldehyde, 37%• sodium thiosulfate, 10mg/ml• sodium carbonate• thermal cycler, model 480 or GeneAmp® system 9600 (Perkin-Elmer)• microcentrifuge• Taq DNA polymerase (GoTaq® DNA Polymerase Cat.# M3001)• Nuclease-Free Water (Cat.# P1193 or equivalent)• Mineral Oil (Cat.# DY1151 or equivalent)• 0.5 ml or 0.2 ml microcentrifuge tubes (compatible with thermal cycler)• 1.5 ml microcentrifuge tubes• BSA Fraction V (optional)• aerosol-resistant pipette tips• crushed ice

The CTT multiplex and GenePrint® Sex Identification System, Amelogenin are opti-mized for use with GeneAmp® reaction tubes and the Perkin-Elmer model 480 ther-mal cycler. When using a thermal cycler on which a system was not optimized, theremay be a loss in product yield or sensitivity, and the balance between loci may changeslightly. Meticulous care must be taken to ensure successful amplification. See ourWeb site or contact Technical Services for help optimizing amplification conditions.

Amplification Setup

We highly recommend that you wear gloves and use aerosol-resistant pipet tips toprevent contamination.

1. Thaw the STR 10X Buffer and 10X Primer Pairs, and place on ice.

Note: Mix reagents by vortexing for 15 seconds before each use.

2. Place one clean, autoclaved 0.5 ml reaction tube for each reaction into arack, and label appropriately.

3. Determine the number of reactions to be set up. This should include a posi-tive and negative control reaction. Add 1 or 2 reactions to this number tocompensate for pipetting error. While this approach does consume a smallamount of each reagent, it ensures that you will have enough PCR mastemix for all samples.

4. Calculate the required amount of each component of the PCR master mix(Table 2). Multiply the volume (µl) per sample by the total number of reac-tions (from Step 3) to obtain the final volume (µl).

Note: This STR-based pro-tocol will not work with thepGL4 plasmids used in theRFLP laboratory (SectionII). You must use single-source human genomicDNA for this laboratory.See Section III.H for sug-gestions for DNA sources.



III.B. Amplification (continued)

5. In the order listed in Table 2, add the final volume of each reagent to a sterile tube. Mix gently (do not vortex), and place on ice.

Note: The volume given assumes a Taq DNA polymerase concentration of5 u/µl. For different enzyme concentrations, the volume of enzyme added-must be adjusted accordingly. If the final volume of Taq DNA polymeraseadded to the master mix is less than 0.5 µl, you may wish to dilute theenzyme with STR 1X Buffer, and add a larger volume. The amount of sterilewater should be adjusted accordingly so that the final volume per reaction is25 µl. Do not store diluted Taq DNA polymerase.

6. Add 22.5 µl of PCR master mix to each tube, and place on ice. Failure tokeep the reagents and samples on ice can produce imbalanced amplifica-tion of multiplexed loci.

7. Pipet 2.5 µl of each sample into the respective tube containing 22.5 µl ofPCR master mix.

8. For the positive amplification control, pipet 2.5 µl (5ng) of K562 DNA (dilutedto 2 ng/µl) into a 0.5ml reaction tube containing 22.5 µl of PCR master mix.

9. For a negative amplification control, pipet 2.5 µl of sterile water (instead oftemplate DNA) into a 0.5ml reaction tube containing 22.5 µl of PCR mastermix.

10. If you are using a thermal cycler with an unheated lid, add 1 drop of mineraloil to each tube. Close the tubes.

Note: Allow the mineral oil to flow down the side of the tube and form anoverlay to limit sample loss or cross-contamination due to splattering.

11. Centrifuge the samples briefly to bring the contents to the bottom of thetube.

Table 2. Combined CTTv Multiplex and Amelogenin Reactions

PCR Master MixComponent

Volume Per Sample(µl)

Number of Reactions Final Volume (µl)

sterile water 14.85

STR 10X Buffer 2.50

CTT Multiplex 10XPrimer Pair 2.50

Amelogenin 10XPrimer Pair 2.50

Taq DNA Polymerase(5u/µl) 0.15 (0.75 u)

Total volume 22.50

× =

Thermal Cycling Protocol

1. Place the tubes in a thermal cycler.

2. Run the protocol below:

Initial Incubation: 96 °C for 2 minutes

Programmed Ramp Times: None

First 10 Cycles94 °C for 1 minute64 °C for 1 minute70 °C for 1.5 minutes

Programed Ramp Times: None

Last 20 Cycles90 °C for 1 minute64 °C for 1 minute70 °C for 1.5 minutes

Extension Step: None

Hold at 4 °C.

3. After completing the thermal cycling protocol, store the samples at –20 °C.

Note: Storing the amplification products at our above 4 °C may result indegradation products.

III.C. Polyacrylamide Gel Preparation

Materials to Be Supplied by the User

• 40% acrylamide:bis (19:1) and TEMED• TBE Buffer, 10X (Cat.# V4251)• Ammonium Persulfate, 10% (Cat.# V3131)• Urea (Cat.# V3171)• bind silane (methacryloxypropyltrimethoxysilane)• Gel Slick® solution (Cambrex Cat.# 50640)• 0.5% acetic acid in 95% ethanol• Nalgene® tissue culture filter (0.2 micron)• polyacrylamide gel electrophoresis apparatus for gels ≥ 30cm (e.g.,

SA32 or S2)• glass plates and side spacers for polyacrylamide gel ≥ 30cm• 14 cm vinyl doublefine sharkstooth comb(s), 49 point, 0.4mm thick; or

square-tooth comb, 35 cm, 60 wells (cut in half for 30 wells/gel), 0.4 mmthick (Owl Scientific Cat.# S2S-60A)

• power supply• Liqui-Nox® detergent (Use of Liqui-Nox® detergent is extremely

important, because other kinds of detergent can build up on the glassplates.)

• clamps (e.g., large office binder clips)• diamond pencil for marking glass plates





Notes

1. Use a 4% gel for separation of the CTTv and amelogenin loci.

2. Unpolymerized acrylamide is a neurotoxin and suspected carcinogen; avoidinhalation and contact with skin. Read the warning label, and take the nec-essary precautions when handling this substance. Always wear gloves andsafety glasses when working with acrylamide powder or solutions.

3. Bind silane is toxic and should be used in a chemical fume hood.

4. The longer glass plate will be treated with Gel Slick® solution to prevent thegel from sticking, and the shorter glass plate will be treated with bind silaneto bind the gel. The two plates must be kept apart at all times to preventcross-contamination.

5. All cleaning utensils (sponges) for the longer glass plates should be keptseparate from those for the shorter glass plates to prevent cross contamina-tion of the binding solution.

6. The shorter glass plate preparation must be repeated for each gel. Thelonger glass plate preparation must be repeated after every four gels.

7. To remove the glass plate treatments (Gel Slick® solution or bind silane)immerse the plate(s) in 10% NaOH solution for 1 hour. Thoroughly rinse theplate(s) with deionized water, and clean with a detergent. The same 10%NaOH solution may be used for multiple gels.

8. New glass plates should be soaked in 10% NaOH for 1 hour, then rinsedthoroughly with deionized water before use. New plates also should beetched with a diamond pencil in the corner of one side to distinguish thesides of the plates in contact with the gel.



Procedure

The following protocol is for the preparation of a denaturing polyacrylamide gel withthe dimensions of 31.0 cm wide × 38.5 cm high × 0.4 mm thick (e.g., S2 sequencinggel electrophoresis apparatus, Whatman Cat.# 21105-010). Use one-half of the vol-umes described here for a gel with the dimensions of 17 cm wide × 32 cm high ×0.4 mm thick (e.g., SA32 sequencing gel apparatus, Whatman Cat.# 31096-019).

1. Thoroughly clean the shorter and longer glass plates twice with 95% ethanoland Kimwipes® tissues.

Note: The gel side is the etched side of the glass plate.

2. Using gloves, apply 3 ml of Gel Slick® solution onto the etched side of thelonger glass plate. With a dry paper towel, spread the Gel Slick® solutionusing a circular motion over the entire surface.

3. Wait 5 minutes for the Gel Slick® solution to dry. Remove the excess GelSlick® solution with a paper towel saturated with deionized water. Finally,dry the glass plate with Kimwipes® tissue.

4. In a chemical fume hood, prepare fresh binding solution by adding 3 µl ofbind silane to 1 ml of 0.5% acetic acid in 95% ethanol in a 1.5 ml tube. Wipethe etched side of the shorter glass plate using a Kimwipes® tissue satu-rated with the freshly prepared binding solution. Be certain to wipe theentire plate surface with the saturated tissue.

5. Wait 5 minutes for the binding solution to dry. Wipe the shorter glass plate3–4 times with 95% ethanol and Kimwipes® tissues to remove the excessbinding solution. Failure to wipe excess binding solution from the shorterglass plate will cause the gel to stick to both plates, and the gel will bedestroyed upon separation of the glass plates after electrophoresis.

6. Take special care not to allow the treated surfaces to touch each other.Assemble the glass plates by placing 0.4 mm side spacers and a 0.4 mmbottom spacer (optional) between the plates and using clamps to hold themin place. Lean the assembled plates against a test tube rack or other similarsupport.

7. Prepare a 4% acrylamide solution (total of 75 ml) by combining the ingredi-ents listed below:

Urea 31.50 gdeionized water 40.00 mlTBE Buffer, 10X 3.75 ml40% acrylamide:bis (19:1) 7.50 mltotal volume 75 ml

8. Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene®

tissue culture filter).

9. Pour the filtered acrylamide solution into a squeeze bottle.

10. Add 50 µl of TEMED and 500 µl of 10% ammonium persulfate to the acrylamide solution, and mix gently.

Note: See comments at theend of this section for infor-mation on precast poly-acrylamide gels.



11. Carefully pour the acrylamide solution between the glass plates. To preventbubble formation, start pouring at one side of the assembled plates andmaintain a constant flow of solution.

12. Position the gel horizontally, resting it on two test tube racks or other similarsupports. Remove any bubbles that may have formed.

13. Insert one or two 14 cm doublefine (49 point) sharkstooth combs, straightside into the gel, between the glass plates (6 mm of the comb should bebetween the two glass plates). If using a square-tooth comb, insert thecomb between the glass plates until the teeth are almost completelyinserted into the gel.

14. Secure the comb(s) with 2 to 3 clamps each.

15. Pour the remaining acrylamide solution into a disposable conical tube as apolymerization control. Rinse the squeeze bottle, including the spout, withwater.

16. Allow polymerization to proceed for at least 1 hour. Check the polymeriza-tion control to be sure that polymerization has occurred.

Note: The gel may be stored overnight if a paper towel saturated with deion-ized water and plastic wrap are placed around the well end of the gel to pre-vent the gel from drying out. If no bottom spacer is used, the bottom of thegel should be wrapped.

III.D. Polyacrylamide Gel Electrophoresis

Gel Pre-Run

1. Remove the clamps from the polymerized acrylamide gel, and clean theglass plates with paper towels saturated with deionized water.

2. Shave any excess polyacrylamide away from the comb. Remove the comband bottom spacer.

3. Add 0.5X TBE to the bottom chamber of the electrophoresis apparatus.

4. Gently lower the gel and glass plates into the buffer with the longer platefacing out and the well side on top.

5. Secure the glass plates to the sequencing gel apparatus.

6. Add 0.5X TBE to the top buffer chamber of the electrophoresis apparatus.

7. Using a 50–100 cc syringe filled with buffer, remove the air bubbles on thetop of the gel. Be certain the well area is devoid of air bubbles and smallpieces of polyacrylamide. Use a syringe with a bent 19-gauge needle toremove the air bubbles between the glass plates on the bottom of the gel.

8. Pre-run the gel to achieve a gel surface temperature of approximately 50 °C.Consult the manufacturer’s instruction manual for the recommended elec-trophoresis conditions.

Note: As a reference, we generally use 60–65 watts for a 40 cm polyacry-lamide gel 40–45 watts for a 32 cm gel. The gel running conditions mayhave to be adjusted to reach a temperature of 50 °C.



Sample Preparation

1. Prepare the PCR samples by mixing 2.5 µl of each sample with 2.5 µl ofSTR 2X Loading Solution.

Note: The sample alleles may appear more intense than ladder alleles onthe gel, but this should not interfere with allele determination. For more evenband intensities, mix 1 µl of each sample with 4 µl of a premix containing2.5 µl of STR 2X Loading Solution and 1.5 µl of STR 1X Buffer.

2. Add 2.5 µl (50 ng) of pGEM® DNA Markers to 2.5 µl of STR 2X LoadingSolution for each marker lane.

Note: We recommend loading pGEM® DNA Markers into the first and lastlanes of the gel.

3. Combine 2.0 µl of the CTT Allelic Ladder and 2.0 µl of Amelogenin ladder.Mix well then combine 2.5 µl of this mixture with 2.5 µl of STR 2X LoadingSolution for each allelic ladder lane. The number of allelic ladder lanes useddepends on personal preference.

4. Briefly centrifuge the samples in a microcentrifuge to bring the contents tothe bottom of the tube.

Sample Loading

1. Denature the samples by heating at 95 °C for 2 minutes, then immediatelychill on crushed ice or in an ice-water bath.

Note: Denature the samples just prior to loading the instrument.

2. After the pre-run, use a 50–100 cc syringe filled with buffer to flush the ureafrom the well area. If using a sharkstooth comb, carefully insert the combteeth into the gel approximately 1–2 mm. Leave the comb inserted in the gelduring both gel loading and electrophoresis.

3. Load 3 µl of each sample into the respective wells. The loading processshould take no longer than 20 minutes to prevent the gel from cooling.

Gel Electrophoresis

1. Once loading is complete, run the gel using the same conditions as for thegel pre-run.

Note: In a 4% gel, bromophenol blue migrates at approximately 40 basesand xylene cyanol migrates at approximately 170 bases.

2. Knowing the size ranges for each locus (Table 4) and migration characteris-tics of the dyes (Step 1, above), stop electrophoresis any time after thelocus of interest has passed the midpoint of the gel. If running more thanone locus or a multiplex, be careful not to run the TH01 locus off the bottomof the gel.

3. Proceed to silver stain detection.



Precast Polyacrylamide Gels

Because so many components of polyacrylamide gels are toxins, you may wish to useprecast gels. Most precast gels that are sold specifically for separation of DNA frag-ments and silver stain detection are designed to run on a specific apparatus. Therefore,to use precast gels, you may need to also purchase an appropriate gel electrophoresisapparatus. Several biotechnology supply companies sell such systems.

Invitrogen sells Novex® precast DNA retardation gels, and they specify the sizerange that you can expect to separate when you use these gels. Their gels aredesigned to run on the XCell SureLock™ MiniCell apparatus.

GE Life Sciences sells the GenePhor™ DNA Separation System and precast gels.

III.E. Silver Staining

This protocol describes silver staining of polyacrylamide gels.

Materials to Be Supplied by the User•SILVER SEQUENCE™ Staining Reagents (Cat.# Q4132)• fix/stop solution• staining solution• developer solution (chilled to 4–10 °C)• Nalgene® wash tubs (54.1 × 43.5 × 13 cm or appropriate size for your system)• orbital shaker or rocker platform

Use 2 liters of each solution per gel for each step (for a 54.1 × 43.5 × 13 cm tray).

Procedure

1. After electrophoresis, empty the buffer chambers and carefully loosen thegel clamps. Remove the glass plates from the apparatus.

2. Place the gel and glass plates on a flat surface. Remove the comb and sidespacers. Use a plastic wedge to carefully separate the two glass plates. Thegel should be strongly affixed to the shorter glass plate.

3. Place the gel (attached to the shorter plate) in a shallow plastic tray (e.g.,Nalgene® wash tub).

4. To silver stain, follow Steps a–h below. Gently agitate during each step.Steps involving solutions containing formaldehyde should be performed in a chemical hood.

fix/stop solution

10% glacial acetic acid

staining solution

1 g/L silver nitrate (AgNO3)0.056% formaldehyde (HCOH)

(1.5 ml of 37% HCOH/liter)

developing solution

30 g/L sodium carbonate (Na2CO3)0.056 M formaldehyde (HCOH)(1.5 ml of

37% HCOH/liter)2 mg/L sodium thiosulfate(Na2S2O3 •

5H2O)



Notes:

1. Save the fix/stop solution from Step 4a, to use in Step 4g.

2. The duration of Step 4e is important. The total time from immersion indeionized water to immersion in developer solution should be less than 20 seconds. If the deionized water rinse step does exceed 20 seconds,repeat Step 4d.

3. Add fix/stop solution directly to developer solution to stop developing reac-tion.

4. Position the gel and shorter plate upright, and allow it to dry overnight. Forbest results, the gel should be completely dried before APC Film develop-ment (Section III.F). Alternatively, to create film prints of the gel immediately,cover the gel with plastic wrap, and expose your film.

Reusing Glass Plates

1. Immerse the plate and affixed gel in a 10% NaOH solution for 1 hour toovernight. Discard the gel, and clean the glass plate with deionized waterand a detergent such as Liqui-Nox® detergent. The 10% NaOH solution may be reused for additional gels.

2. All cleaning utensils and sponges for the longer glass plates should be keptseparate from those for the shorter glass plates to prevent cross-contamina-tion of the binding solution.

Step Solution Time

a. fix/stop solution (See Note 1) 20 minutes

b. deionized water 2 minutes

c. repeat Step b, twice 2 × 2 minutes

d. staining solution 30 minutes

e. deionized water (See Note 2) 10 seconds

f. developer solutionup to 5 minutes (untill alleles and ladders are visible)

g. fix/stop solution (See Note 3) 5 minutes

h. deionized water 2 minutes



III.F. Generating Film Images

A direct image may be produced using Automatic Processor Compatible (APC) Film. Theimage produced on APC Film is the mirror image of the gel. Use of film allows the gener-ation of multiple permanent images with more control over band and background inten-sity than does development of the gel alone. Handle all plates with gloved hands to avoidfingerprints.

Materials to Be Supplied by the User• white light box• automatic film processor or film developing tanks• Automatic Processor Compatible (APC) Film (Cat.# Q4411)

1. In the darkroom with a safelight on, place the dry, stained gel attached tothe shorter plate (gel side up) on a white fluorescent light box.

Note: For best results, the gel should be completely dry before the image iscaptured with APC film. If capturing an image from a gel that has not beendried, cover the gel with plastic wrap.

2. Position the APC Film, emulsion side down, over the gel to be copied.

Note: The emulsion side of the film can be identified as the glossy whitesurface; the nonemulsion side has a gray tint.

3. Place a clean glass plate on top of the film to maintain contact between thegel and film. Turn on the white light box, and expose the film for 1–2 min-utes, depending on the gel background level and the intensity of the whitelight. (This step must be optimized for individual light boxes.)

4. Develop the film as recommended by the manufacturer. APC film may beprocessed manually or with an automatic film processor. For automatic filmprocessors, follow the manufacturer’s instructions.

Note: The image produced on APC Film is the mirror image of the gel.

5. If there is very little signal, decrease the exposure time used in Step 3. If thefilm appears brown or black, increase the exposure time.



III.G. Analyzing Data

Run your allelic ladders in lanes adjacent to each sample to ease interpretation ofresults. Direct comparison between the allelic ladders and amplified samples of the samelocus allows easier assignment of alleles. The TH01 allele 9.3 is a microvariant allele anddoes not comigrate with allelic ladder fragments. In addition, mutations or rare allelesmay be seen occasionally. The migration of such “off-ladder” alleles cannot be predicted.

With silver stain detection, both DNA strands are detected. For some loci, such as TH01,the difference in the sequence of the opposing strands causes them to migrate at differ-ent rates. This results in doublets for each allele (Figure 1). This strand separation maybe more pronounced with longer electrophoresis of gels.

Artifact bands also may be detected with these systems. Shadow banding (1–3) or repeatslippage appears as faint bands one repeat unit (i.e., 4 bases) below the true alleles.

Terminal nucleotide addition occurs when Taq DNA polymerase catalyzes template-inde-pendent addition of a nucleotide to the 3´-termini of amplified DNA fragments (3–5). Aband that is one base shorter than the expected allele may result from the inefficiency ofthe terminal nucleotide addition. An artifact band is generated when this terminal additiondoes not occur with 100% efficiency. This may be visualized as an extra band.

pGEM® DNA Markers

The pGEM® DNA Markers are visual standards used to confirm allelic size ranges for theloci. The markers consist of fifteen DNA fragments with the following sizes (in basepairs):

2,645 460 126

1,605 396 75

1,198 350 65

676 222 51

517 179 36

Controls

Observe the lanes containing the negative controls. They should be devoid of amplifica-tion products. Observe the lanes containing the positive K562 DNA positive controls.Compare the K562 DNA allelic repeat sizes with the locus-specific allelic ladder. Theexpected K562 DNA allele size(s) for each locus are listed in Table 4.

STR Ladders

Each locus or multiplex has a characteristic allelic ladder. Please refer to Table 4 forlocus-specific allelic ladder information. In general, the allelic ladders contain fragmentsof the same lengths as either several or all known alleles for the locus. Visual comparisonbetween the allelic ladder and amplified samples of the same locus allows preciseassignment of alleles.

Representative Data

CSF1PO

TPOX

TH01

L 1 2 L 3 4 L

– 15

– 7

– 13

– 6

– 11

– 5

CTTMultiplex

L 1 2 L 3 4 L

F13A01

FESFPS

vWA

– 16

– 14

– 7

– 20

– 13

– 4

FFvMultiplex

L 1 2 L 3 4 L

D16S539

D7S820

D13S317

– 15

– 5

– 14

– 6

– 15

– 7

SilverSTR® IIIMultiplex

5808

TA



Figure 1. Representative data. Individual genomic DNA samples(lanes 1–4) were amplified using GenePrint® STR Systems as indi-cated and detected using silver staining as described in this manual.The amplification products were separated using a 4% denaturingpolyacrylamide gel.

Numbers to the right of each image indicate the smallest and largestnumber of repeat units present in corresponding fragments of theallelic ladder.

0753

TM

10_4

B

218bp (Y)

212bp (X)

1 2 3 4 6 L5L

Amelogenin

Figure 2. Amplification of varying concentrations ofK562 template DNA at the Amelogenin locus. DNAwas amplified using a Perkin-Elmer model 480 thermalcycler. Lanes 1 and 8 contain the locus-specific allelicladder; lanes 2–6 contain amplified K562 DNA using250, 25, 5, 1 and 0.5 ng of starting tem-plate,respectively.

Table 3. Locus-Specific Information for CTT plus Amelogenin Multiplex.

STR Locus ChromosomalLocation

GenBank® Locus andLocus Definition

Repeat Sequence5´–3´

Amelogenin1 Xp22.1–22.3 and Y

HUMANEL, Human Ychromosomal gene foramelogenin-like protein NA

CSF1PO1 5q33.3–34

HUMCSF1PO, Humanc-fms proto-oncogenefor CSF-1 receptorgene AGAT2

TH01 11p15.5 HUMTH01,Human tyro-sine hydroxylase gene AATG2

TPOX 2p25.1–pter

HUMTPOX, Humanthyroid peroxidasegene AATG2

1Amelogenin is not an STR, but displays a 212-base, X-specific band and a 218-base, Y-specificband. K562 DNA (female) displays only the 212-base, X-specific band.2Repeat sequences represent all four possible permutations (e.g., AGAT is used for AGAT, GATA,ATAG or TAGA). The first alphabetic representation of the repeat (e.g., AGAT) is used according tothe precedent of Edwards et al. (6).

Comments

1. PCR amplification sometimes generates artifacts that appear as faint bands belowthe alleles. These products probably result from a process known as slippage, com-monly observed in PCR amplification of regions that contain tandem repeats of shortsequences (1–3).

2. A strong extra band may be observed below the 212 bp Amelogenin allele whenmore than 25 ng of template DNA is amplified.

3. Locus TH01 has a common 9.3 allele (7). A one-base deletion is present in the allelethat contains 10 repeats. Note that reference 6 refers to this allele as 10.1 ratherthan 9.3. This allele was renamed 9.3 at the ISFH Conference in Venice, Italy, inOctober 1993.

Power of Discrimination

The following tables provide information about the power of discrimination (matchingprobability, paternity index [PI], and power of exclusion) within a variety of populationsusing the alleles in the CTT Multiplex (not including Amelogenin). A measure of discrimi-nation often used in paternity analyses is the paternity index (PI), a means for presentingthe genetic odds in favor of paternity given the genotypes for the mother, child and atested man (8). An alternative calculation used in paternity analyses is the power ofexclusion (8).



Table 4. Additional Locus-Specific Information for the CTT plus Amelogenin Multiplex.

STR Locus

Allelic LadderSize Range1

(bases)

STR LadderAlleles (# ofrepeats)2

Other KnownAlleles3 (# ofrepeats)

K562 DNAAllele Sizes (#of repeats) Comments

Amelogenin4 212–218 NA None 212,212 1,2

CSF1PO 295–3277,8,9,10,11,12,13,14,15 6 9,10 1

TH01 179–203 5,6,7,8,9,10,11 9.3 9.3,9.3 1,3

TPOX 224–2526,7,8,9,10,11,12,13 None 8,9 1

1Lengths of each allele in the allelic ladders have been confirmed by sequence analyses.2Alleles in bold are present in greater amounts than other alleles. This simplifies interpretation.3Alleles that represent <0.2% of the population may not be listed in this table. 4Amelogenin is not an STR, but displays a 212-base X-specific band and a 218-base Y-specificband. K562 DNA (female) displays only the 212-base X-specific band.

Table 5. Population Statistics for the CTT Triplex.

African-American Caucasian-American Hispanic-American

Matching Probability 1 in 1,590 1 in 435 1 in 549

Paternity Index 10.2 6.8 5.2

Power of Exclusion 0.906 0.869 0.830



Note: Instructors areresponsible for ensuringthat your institution’s guide-lines regarding student-provided samples and workwith human biologicalmaterials are followed.

Be sure you know andunderstand your institu-tion’s rules and regulationsif you decide to includehuman samples in yourgenotyping exercise.

III.H. Obtaining Single-Source Human Genomic DNA

STR mapping requires single-source human genomic DNA. You can have your stu-dents use the K562 DNA provided with the CTT Multiplex System. However, if youwould like your students to compare two different DNA samples using STR mapping,human cultured cells can be used as an source for genomic DNA. There are manycell lines available from the American Type Culture Collection (ATCC), and the geno-types of lines supplied by ATCC are known. However, use of cell-line-derived DNAmay result in allelic imbalance and imbalance between STR loci.

To obtain DNA from tissue culture cells, use the Wizard® SV Genomic DNA PurificationSystem. This system uses either a spin or a vacuum protocol. Details about genomicDNA isolation using this kit are available in the Wizard® SV Genomic DNA PurificationSystem Technical Bulletin #TB302, available at:www.promega.com/tbs/tb302/tb302.html. This protocol is also available in Unit 4 ofthe Education Resources Web site (www.promega.com/education/default004.htm).

Many institutions have restrictions regarding student-provided samples for teachinglabs because of ethical and safety considerations. Often such teaching laboratorieswill be subject to review by an internal review board (IRB). Be sure that you thor-oughly investigate and follow your institution’s guidelines regarding student-providedsamples and working with human biological material if you decide to have the stu-dents genotype their own DNA.

The DNA IQ™ System (Cat.# DC6700) is a DNA isolation and quantitation systemdesigned specifically for forensic and paternity samples (8). This novel system usesparamagnetic particles to prepare clean samples for STR analysis easily and effi-ciently. The DNA IQ™ Resin eliminates PCR inhibitors and contaminants frequentlyencountered in casework samples. With larger samples, the DNA IQ. System deliv-ers a consistent amount of total DNA. The system has been used to isolate andquantify DNA from routine sample types including buccal swabs, stains on FTA®

paper and liquid blood. Additionally, DNA has been isolated from casework samplessuch as tissue, differentially separated sexual assault samples and stains on supportmaterials.



IV. STR Analysis of DNA Using the PowerPlex® 16 System

Modern forensic and paternity labs analyze DNA samples using the multiplex amplifica-tion and capillary electrophoresis. However, this level of analysis requires significant capi-tal investment, and the required equipment and facilities are not often available for teach-ing labs. However, instructors at large insitutions may have access to the requiredequipment through a core laboratory facility.

The PowerPlex® 16 System (10,11) allows co-amplification and three-color detection ofsixteen loci (fifteen STR loci and Amelogenin), including Penta E, D18S51, D21S11,TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D, CSF1PO,D16S539, D7S820, D13S317 and D5S818. One primer for each of the Penta E, D18S51,D21S11, TH01 and D3S1358 loci is labeled with fluorescein (FL); one primer for each ofthe FGA, TPOX, D8S1179, vWA and Amelogenin loci is labeled with carboxy-tetramethyl-rhodamine (TMR); and one primer for each of the Penta D, CSF1PO, D16S539, D7S820,D13S317 and D5S818 loci is labeled with 6-carboxy-4´,5´- dichloro-2´,7´-dimethoxy-fluo-rescein (JOE). All sixteen loci are amplified simultaneously in a single tube and analyzedin a single injection or gel lane.

The PowerPlex® 16 Monoplex System, Penta E (Fluorescein) (Cat.# DC6591) andPowerPlex® 16 Monoplex System, Penta D (JOE) (Cat.# DC6651) are available toamplify the Penta E and Penta D loci, respectively. Each monoplex system allows amplifi-cation of a single locus to confirm results obtained with the PowerPlex® 16 System. Themonoplex systems can be also used to re-amplify DNA samples when one or more of theloci do not amplify initially due to nonoptimal amplification conditions or poor DNA tem-plate quality.

The PowerPlex® 16 System is compatible with the ABI PRISM® 310, 3100 and 3100-Avant Genetic Analyzers, and Applied Biosystems 3130 and 3130xl Genetic Analyzers.

If you would like to pursue a forensics laboratory using these state-of-the-art systems,please see the PowerPlex® 16 System Technical Manual #TMD012 available at:www.promega.com/tbs/tmd/tmd012.html

Alternatively the BioPharmaceutical Center Institute (BTCI) in Fitchburg, Wisconsin, con-ducts on-site workshops. Visit their Web site: www.btci.org for more information.



V. Supplier and Ordering Information

Please see the “Materials Required” list that preceeds each protocol for a list of requiredequipment, props and reagents required for the laboratory that you are performing.

RFLP Laboratory

Product Cat.#XmnI R7271HincII R6031pGL4.11 [luc2P] Vector E6661pGL4.12 [luc2CP] Vector E6671Agarose, LE, Analytical Grade V3121TBE Buffer, 10X V4251BenchTop pGEM® DNA Markers G7521

STR/Silver Staining Laboratory

Product Cat.#Nuclease-Free Water P1193GoTaq® DNA PolymeraseMineral Oil DY1151GenePrint® SilverSTR® CTT Multiplex DC6001GenePrint® Sex Identification Amelogenin (Silver Detection) DC4081SILVERSEQUENCE™ Staining Reagents Q4132

TBE Buffer, 10X V4251



VI. Resources

References

1. Levinson, G. and Gutman, G.A. (1987) Slipped-strand mispairing: A major mech-anism for DNA sequence evolution. Mol. Biol. Evol. 4, 203–21.

2. Schlotterer, C. and Tautz, D. (1992) Slippage synthesis of simple sequence DNA.Nucleic Acids Res. 20, 211–5.

3. Walsh, P.S., Fildes, N.J. and Reynolds, R. (1996) Sequence analysis and charac-terization of stutter products at the tetranucleotide repeat locus vWA. NucleicAcids Res. 24, 2807–12.

4. Smith, J.R. et al. (1995) Approach to genotyping errors caused by nontemplatednucleotide addition by Taq DNA polymerase. Genome Res. 5, 312–7.

5. Magnuson, V.L. et al. (1996) Substrate nucleotide-determined non-templatedaddition of adenine by Taq DNA polymerase: Implications for PCR-based geno-typing. BioTechniques 21, 700–9.

6. Edwards, A. et al. (1991) DNA typing and genetic mapping with trimeric andtetrameric tandem repeats. Am. J. Hum. Genet. 49, 74–56.

7. Puers, C. et al. (1993) Identification of repeat sequence heterogeneity at thepolymorphic STR locus HUMTH01[AATG]n and reassignment of alleles in popu-lation analysis using a locus-specific allelic ladder. Am. J. Hum. Genet. 53,953–8.

8. Brenner, C. and Morris, J.W. (1990) In: Proceedings from the InternationalSymposium on Human Identification 1989, Promega Corporation, 21–53.

9. Mandrekar, P.V., Krenke, B.E. and Tereba, A. (2001) DNA IQ™: The intelligentway to purify DNA. Profiles in DNA 4(3), 16.

10. Krenke, B. et al. (2002) Validation of a 16-locus fluorescent multiplex system. J. Forensic Sci. 47, 773–85.

11. Budowle, B. et al. (2001) STR primer concordance study. Forensic Sci. Int. 124,47–54.

Protocols

PowerPlex® 16 System Technical Manual #TMD012(www.promega.com/tbs/tmd012/tmd012.html)

GenePrint® STR Systems (Silver Stain Detection) Technical Manual #TMD004(www.promega.com/tbs/tmd004/tmd004.html)

Wizard® SV Genomic DNA Purification System Technical Bulletin #TB302(www.promega.com/tbs/tb302/tb302.html)

Profiles in DNA (www.promega.com/profiles)

instructor's lab manual - promega

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