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Acknowledgement

These research projects were funded by the National Institute of Justice, through the

Midwest Forensics Resource Center at Ames Laboratory under interagency

agreement number 2002-LP-R-083. The Ames Laboratory is operated for the US

Department of Energy by Iowa State University, under contract No. W-7405-Eng-82.

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Midwest Forensics Resource Center (MFRC)

Research and Development Program

Introduction

The mission of the MFRC Research and Development Program, is to provide technological

advances in forensic science for the benefit of our regional partners as well as the forensic

community at large. Key areas of forensic science need are identified through our interactions

with our Midwest partners and our R&D advisory group, as well as through our participation in

national meetings in forensic science. Under the sponsorship of the National Institute of

Justice, the MFRC solicits proposals for the development of practical and useful technology,

instrumentation, and methodology that address needs in areas related to forensic science and

its application to operational crime laboratories. The MFRC facilitates proposal development

by working to establish partnerships between researchers and our regional partners. The

MFRC administers a peer-review of the proposals and then funds the selected projects at acost of approximately $55,000 each, with a 12-month period of performance.

The process for selection of these projects includes the following steps: 1)drafting of a call for

proposals by MFRC staff, 2) review of the draft call by members of the R&D advisory

committee, 3) review and approval of the call by NIJ, 4) issuance of the call to ISU, Ames

Laboratory, regional partners, and research organizations,5) receipt of proposals,6 ) eview of

proposals by R&D advisory committee, 7) ranking and selection by MFRC staff using advisory

committee reviews, with concurrence by NIJ, 8) notification of proposers,9) eceipt and review

of progress reports by MFRC, IO) receipt and review of final reports by MFRC, R&D advisory

committee, and NIJ.

The decision to fund any specific project is based upon a peer-reviewed call-for-proposal

system administered by the MFRC. The reviewers are crime laboratory specialists and

scientists who are asked to rate the proposals on four criteria areas including: 1) relevance to

the mission of the MFRC, 2) technical approach and procedures, 3) capabilities, teaming, and

leveraging, and 4) implementation plan. A successful proposal demonstrates knowledge of

the background for the research and related work in the field and includes a research plan with

a defined plan to implement the technology to benefit our partners at the crime laboratories.

Program Summary Technical Sheets

The following project summaries are meant to demonstrate the rangeof research funded by

the MFRC including chemistry, DNA, and patterned evidence. The project summaries

describe the forensic need the projects serve as well as the benefits derived from the

technology. The summaries provide a brief description of the technology and the

accomplishmentsto date. In addition, the collaboration with regional partners and the status

of the implementation of the technology are highlighted. These technical summaries represent

the development and implementation of practical and useful technology for crime laboratories

that the MFRC hopes to accomplish.

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TABLE of CONTENTS

PAlTERNED EVIDENCE

Hall-Effect Measurements Under Alternating-CurrentExcitation for the Reconstruction of ObliteratedSerial Numbers in Magnetic Steels

Magnetic Particle Recovery of Serial Numbers

Quantitative Characterization of Machin ing Marks forComparative Identification

Quantitative/StatisticalApproach to Bullet-to-FirearmIdenti fication Using Consecutively Manufactured Barrels

DNA

Evaluation of the PROMEGA DNA lQTM ystem and QiagenEZ lm System as an Extraction Method for Mitochondrial

DNA Analysis

Validation of DNA Quantitation and Species Tests

Validation of Y-Plex 12 Database Study

CHEMISTRY

Developing Aptamers to Methamphetamine as Nucleic

Acid Sensors

Laser Ablation-Inductively Coupled Plasma-Mass

Spectrometry of Forensic Glass Samples

Forensic Analysis of Trace Explosives

1

3

6

8

12

15

19

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HALL-EFFECT MEASUREMENTS UNDER ALTERNATING-CURRENT EXCITATION FOR THE RECONSTRUCTION OF

OBLITERATED SERIAL NUMBERS IN MAGNETIC STEELS

FORENSIC TECHNOLOGY NEED

Existing forensic techniques employed torecover obliterated serial numbers fall mainlyinto one of two categories, those requiringextensive sample preparation including the useof acid etchants, and those utilizing magneticparticles to image irregularities in surfacemagnetic properties. The former is timeconsuming and utilizes potentially harmfulchemicals while the latter messy andpotentially low insensitivity. A new approach isbeing investigated whereby the stray magneticfield from a magnetized sample is measuredusing a Hall effect sensor. The sample ismagnetized using an electromagnetic c-coreyoke. For increased sensitivity, an ACapproach is being utilized that benefits from theuse of a lock-in amplifier. The approach hasbeen tested on some artificial specimens andpart of a real gun with the serial numbersremoved by surface grinding.

TECHNOLOGY DESCRIPTION

The objective of this work is to develop amagnetic imaging technique for thenondestructive restoration of obliterated serialnumbers in ferromagnetic metals. This can beachieved by imaging the magnetic signaturesthat result from residual plastic deformations.To obtain both the high sensitivity and highspatial resolution necessary for recovering the

serial numbers, advanced Hall-effect sensorsbased on indium-antimonide echnology havebeen utilized. By scanning the Hall sensoracross the obliterated serial numbers it ispossible to obtain a two dimensional imageof

the stray magnetic field distribution, andbecause of magnetoelastic coupling, thisimage contains information about the plasticallydeformed regions under the stampedcharacters and can therefore be used toreconstruct the serial numbers.

Inorder to build up an image of the scatteredfield from a magnetized specimen it isnecessary to scan a solid state field sensor (aHall device, for example) over the surface ofthe specimen while acquiring readings. This i:

achieved by mounting the Hall Device on an x.y linear motion stage sitting ust above thespecimen. The Hall device is mounted on acantilever to ensure consistent surfacecontact. Typically, Hall sensors have betterperformance when operated in constant-

current mode. A magnetic imaging systemfor serial number reconstruction has beenassembled and tested in a laboratoryenvironment. Some simple tests have shownthe technique to be effective at serial numberreconstruction. In many cases the magneticsignature as detected by Hall or GMR sensorsis stronger than the visual image provided bytechniques such as magnetic particle imaging,Figure 1 for example. The approach was alsoapplied to some more realistic specimens.

The results from a shot gun are shown inFigure2.

Figure 1. Top Left: Reconstructed mage of the letter

'M' following a Hall-device scan. BottomRight: Reconstruction of the same

character using fluorescent magnetic

aerosol particles following magnetizationof the part ina highcurrent DC coil.

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ITECHNOLOGY BENEFITS

Improved sensitivity through the use ofadvanced Hall sensors. This couldmean detection of serial numbersfollowing significant material removal orin metals that are weakly magnetic.

Improved reliability through automatedimage-analysis procedures.

Greater versatility through customizableinstrumentation, i.e. ability to tunemeasurement conditions in order to

maximize sensitivity for a particular

material or geometry.

COLLABORATlONnMPLEMENTATlON

Publications and PresentationsJohnson,M.J., C.C.H. Lo, and L. Naidu,“Hall-Effect Measurements Under ACExcitation for the Reconstruction ofObliterated Serial Numbers in MagneticSteels,” Review of Progress inQuantitative Nondestructive Evaluation,Vol. 23, New York: AIP, 2004: 1445

This work has been largely conducted inthe laboratory and to date no field trials

ACCOMPLISHMENTS/ STATUS

Results to date demonstrate the potential of theHall-device based magnetic imaging system forserial number reconstruction. Issues remainwith the ability to resolve serial numbers that areclose together. One way to improve resultswould be to use a Hall device with a smalleractive area such as 50 by 50 mm.

CONTACTS

Dr. Marcus JohnsonCenter for Nondestructive Evaluation285 ASC Building II1915 Scholl RoadIowa State UniversityAmes, IA 5001 1-3042(515) 294-6802 fax: [email protected]

Dr. Chester LoCenter for Nondestructive Evaluation205 Metals Development Bldg.Arnes LaboratoryIowa State UniversityAmes, IA 5001 1-3042(515) 294-4312 fax: [email protected]

have been carried out.2

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MAGNETIC PARTICLE RECOVERY of SERIAL NUMBERS


One method used by crime labs to recoverobliterated serial numbers in steel firearms isthe magnetic particle technique. The use ofthis method is based on the detection of metaldeformation present under stamped serialnumbers after the visible stamp has beenremoved. The purpose of this project was todefine conditions that would increase thelikelihood that the technique could besuccessfully applied in forensic work.

This work was performed at Ames Laboratory’sCenter for Non Destructive Evaluation incollaboration with the Iowa Division of CriminalInvestigation (DCI). Tests were made onactual firearm components provided throughthe assistance of the DCI.


The aim of this project was to investigatespecific aspects of magnetic particle inspectionfor serial number recovery. This includes teststo understand the magnetic characteristics ofdifferent firearm steels that affect theirperformance in the test, such as varying resultsfor carbon steels and alloy steels after differentthermal and forming treatments. Alsoinvestigated were the effects of the nature ofthe sample magnetization (AC, rectified DC,and true DC) and the effects of various

detection media, such as visible powders andfluorescent sprays, on test outcome. Surfacepreparation of the firearms prior to numberrecovery work was examined.

Visual observations were made aftermagnetization of the samples and theapplication of the magnetic suspension. Thesamples were tested in a horizontal wetmagnetic stand using a large coil, as well as aportable yoke. Both AC and rectified DC

results on metal test samples indicated that

the use of AC current provided inferior results.Serial number and name recovery were thenmade by visual observation with actual firearms.Both the serial number and part of the“Remington” name were ground away from aRemington 12-gauge shotgun using an electricgrinding tool. Recorded images were madeusing a Minolta DiMAGE7 digital camera asshown in Figures 1-4.

TECHNOLOGY BENEFITS

This study showed that serial number recoverywould be improved by:

Using fluorescent magnetic particles in

the recovery attempt,Using DC power supply for control of the

magnetic yokes used,Modifying the design of such yokes to

permit better contact with firearms,Performing some surface preparationprior to magnetic particle recoveryattempts,Attempting magnetic particle recoveryfirst, avoiding damage to the surfacethrough chemicals,Using digital photography to captureimages.

COLLABORATION/ IMPLEMENTATION

This work was performed at the IowaDemonstration Lab of the Center for NonDestructive Evaluation at Iowa State University/Ames Laboratory. Discussions on the merits of

this work have been held with Vic Murillo andBob Harvey of the Division of CriminalInvestigation crime laboratory in Des Moines,Iowa and with John Collins of the DuPageCounty crime laboratory in Wheaton, Illinois.The final project report is posted on the MFRCwebsite for dissemination to crime laboratory

excitation to the coils were used initially. Earlypartners.

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Publications and Presentations

Utrata, D. and M.J. Johnson.,“Magnetic Particle Recovery of SerialNumbers,” Midwest ForensicsResource Center/Ames LaboratoryFinal Report Ames, IA: Ames

Laboratory, 2004, IS-5159.

Utrata, D. and M.J. Johnson,“Development of Magnetic ParticleMethod for Forensic Recovery ofSerial Numbers,” Review of Progressin Quantitative NondestructiveEvaluation,Vol. 23, edited by D.O.Thompson and D. E. Chimenti,Melville, New York: American Instituteof Physics, 2004: 1438-1444.

ACCOMPLISHMENTS AND ONGOING

WORK

Several conclusions were reached during theevaluation of the magnetic recovery ofobliterated serial numbers. These included:DC power should be used for magnetization,giving better results; the use of fluorescentparticles and ultraviolet lighting providedenhanced viewing of recovery efforts; andthat some degree of surface preparationimproved efficacy of recovery results.

CONTACTS

Mr. Dave UtrataCenter for Nondestructive Evaluation177 ASC Building II1915 Scholl RoadIowa State University

Ames, IA 50011-3042(515) 294-6095 fax: -7771heydavea astate.edu

Dr. Marcus JohnsonCenter for Nondestructive Evaluation285 ASC Building I11915 Scholl RoadIowa State UniversityAmes, IA 50011-3042(515) 294-6802 fax: -7771

marcusj iastate.edu

Figure 1 . Original “Remington” name and serialnumber before obliteration.

Figure 2. The “Remington” name and serial numberafter obliteration.

Figure 3. Magnetic images after testing in a yokeusing “ A C power.I

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Figure 4. Magnetic images after testing in a yoke using“ D C ower.

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QUANTITATIVE CHARACTERIZATON OF MACHININGMARKS FOR COMPARATIVE IDENTIFICATION


The analysis of tool marks at a crime scenepresent a special challenge in that the markleft is a function of not only what kind ofinstrument but is also a function of how thatinstrument was used by a particular individual.Toolmarks are difficult to characterize in that,unlike bullets or cartridge cases where thegeometry is fairly predictable, they present awide range of possible shapes and sizes. Theforce applied and the angle of attack create

differences in the appearance of the markings.This project seeks to extend the quantificationof tool marks from two-dimensional to three-dimensional examinations.

This work was performed by the AmesLaboratory in collaboration with Jim Kreiser,retired forensic scientist of the Illinois StatePolice Crime Laboratory.


The objective of this project is to develop amethod to quantitatively measure surfaceroughness as a means of identifying featuressuch as toolmarks. The method will beapplicable to any shaped surface and willinvolve a statistical analysis of the data todetermine the probability of a match betweenan unknown and a standard sample. Theoverall goal is to provide local, state, andfederal law-enforcement officials with

statistically valid data that is suitable forcourtroom presentation.

A series of screwdrivers were purchased andboth sides were used to produce markings ona series of small brass and lead plates (allsamples were produced by Jim Kreiser). Oftenthe toolmark may be left on the surface of adoor or some structural member, making itimpossible to remove the actual evidentialmark for examination. In such cases replicas

are made of regions of interest, typically using

either an epoxy or silicone based resin. Thesereplicas can then be removed and opticallyexamined and compared to similar markingsproduced using suspected tools. Commerciallyavailable material was used to produce replicasof the surface. The surface relief of both thereplicas and the original marks was examinedusing a Detroit Precision Hommel profilometer.The surface profile was found by moving adelicately balanced diamond stylus across thesurface of the toolmark perpendicular to the

mark. Height measurements are taken atperiodic intervals and the data output of theprofilometer is obtained as an array (or matrix).Normally, a three-dimensional mage will beproduced by making a series of parallel passesacross the surface, a typical scan involving upto 6000 lines with 9600 data points along eachline. The collected data were analyzed using acomputer spreadsheet.

Once a working routine was in place it was

tested by comparing scans between knownmatch and non-match areas as identified by Mr.Kreiser (Figure 1). In over 90% of the casesthe known match areas were identified with ahigh degree of confidence.

TECHNOLOGY BENEFITS

The process yields a forensic techniquethat is rapid, easy to perform, applicableto any shaped surface, and has the

potential for automated matching

This method provides quantitativesurface data to answer challengesposed by Daubert. The currentdevelopment of a computer matchroutine will take the quantitative data,automatically align the ‘evidence’ and‘standard’ scans, and then comparethem statistically.

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igure 1. Plot of depth data obtained from scans

(each scan presented vertically here) of the

profilometer across the evidence sample

(left) and the standard sample (right). Thisdata is presented with the scan data from

the two samples abutting along the

discontinuity at the right portion of the

image, to simulate the hairline in a stereo-

microscope. The area indicated n the box

corresponds to the boxed area in Figure 2.

OLLABORATION/ IMPLEMENTATION

IIsamples were produced by Jim Kreiser,!tired forensic scientist of the Illinois Staterime Laboratory.

ublications and Presentations

Churnbley, L.S., L.J. Genalo,“Quantitative Characterization ofMachining Marks for ComparativeIdent f cation Midwest Forensics

Resource Center/Am es Laboratory FinalReport,Ames, IA: Ames Laboratory,2004, IS-5165.

Bossard, C.A., L.S. Chumbley, L. J.

Genalo, and M.F.Besser, ‘Examinationof Toolrnark Replicas Using a SurfaceProfilometer.” Journal of ForensicScience, 50 (January 2005):143-156.

Churnbley, L.S. and L.J. Genalo,“Quantitative Characterization of Tool

Marks for Comparative Identification”,Proceedings: American Academ y of

Forensic Sciences, 10, Paper D31,Denver, CO: Publication Printers

CorDoration. 2004: 170.


WORK

The initial results were used as the basis for aproposal submitted and funded by the NationalInstitute of Justice to continue work on the

routine for comparing scans between knownmatched and unmatched surface areas. Apaper summarizing the initial results wassubmitted to the Journal of Forensic Sciences.

CONTACTS

Dr. L. Scott ChumbleyProfessorMaterials Science & Eng. Dept.Iowa State University/Arnes Lab

214 Wilhelm HallAmes, IA 50011(515) 294-7903 fax: -4291chumbley@ astate.edu

Dr. Larry GenaloProfessorMaterials Science & Eng. DeptIowa State University/Ames Lab2220A Hoover HallArnes, IA 5001 1

[email protected](515) 294-5444 fax: -4722

Figure 2. Small toolmark match region between anevidence sample on the left and a

standard sample on the right identified byan examiner using comparison

microscope.

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Quantitativdstatistical Approach to Bullet-to-FirearmIdentification Using Consecutively Manufactured Barrels


Firearms identification is one of the“identification” sciences that rely on patterncomparisons to reach conclusions as toidentity or difference. There is very widespreadagreement on the validity of the identificationsciences and on their conclusions. However,some challenges to these “identification” orpattern-evidence sciences have been mountedsince the US. Supreme Court handed down itsdecision in Daubert v. Merrell Dow

Pharmaceuticals 111. The challenges do not somuch allege that pattern evidence cannot beindividualized as that there is not anappropriate, objective scientific basis for theassertion that it can. This situation hasprompted research that will help establish on afirm, scientific basis for what most forensicscientists already think is true -that patternevidence can be shown to be individualizable.

In the firearms identification arena, these more

recent studies [2-61take different approaches,but all are focused on establishinggeneralizable principles. There have beensome prior studies on consecutivelymanufactured rifled barrels. However, none of

Figure 1. Schematic of the scanned circumference

of the bullet.

the prior studies have taken advantage of the

analytical tools afforded by either the SciClopsscanning system or densitometric conversionsof IBIS images. The data gathered by thesesystems can be analyzed using appropriatenon-proprietary software, such as Un-Scan-It,SigmaScanPro, PeakFit, etc. Analysis of data issimilar to analyzing similarities / differences ingas chromatograms, mass spectra, or otherfamiliar patterns. Data analysis permits theapplication of appropriate statistics to helpestablish the expected low probabilities of

“chance match” in comparison with the size ofthe test population.


The specific aim of this project was theapplication of quantitative scoring methods tothe striation markings used by firearmsexaminers for bullet-to-firearm dentifications toobtain data in a format amenable to frequency/statistical analysis. This approach used

consecutively manufactured barrels from twodifferent manufacturers, that is, underconditions where maximal similarity in theidentification markings is expected. Bulletsfired from consecutively manufactured barrelson the same assembly line should be as similaras two bullets that were fired from differentbarrels can be. The idea is that if these bulletscan be told apart, there should not be aproblem differentiating bullets from barrels thatare expected to be different.

This project tested the prospect of producing aquantitative picture of similarities /differencesamong the striation markings normally used byfirearms examiners in determining whether aparticular bullet was fired by a particular firearm(with a rifled barrel). This method could helpidentify an optimal approach to generatingobjective data that attempts to partially simulatethe pattern identification process, and thus helpprovide a scientifically defensible basis forDaubert-type challenges.

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The goal of this project was to determinewhether instrumental analyses of the individualstriation markings on fired bullets, or images offired bullets, could yield quantitative data thatmirrored the comparisons examiners performwith their eyes. Figure 1 shows a schematic ofa scanned bullet with striation markings around

its circumference. This project was designedto help validate firearms identification byinstrument analyses under Daubert guidelines.Two approaches to scanning bullet striationmarkings were used and the results compared.First, fired bullets were imaged using the(Forensic Technology, Inc.) FTI IBIS system,the standard imaging device for the NlBlN

database. Second, fired bullets were analyzedusing a measuring device called SciClops&manufactured by Intelligent Automation Inc.

(Figure2). A third task consists of a validationtest by human firearms examiners.

The experiment which fired bullets fromconsecutively manufactured barrels has beendone before with human examiners, andyielded 100% correct identifications. The priorstudy was done with 9 mm bullets. We chose.45 cal for our study. The expectation was thathuman examiners could correctly match upeven these extremely similar, yet different,bullets in a blind trial type test. We exploredwhether a measuring device that attempts tomeasure what examiners look at, or an imagescan analysis of bullet surface scans, coulddemonstrate quantitatively what examiners doby eye -getting “match scores” of some kindthat were higher among bullets fired from thesame barrel than from those fired fromdifferent, or even consecutively manufactured,barrels.

TECHNOLOGY BENEFITS

This project looked at validation approachesthat might satisfy the criteria of the Daubertdecision. These can be summarized as: Isthere a scientific basis for the proposition? Theproposition, in this case, is that bullet to barrelmatching is possible and accurate. To test theproposition, one turns to examples of thepattern that are maximally similar among the“differenf‘ category. With fingerprints, for

Figure 2. Th e SciClops laser-based profilometer byIntelligent Automation, Inc.

example, one might collect the prints ofidentical twins. With fired bullet evidence, thecorrelate is bullets fired from consecutivelymanufactured barrels.

The primary purpose of IBIS is to collect andstore images of the land impressions on bullets,and the firing pin indentation and othermarkings on cartridge cases. Given a barrelmodel with some number of lands, say six forexample, there would be six images associatedwith an exemplar bullet from that barrel -one

image for each land impression (sometimescalled a land engraved area or LEA).Aquestioned bullet can then be imaged, and theimage compared with existing image files. If thequestioned bullet is in good condition, and sixland impressions can be imaged, and they caneach be pair-wise compared with the six on anyexemplar. The system thus enables connectingfired evidence from different cases, and in thisway assisting investigations. It is not and hasnever been intended to substitute for a human

firearms examiner, but only to sort through largcquantities of evidence and generate amanageable list of “possibles.”

When two bullets are compared in the IBIS

system, three different scores result. These aremax phase, peak phase, and peak score. Onbullets with, for example, six twist and thereforesix LEAS, here are 36 comparison scores:LEAl of bullet 1 with LEAl of bullet 2, thenLEA2 of bullet 2, then LEA3 of bullet 2, and soon. The highest score here will indicate that the

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proper LEAS are being compared (highestpeak phase). Then a peak score is computed,and the highest peak scores among a group ofbullets being compared are noted.

Once all bullets are entered, a correlation isperformed for each bullet against all otherbullets in the test group. For each bullet a

correlation result is produced. For eachcorrelation result produced, the three scorearrays noted above are generated. Each scorearray is sorted, and knowing which bullet is the“sister,” each array is consulted to determineits position. Out of the three arrays the lowestposition is noted. And the lowest positionoccupied among all the arrays is recorded.From these data, a probability density vs.position can be computed -the probability thatthe sister bullet of any given bullet being

compared will be one of those in the top npositions, where n is the position number beingplotted- ndicating a perfect match.

This study showed that the FTI IBIS imagecomparison can correctly distinguish betweenall Springfield barrel bullets. The comparisonresults provide some evidence that barrel tobullet matching is objectively reliable.


There are other, independent efforts underwayto help establish a sound scientific basis for thecomparison of primary pattern evidence types:bullet and cartridge case markings,fingerprints, and handwriting. We collaboratedwith (Forensic Technology, Inc.) FTI, themanufacturer of IBIS systems for the imagingand image-databasing of fired evidence(bullets and cartridge cases). FTI agreed to

image the study bullets, and provided bitmapimage results. To protect their technology, theycould not share the details of their comparisonalgorithms.

For the human examiner validation test, werecruited examiners to do 55 tests. Theexpectation was that human examiners wouldbe able to make identifications with most of theQ specimens (i.e., match the Q to a K) andthat there would be no mis-identifications.Indeed, the human examiner validation test

results showed that there were no mis-identifications. This result on .45 cal. bulletsconfirmed the earlier “consecutivelymanufactured barrel” exercise with firearmexaminers on 9 mm bullets.

Publications and PresentationsStriupaitis, P. and Gaensslen, R.E.

“Quantitative /Statistical Approach toBullet to Firearm Identification withConsecutively Manufactured Barrels-Final Report,” Midwest ForensicsResource Center/Ames Laboratory FinalReport,Ames, IA: Ames Laboratory,2004,S-5170.


WORK

Probability density versus position forSpringfield bullets was generated using the IBISTechnology image comparisons. Thiscalculation gives the probability that the sisterbullet of any given bullet being compared will beone of those in the top n positions, where n isthe position number plotted- ndicatingaperfect match. These FTI IBIS imagecomparison tests correctly distinguishedbetween all Springfield barrel bullets andbetween most, bu t not all, of the HiPoint Barrel

bullets. In the HiPoint cases that were notdistinguished all of the time, they would bedistinguished at least 83% of the time. Theresults provide some evidence that barrel tobullet matching by image comparison is reliable.

The early version of the SciClops measuringsystem used in this study was not able to fullyresolve either manufacturer barrel bullets aswell as the FTI IBIS image comparisontechnology.

10

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References

~~~~

CONTACTS

1.

2.

3.

4.

5.

6.

Daubert v. Merrell Dow Pharmaceuticals,US. Supreme Court, 509 US. 579(1993)Bachrach B. “Development of a 3-0Automated Firearms EvidenceComparison System.” J. Forensic Sci.

47 [6] (2002):l-12. [paper #JFS2002054-476]Nichols, RG. “Firearm and ToolmarkIdentification Criteria:A Review of theLiterature.”J. Forensic Sci.42 [3]

Nichols, RG. “Firearm and ToolmarkIdentification Criteria: A Review of theLiterature, Part 11.” J ForensicSci.48 [2](2003):318-27. [paper # JFS2002246-4821Brundage DJ. ‘The Identification ofConsecutively Rifled Gun Barrels.” AFT€J (summer 1998): 30.Miller J. “An Examination of TwoConsecutively Rifled Barrels and aReview of the Literature.”AFT€ J

(summer 2000): 32.

(1997): 466-74.

R.E. GaensslenForensic Science GroupDept of Biopharmaceutical SciencesUniversityof Illinois at Chicago833 S. Wood St.

Chicago IL 60612

[email protected]

Peter StriupaitisFirearms Examiner(Retired from Illinois State Police ForensicScience Command)c/o University of Illinois at Chicago833 S. Wood St.Chicago IL 60612

[email protected]

(312) 996-2250

(312 ) 996-0888





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EVALUATIONOF THE PROMEGA DNA IQTMSYSTEM ANDQIAGEN BIOROBOT EZITM YSTEM AS AN EXTRACTION

METHOD FOR MITOCHONDRIAL DNA ANALYSIS


The expanding demand for DNA analyses iscreating the need for automation to improvethe efficiency and quality of the product, and to

save time. The use of automation foramplification and genetic analysis is not newfor forensic DNA analysis. DNA extraction andquantitation are two methods for whichautomation is relatively new. The need to

automate the extraction process is two-fold.One need is for the reduction of the time forDNA extraction from a day or two to an hour orless. The DNA analyst would in turn be free towork more cases and reduce case backlogsand have shorter case turn-around times. Thesecond need is to minimize analyst “hands-on”time with the evidence, which will minimizeevidence contamination and will reduce theanalytical space required for manual extractionmethods.

DNA automation is a common desire for manycrime laboratories and forensic analysts. Theautomated systems available can be large-scale and all-encompassing such as theBeckman-Coulter BiomekQ2000 to a smallscale extraction system like the QiagenBioRobot EZITM.The Northern Illinois PoliceCrime Laboratory and the DuPage CountySheriffs Office Crime Laboratory havecollaborated on the evaluation and applicationof the Qiagen BioRobot EZITM, ot only as amethod for automated DNA extraction ofnuclear DNA, but also as an automatedmethod for extracting mitochondrial DNA.


The Qiagen BioRobot EZITMSystem (Figure1) is a self-contained automated DNAextraction system utilizing magnetic particlescontained within individual reagent cartridges.The reagent cartridges are supplied in a

Figure 1. The Qiagen BioRobot EZI TMSystem.

sealed condition and are only opened within theinstrument, therefore minimizing contaminationfrom the outside environment. Also containedwithin the instrument are a heating block, amagnet, and a pipette head attachment fordispensing reagents.

Although the Qiagen BioRobot EZ1TM System is

versatile to employ various extraction methodsfor a variety of biological substances, its primanuse will be for the extraction of nuclear andmitochondrial DNA from forensic samples.

Presently, the Northern Illinois Police CrimeLaboratory, the DuPage County Sheriffs OfficeCrime Laboratory, and many other forensiclaboratories utilize a phenol/chloroform(organic) extraction for the extraction of DNA.This method, although suitable, requires theanalyst to manipulate many tubes for theextraction and purification of the DNA, whichmay lead to potential sample loss orcontamination. The organic extraction alsoutilizes a fume hood to dispel any vapors fromthe phenol, which requires more spaceallocation for extraction areas.

The Qiagen BioRobot EZITM ystem has all ofthe necessary components for the extraction

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and purification of DNA contained within thesystem. Minimal “hands-on” time is needed forsample preparation. The procedure requires a15-minute incubation period before the sampleis placed in the robot. Sample incubation foran organic extraction may typically take 6hours to overnight. All of the necessaryconsumables (buffer, tips, tubes, and reagentcartridges) are included in the same container.An organic extraction may require variousconsumables from various vendors. Finally,overall time from placing the sample cutting ina tube to final DNA extraction typically takes 35

to 40 minutes for the BioRobot EZITM.Anorganic extraction may take from 1 to 1 %

days.

TECHNOLOGY BENEFITS

Contamination and sample loss

incidents should be minimized due toDNA extraction and purification in anenclosed system and individual reagentcartridges. Once the sample is placedin the BioRobot EZITM,DNA extractionand purification is performed in parallel(one sample per one tip per one reagenicartridge).

Automation of DNA extraction will bemost time effective by reducing “hands-on” time an analyst has to spend with asample. The process of extracting up tosix samples at a time can take up to 35 -

40 minutes using the BioRobot EZITMand can be repeated for however manysamples need to be extracted. Thecurrent organic method typically cantake from 6 hours to overnight.Additionally, purification of the DNA bythe organic method takes an additional1-1 Y2 hours using separate tubes.


The data obtained in the current project waspresented as a poster presentation at the 15IhInternational Symposium on HumanIdentification2004 sponsored by Promega inPhoenix, Arizona.

Figure 2. The amplification of the mitochondrial

HVI as well as HVI regions of the blood,

urine, saliva, semen, and perspiration


Pfoser, K., and M.K.Gannon,“Validation of the Qiagen BioRobotEZ1:A Quick, Automated Method forExtracting DNA,” Poster Presentation,15IhInternational Symposium on

Human Identification.

Data from the current project will alsobe compiled by Qiagen for anupcoming paper. Initial plans are touse the BioRobot EZ1 M for theextraction of nuclear DNA at theNorthern Illinois Police CrimeLaboratory. Future collaboration withthe DuPage County Sheriff’s OfficeCrime Laboratory and the Illinois State

Police is being considered for theanalysis of mitochondrial DNA.

Other organizations utilizing theBioRobot EZ1TM with publications forforensic use are from the San DiegoPolice Department, the lnstitut furRechtsmedizin der Charite Berlin, andthe Armed Forces Scientific Institute forProtection Technologies.

ACCOMPLISHMENTS AND ONGOINGWORK

Primarily, the major accomplishmentsachieved from this research have been toobtain valuable data for both nuclear DNA andmitochondrial DNA in forensic samples. Thebody fluid study utilized samples typicallytested in forensic casework. Table 1 illustratesthe results of the amplification of the nuclearDNA. Figure2 illustrates an agarose gel

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I I IBlood I 100 I 5.0 13

3rine

Saliva 100 5.0 13

Semen 100 1.25 13

13

Buccal swab N/A 5.0 13

Belowdetection

0

Skin/ BelowN/A

Perspiration detection

Table 1. The Quantity and STR loci profile results of

the amplification of the nuclear DNA for

given test volumes of various sample

materials.

quantifying the amplification of the

mitochondrial HVI and HVll regions of theblood, urine, saliva, semen, and perspirationsamples.

Future work will include the additionalvalidation and utilization of protocols for lowcopy DNA samples typically tested formitochondrial DNA (bone, hair, teeth, etc.).Also, a new method for mitochondrial DNAtesting by Roche, LINEAR ARRAYMitochondrial DNA HVVHVII Sequence Typing

Kit will be investigated using samples from thecurrent study.

CONTACTS

Kenneth Pfoser 8, M. Kelly GannonNorthern Illinois Police Crime Laboratory1000 ButterfieldSuite 1009Vernon Hills, IL 60061(847) 362-0676 fax: -0712

dna@?nipcl.org

14

mailto:dna@?nipcl.org

mailto:dna@?nipcl.org

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VALIDATION OF DNA QUANTITATION AND SPECIES TESTS


DNA QuantificationOne of the least precise technical steps in DNApreparation and analysis is quantitation.Originally, both UV absorbance spectroscopy(260/280 m ratios) and mini-gels were used toestimate the total DNAfrom a preparation. UVis a tried-and-true method, but is limited by thelower range of the assay (-5 ng/pL) and by thevolume required for most cuvettes (-200 kL).Sometimes, there is not enough total samplefor this assay. Mini-gel estimates are limited

because they rely on visual estimation offluorescence intensity and are subject to 3- to5-fold variation. Further, both assays estimatetotal DNA. In “clean” specimens (DNAprepared from blood cells, for instance), totalDNA equals human DNA. But in most forensicspecimens, including DNA from buccal orvaginal swabs, there is considerable non-human DNA. What is needed is an estimate ofhuman DNA.

DNA-Based Species TestingLabs can receive non-human blood or bodyfluid evidence. Traditionally, dried bloodevidence was tested for species of origin byimmunological techniques. Some labs still dothis. But there is a tendency to do all testingusing DNA- or RNA-based methods. It is stillimportant to test evidence for species prior tosubjecting it to DNA analysis.


The universally used PCR-amplification kits arevalidated for 1-2 ng template human DNA. It isprobably fair to say that a target range of 500pg to 4 ng is acceptable- about the sameresults are expected within that range of inputtemplate. Outside this range, PCR artifacts thatmake interpretation of the profile difficult orimpossible start to become a significant factor.

The current widely used method for estimating

contained in the QuantiBlot kit from AB1(Applied Biosystems Group). The estimate

relies on a visual comparison of color orluminescence intensity between standards andspecimens. We have investigated the validity oiBodeQuant, a sensitive, spectrofluorimetricassay for human DNA estimation.

At the outset of our quantitation project,BodeQuant was in use only at Bode (The BodeTechnology Group, Inc.), where it was devised.More widespread use requires validation.Sufficient validation by us and by others, and

willingness of labs to acquire the necessaryhardware, could result in widespread use of thismethod. Currently, a different technology,based on RT-PCR, is available for human DNAquantification. It is not clear whetherBodeQuant would be materially cheaper thanRT-PCR, but probablyso. In addition tohardware costs, RT-PCR will be done withexpensive test kits. There are no test kitsmarketed for BodeQuant.

Our project was designed to validate the use ofa PCR-based amplification of a mitochondrialsequence called Cox-I for species testing. Theproject was designed as “proof of concept”rather than operational assay. The idea was todemonstrate that a species test could be basedon the amplification of Cox-I using fluorescent-tagged primers- he same fluorescent tagsused for CODIS loci. If the PCR amplicons fordifferent species were of different size, theywould separate as clean peaks by CE in an AB1

analyzer. Most labs use such instruments forCODIS locus profiling. This would be justanother injection.

TECHNOLOGY BENEFITS

Unless there is some independent reason toknow that a bloodstain is human (such as itwas observed dripping from a body at a scene):testing is necessary to verify species since it ispossible that blood or body fluids from animalshuman DNA is based on a dot-blot technique

I

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IS0 Run 1 ZMarO4 EO Run2: 29Mam4

Quantiblol Quantiblot Well 1 , Well 2, Well 1, Well 2, BQ 1 Well 1, Well 2, Well 1, Well2, BO 2

Specimen Analyst 1 Analyst 2 Scan 1 Scan 1 Scan2 Scan2 Average Scan 1 Scan 1 Scan2 Scan2 Average

9A 10.00 10.00 13.07 11.75 13.10 11.57 12.37 11.32 12.64 11.22 12.62 11.95

1 OA 10.00 8.00 9.76 8.83 9.73 8.78 9.28 8.57 no data 8.69 no data 8.63

11A 8.00 10.00 9.10 7.94 9.02 7.82 8.47 10.21 9.97 10.15 10.02 10.09

12A 2.50 3.00 3.67 3.67 3.78 3.71 3.71 3.16 3.19 3.17 3.18 3.18ME-A none none 0.34 <0.1 0.33 cO.1 N/A 0.12 cO.1 0.18 cO.1 NfA

18 0.13 0.13 0.61 cO.1 0.59 cO.1 - - - 0.11 0.12 0.16 0.19 0.15

28 5.00 5.00 15.98 13.71 16.05 13.80 14.89 5.61 5.73 5.78 5.85 5.74

38 1.25 1.25 2.02 1.14 2.00 1.13 1.57 1.76 1.44 1.89 1.47 1.64

48 16.00 12.00 20.78 23.98 20.83 24.33 22.48 10.59 11.99 10.76 12.09 11.36

58 5.00 5.00 7.21 7.26 7.13 7.20 7.20 6.67 6.95 6.71 7.11 6.86

68 5.00 5.00 13.06 12.51 13.14 12.67 12.85 5.44 6.06 5.58 6.28 5.84

78 12.00 12.00 16.64 13.76 16.81 13.89 15.28 15.03 14.64 14.92 14.73 14.83

88 12.00 10.00 11.55 9.94 11.56 9.89 10.74 9.30 8.79 9.37 8.90 9.09

98 7.00 8.00 10.78 9.29 10.86 9.20 10.03 8.57 8.92 8.77 9.06 8.83

108 16.00 14.00 9.77 11.42 9.80 11.40 10.60 11.37 11.04 11.38 11.19 11.25

118 11.42 9.14 11.98 10.31 12.01 10.37 11.17 10.10 10.79 10.07 10.85 10.45

ME-8 none none 0.44 0.24 0.46 cO.1 N/A <O.l <O.l <O.l <0.1 N/A

Table 1. QuantiBlotvs BodeQuant (BQ) (Numbers are DNA quantities in nglFL)

kept as pets could find their way ontoevidential specimens. And in some cases,species is the issue- llegal game killing, forexample. In addition, stains found on textilesor other items could be of non-human origin.Our results suggest that this methodologycould be developed without much difficulty.Forensic labs are seeking standardization andcomparatively easier validation. Thisapproach uses amplicon detection technologyidentical to Forensic Lab technology for DNAprofiling amplicon detection.

COLLABORATION/IMPLEMENTATlON

We have collaborated with lab personnel in theChicago Center of the Illinois State PoliceForensic Sciences Command. We are askingthe Command’s R&D section to explore Bode-quant as a possible alternative to QuantiBlot.

We are seeking others with an interest in thespecies methodology. The data is given inappendices of the final report.

Publications and PresentationsGaensslen, R.E. and Llewellyn, B.,“Further Development and Validation oiDNA-Based Methods for Species of OriginDetermination and Human DNAQuantitation in Forensic CaseworkSpecimens- Fin aI Report, ” MidwesiForensics Resource Center/Arne5Laboratory Report, Ames, IA: AmesLaboratory, 2005, IS-5169.


WORK

DNAQuantificationThe standard curve was optimized by titrationof the amount of the THO1 primer set used inthe PCR. Once the standard curve wasoptimized, preliminary validation of the assaywas initiated. A small number of samples wererun with the functional BodeQuant standardcurve assay and compared directly withQuantiBlot. The resulting DNA concentrationsof each of the specimens were first estimatedby running a preliminary yield (mini) gel and

6

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then employing QuantiBlot withchemiluminescence detection on x-ray film.Specimens used for validation -side by sidecomparison with QuantiBlot-were extractedby the standard phenol-chloroform-isoamylalcohol isolation method. To determine thevariability in the subjective band intensityjudgments required by QuantiBlot, DNAquantities were separately determined by theresearch assistant, and by a DNA analyst-supervisor at the lab (referred to as “Analyst 1”and “Analyst 2). The results are shown inTable 1.

Although there are some variations in thesubjective band intensity judgments of the two

analysts with the specimens, the QuantiBlotresults are close to the BodeQuant average inall cases. The results in Table 1 show enough

differences that further validation would benecessary before BodeQuant replacedQuantiBlot as the sole quantitation technique.The data suggest that it probably could beaccomplished, however.

QuantiBlot may have an advantage in that ithas been used extensively for a number ofyears. Yet, even our limited data show thatthere is variability in the estimates resultingfrom the subjective judgment of band intensityin QuantiBlot.

In practice BodeQuant is faster than

QuantiBlot in the sense that the analyst hasdown time during BodeQuantto be doing otherthings. In addition, more specimens can berun, and the range of the assay is greater.Another advantage to BodeQuant is that theassay must be standardized using curve fittingmethods. This characteristic of BodeQuantprovides a statistical basis for the results. Wefollowed the lead of the original paper [Fox,J.C., C.A. Cave, J.W. Schumm,BioTechniques,31 4-22(2003)], and used

Microsoft Excel for this purpose.

I Horse Human Dog Cat

Figure 1. Composite Photos of Post-Amplification Gels for All Four Species. The left-most lane is a OX-174-Haelll ladder. To the right is a diagrammatic representation of the sizing bands in thisladder. The ladder bands do not always resolve completely, but it is generally possible toverify that the size of the amplicon is approximately as expected. In the Horse, Human andCat gels, the middle lane is a A-DNA PCR control product of 500 bp. Because it is genomic,the numberof PCR cycles used for the mt-DNA result in overamplification of the A control.

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n

Figure 2. Single-plex results showing a compositeof all four individual single-plex results from top tobottom: dog, cat, human, horse.

DNA-Based Species Testing

A PCR-based species can be devised basedon mt Cox-I locus. The concept of doingspecies tests using amplification of Cox-I loci

from human, horse, cat, and dog with JOE-labeled primers has been shown to work usinggel separations. (Figure 1) In this work weestablished which variant of Taq polymerasewould do best-and it was Taq Gold. Theenzyme is robust, but requires a hot start (thisis not a problem). As proof of concept, labeledPCR products from all the single-plexes andseveral multiplexes were run on the AB1 310.(Figure2)

Although the final electropherograms show thatthere is still a need for refinement, we aresatisfied that the concept we set out to test hasbeen successfully demonstrated- namely, thata PCR-based species test can be devised,based on the mt Cox-I locus. Although fourspecies were somewhat arbitrarily chosen forthis proof of concept project, the technologycould easily be extended to any species forwhich sequence information is available for themt Cox-I locus.

The appeal of this approach is that it usesamplicon detection technology identical to thatroutinely employed in forensic labs for thedetection of DNA profiling amplicon detection.Accordingly, the specimen preparation s verysimilar, and the software is set up to providepeak sizings. In practice, the use of anapproach like this would entail running oneextra tube per specimen of interest. Assumingthere was no information from the case file tosuggest non-human DNA, one could use ahuman-animal duplex, or human-animal-animaltriplex, or even human-three other animalquadriplex. The expectation would be a humanDNA amplicon but no animal ones. The resultwould then explicitly confirm human origin.

CONTACTS

R.E. GaensslenForensic Science GroupDept of Biopharmaceutical SciencesUniversityof Illinois at Chicago833 S. Wood St.,Chicago IL 60612

[email protected] (312) 996-2250

Barbara LlewellynIllinois State PoliceForensic Science Center at Chicago1941 W. Roosevelt RoadChicago IL 60608

llewelb@ isp.state.il.us(31 2) 433-8000

8



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VALIDATION Of Y-PLEX 12 DATABASE STUDY


Forensic laboratories throughout the UnitedStates are grappling with how to manage anever increasing backlog of forensic caseworkrequiring DNA analysis and the costsassociated with new methodologies in DNAanalysis. DNA analyses conducted onevidence from criminal sexual assault casesare routinely used in forensic laboratories. Theinterpretation of DNA data on cases where

more than one semen donor is detected is verydifficult due to lack of automated techniques forthe separation of female victim DNA from malesperm DNA. Y-Chromosome STR systems areused to resolve mixed DNA profiles in sexualassault cases.

The work was performed by Illinois State PoliceForensic Science Command in collaborationwith ReliaGene Technologies, Inc, multiplexY-STR systems for use in forensic casework and

in the development of a population database.


The first goal of this project is to evaluate andvalidate commercially available Y-STR systemswhich have the potential to resolve mixed DNAprofiles in sexual assault cases. The secondgoal of this project is to generate a populationdatabase for Y-Chromosome STRs in the threemajor US. population categories: Caucasians,

African-Americans, and Hispanics.

Y-Chromosome STRs are short tandem repeatloci on the Y-chromosome (found only inmales) that have been found to exhibitdetectable polymorphism. STR genotyping isperformed by comparison of sample data toallelic ladders. In order to implement the useof Y-STRs in forensic casework, populationdata for the various loci need to be collected to

establish a population database. Because ofthe Y-Chromosome is inherited from father toson without recombination, there is lessvariability between individuals.

At the present time, the only statisticalevaluation that can be conducted to giveweight to a DNA match is the countingmethod. The statistical value obtained fromthe counting method is dependent on thenumber of samples that have been analyzed;

therefore, it is imperative to have a largepopulation database. These databases canonly be generated by evaluating a largenumber of samples in three major categories:Caucasians, African-Americans, andHispanics.

These Y-STR systems evaluations wereconducted under the validation guidelinesrecommended by the Scientific Working Groupon DNAAnalysis Methods (SWGDAM).

During the study, approximately 450 sampleswere collected and analyzed from the threemajor population categories utilizingReIiageneB Technologies Y-PLEX 12

amplification kit. Figure 1 shows anelectropherogram example from the Y-PLEX12 database population validation. Theresulting profiles were added to an existingdatabase maintained by ReliageneBTechnologies.

TECHNOLOGY BENEFITS

This Y-Plex 12database study validated

commercially available Y-STR systemsfor resolving mixed DNA profiles in sexualassault cases

The inclusion of this additional data to

the established Y-STR haplotypereference database will provide the

1

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, , , 5p I , , ,y . , , I70, , , 2 y . , , , 2 y . , . . 3 y . . . ,370, . . ,470, . . , 4 7 0 , , . . p0. . I I590. 1

50 IO0 15 0 200 250 300 350 400 45 0 500 5 5 0

,I 1-HOB 4 2 7 Blue HDB 4 2

2000

500

61 l U Q B 4 2 7 Green HOB 4 2

3000

2000

1000

i l l -HDB 42 7 Yellow HOB 4 2

I20001500

1M)0500

Figure 1. An exam ple of an electropherogram from the Y-STR analysis showing microvariantsample alleles that can be compared to the allelic ladders for STR genotyping.

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forensic science community with anadditional analytical tool that willenhance DNA Services to the benefit oflaw enforcement and criminal justice.

0 Forensic Laboratories can now utilize Y-

STR analysis in forensic casework for the

separation of female victim DNA frommale sperm DNA


The resulting profiles were added to anexisting database maintained by ReliageneBTechnologies. The inclusion of this additionaldata to the established Y-STR haplotypereference database provides forensiclaboratories with an additional analytical DNA

tool.

Publications and Presentations0 Frank, W.E., and S.N. Brown,

“Validation of Y-PLEX 12 Database,”Midwest Forensics Resource Center/Ames Laboratory Final Report, Ames,IA: Ames Laboratory, 2004, IS-5166.

0 Frank,W.E., and S.N. Brown,“Validation of Y-PLEX 12 Database,”

Presentation at Midwest ForensicsResource Center Annual Meeting, July15, 2004, Ames, IA, 50014.


WORK

This Y-Plex 12 database study generated theneeded population data (Caucasians, African-Americans, and Hispanics) for the establishedY-STR haplotype reference database.

With this validated data, Forensic Laboratoriescan now utilize Y-STR analysis in forensiccasework for the separation of female victimDNA from male sperm DNA.

Currently, Y-Plex 12 data is being collectedand reviewed for Y-Plex Validation. Uponcompletion of this review, the results of thepopulation database validation study will besubmitted to the Journal of Forensic Sciences.

CONTACTS

Mr. William FrankDNA Research CoordinatorIllinois State PoliceDivision of Forensic ServicesForensic Sciences Command

Research and Development Laboratory2060 Hill Meadows DriveSpringfield, Illinois 62702

William-Frank@ isp.state.il.us

Ms. Sandra N. BrownLaboratory DirectorIllinois State PoliceDivision of Forensic ServicesForensic Sciences CommandResearch and Development Laboratory2060 Hill Meadows DriveSpringfield, Illinois 62702

Sandy-Brown @ isp.state.il.us

(217) 557-2399 fax: -3989

(217) 557-2399 fax: -3989

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DEVELOPING APTAMERS TO METHAMPHETAMINEAS NUCLEIC ACID SENSORS

FORENSIC TECHNOLOGY NEED NO2 0

The abuse of methamphetamine in the USA isdescribed in a recent government publication

Abuse (NIDA) as “an extremely serious and

has increased significantly in the Midwest. ADepartment of Justice report states “an expertassociated with Juvenile Court Services in

HO&H - .N”O’bo-NKH

by the director of the National Institute on Drug I H O b H M e

Megrowing problem”. Methamphetamine traffic A

Deprotect

0

IMarshall County, Iowa, estimated in 1998 thatone-third of the 1.600 students at Marshalltown H2N+o+eN+ NHMe

NO2

High School had tried methamphetamine”.From 1994 to 1999 methamphetamine arrestsrose almost 4-fold and meth lab seizuresincreased about 8-fold. Methamphetamineeffects are long lasting and continue to causedamage long after the user has stopped takingthe drug. Long-term effects can include fatalkidney and lung disorders, possible braindamage, depression, hallucinations, violentand aggressive behavior, weight loss,insomnia, behavior resembling paranoidschizophrenia, malnutrition, poor copingabilities, lowered resistance to illnesses, liverdamage, stroke, and death. Societal impactsinclude crimes, fires due to explosions fromthe illegal manufacture of meth, andhazardous waste.

Aptamer-based assays will have severaladvantages over immunoassays for the rapidscreening of biological samples. Chiefamongst these advantages are that: 1) theaptamers are stable even when dehydratedand will be adaptable to mobile equipment,and2) new aptamers can be rapidly “evolvedin the laboratory. Thus, the aptamertechnology will be able to keep up with theintroduction of new drugs and changes in druguse over time.

w H wMe

Figure 1. The meth analog A with a carboxylic

acid tether that was prepared for this

project. It has been connected to a UVactive segment so that quantitation ofthe meth analog can be readily done.


The long-term goal of this project is to produceaptamers that can be used as screening toolsfor a variety of drugs to provide forensicinvestigators with rapid accurate screeningprocedures for common drugs. The followingspecific aims were proposed: 1 select for DNAaptamer(s) that recognizes methamphetamine2) clone and characterize the isolatedaptamer(s).

Aptamers are small nucleic acids that avidly

and selectively bind small ligands with affinitiesin the nanomolar to picomolar range and withexquisite specificities. A column ofmethamphetamine-linked sepharose is used tcselect for the aptamer. To prepare the column,the compound can be prepared from the meth!ester of para-formylbenzoic acid andnitroethane and a primary amine in 78% yield(Kraus, unpub-lished). The crystalline productis then reduced in two steps to the amino esterwhich is hydrolyzed to the acid with lithium

hydroxide~(49% ield). The acid is mixed with22

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EAH Sepharose 48 (Pharmacid Amersham) toselectively form a covalent linkage between thecarboxylic acid group and the functional groupson the EAH Sepharose 48 which is designedto link with compounds containing free carboxylgroups in order to produce affinity resins(Figure 1).

The procedures for making single strandedDNA (ssDNA) aptamers are well described [ l21. Methamphetamine-linked magnetic beadsare prepared for the SELEX procedure. Amixture of randomly varied ssDNAs is passedmultiple times through a methamphetamine-agarose column with intervening amplificationsteps that also introduce new variations into thesequence. We began with a pool of singlestranded DNAcontaining about 1014 randomsequences. (Figure2) Several rounds of

SELEX are needed to select aptamers.

Methamphetamine is used to elute themethamphetamine-specific ssDNAs from theaffinity column. After each round, the pool sizeof the DNA in the mixture will be monitored bydetermining the percent of total loaded ssDNAthat is adsorbed and eluted from the affinitycolumn. Negative selections againstSepharose and inactive methamphetamine

analogues are also included. The winningssDNAs (that bind to the column) are clonedand sequenced to identify consensussequences for aptamer structure. Consensussequences for aptamers are synthesizedchemically and tested for the ability to bind tomethamphetamine-sepharose.The selectedaptamers are also tested for their affinity to

meth using a filter-based assay.

References1. Tuerk, C. and L. Gold, “Systematic

evolution of ligands by exponentialenrichment: RNA ligands tobacteriophage T4 DNA polymerase.”Science 249 [4968] (1990): 505-10.

Schneider, D.J., et al., “High-affinityssDNA inhibitors of the reversetranscriptase of type 1 human

immunodeficiency virus.” Biochemistry

2.

34[29] (1995): 9599-610.

TECHNOLOGY BENEFITS

Procedures, such as GC, HPLC, and MS thatare currently used to analyze urine, hair, andother tissue samples for amphetamines andother drugs are costly and time consuming.

randomssDNA pool

r dsDNA

. ...... ..r..~

fretained

ssDNA eluted

------Figure 2. SELEX procedure used to optimize the first generation of aptamers.

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ACCOMPLiSHMENTSeally, the initial screening for drugs, includingnethamphetamine, should be rapid andiccurate.

iLlSAs are currently being developed fornitial screening because of their ease ofierformance and because these assays canle automated. Antibody microarrays areieing developed to move the immuno-basednethods of drug detection to a high-hroughput, large-range assay. However, theIbstacle remains that antibody responses arelot constant between animals, suffer fromlatch-to-batch inconsistencies, and require:hat significant effort be dedicated tostandardization.

4ptamers are nucleic acids with similar target-~indingroperties to antibodies. Unlike

mtibodies, aptamers are synthesized:hemically and therefore have little batch toDatch variation. Aptamers are ideally suited touse in analytical applications due to their smallsize and their stability to dehydration and hightemperatures.

COLLABORATION

The results of the current proposed researchwill be disseminated by publication in research

journals. The long-term goal of these studiesis to develop aptamers that specificallyrecognize methamphetamine and other drugsand that can be used for the detection of thesedrugs at the crime scene and in the laboratory.We have contacted Sandy Stoltenow and NilaBremer, scientists at the Iowa Divisionof

Criminal Investigation and will work with themonce we have obtained an aptamer that canbe used to sense methamphetamine. Onceestablished in Iowa, the use of aptamers for

sensing drugs will be readily applicable toother states.

Publications and PresentationsKraus, G.A., and M. Nilsen-Hamilton.“Developing Aptamers toMethamphetamine as Nucleic AcidSensors,” Presentation at MidwestForensics Resource Center AnnualMeeting, July 15, 2004, Ames, IA,5001 4.

We have now successfully passed through fiverounds of positive selection in SELEX and oneround of negative selection. Although apopulation has not yet evolved, we are carryinga subset of our oligonucleotides through these

rounds and anticipate soon observing anevolving population. Frequently 12 to 20rounds of SELEX are required to selectaptamers. Once an aptamer population hasevolved, the selected oligonucleotides will becloned and consensus sequences identified forfurther development of the aptamers.

In addition to the SELEX, we also linkedmethamphetamine to a biphenyl diamine as asecond methamphetamine derivative to use astarget tor the aptamers. By using this secondtarget in later rounds, we eliminate from thepool potential aptamers that interact with thearm (resorcinol or biphenyl) that links themethamphetamine to the magnetic beads. Themethamphetamine-(4-Bis (aminomethyl)biphenyl) will be used to preparemethamphetamine-linkedmagnetic beads forSELEX procedures.

CONTACTS

Marit Nilsen-HamiltonDepartment of Biochemistry, Biophysicsand Molecular BiologyIowa State University3206 Molecular Biology Blding

[email protected]

George KrausDepartment of ChemistryIowa State University

2759 Gilman HallAmes, IA 5001 1

[email protected]

(51 5) 294-9996

(51 5) 294-7794

4

mailto:iastate.edu

http://iastate.edu/

http://iastate.edu/

mailto:iastate.edu

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LASER ABLATION-INDUCTIVELY COUPLED PLASMA-MASSSPECTROMETRY OF FORENSIC GLASS SAMPLES


Glass fragments are often recovered as traceevidence during criminal investigations.Because of the many different types ofglasses manufactured, the challenge for theforensic examiner is to uniquely identify andcharacterize the glass trace evidence materialfrom a crime scene with a high level ofcertainty that is defensible. Characterizationof glass fragments is normally accomplishedby measuring the physical and opticalproperties of density and refractive index.However, further discrimination, such asidentification of a suspected source, hasbecome more difficult as the range of

refractive indices has narrowed within glasssubtypes because of advances in glassmanufacturing technology. Glasses from thesame subtype, which have the same grosselemental composition, can have differenttrace and ultra-trace elemental signatures.

An obvious major source of this variation liesin the trace and ultra-trace elementalcomposition of raw materials consumed in themanufacturing process, which is dependent onwhere the raw materials are mined. Thepresence, absence, and relative abundance ofelements in specific association patternsprovides a unique means of display andcomparison of the trace elemental signaturefor samples and is easily understandable.

Therefore elemental compositional analysishas frequently been considered as anapproach to classify glass fragments anddifferentiate between glasses within a class.

As a result, scientists have investigated theuse of elemental analysis techniques,particularly inductively coupled plasma-atomicemission spectrometry and -massspectrometry, for discrimination ordifferentiation between glasses within a

particular class (e.g., window glass) based ontheir trace elemental content. Theimplementation of laser ablation as a samplingtechnique extends this elemental analysis tomuch smaller sizes, typical of trace samplesencountered in forensic cases.


This project involves the evaluation of laserablation-inductively coupled plasma-massspectrometry as an analysis technique thatcan differentiate glass fragments, which havesimilar refractive indices, based on the uniquetrace elemental signatures of the glasssamples. Additionally, criteria and protocolsfor the comparison and differentiation of glassfragments from different sources, based onmultivariate analysis techniques, have beendeveloped. Multivariate analysis techniquesallow use of the full acquired mass spectrumwithout any prior knowledge of the chemical

composition of the sample.

A number of trace elemental analysis methodshave been investigated for discrimination ofglass samples. These methods includeatomic absorption spectrometry, neutronactivation, mass spectrometry, X-rayfluorescence, as well as inductively coupledplasma-atomic emission spectroscopy and -mass spectroscopy (ICP-AES and ICP-MS).Each method has its strengths, but only ICP

methods have the precision, sensitivity, multi-element detection capability, and dynamicrange suitable for trace elemental analysis offorensic glass samples. Although glassanalysis methods by ICP-AES and ICP-MSare similar, ICP-MS has better sensitivity (by afactor of 100 for some elements) and has thecapability to provide isotopic information formost elements.

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TECHNOLOGY BENEFITS

ICP-MS provides a high level of discriminationfor glass samples due to excellent detectionlimits (10-100 times better than AES),unparalleled elemental coverage, and isotopicinformation. Laser ablation-ICP-MS is rapid,

eliminates the need for extensive samplepreparation, and is a virtually nondestructivetechnique allowing the questioned samples tobe further analyzed by corroborativetechniques. Furthermore, laser ablationpromises to increase the number of analyticallyuseful elements detectable by standard ICP-MS techniques by eliminating problems withsome elements due to poor dissolution andcontamination. Additionally, smaller samplesmay be analyzed making the technique

applicable to more cases.

A novel approach for sample comparison inthis project is the utilization of multivariateanalysis techniques, in particular PrincipleComponent Analysis (PCA). PCA allows theuse of the full mass spectrum withoutrequiring any pre-selection or elimination ofelements. PCA is a multivariate data reductionmethod that examines the variance patternswithin a multidimensional dataset. PCA

reduces the dimensionality of the dataset to afew simple variables while retaining a majorportion of the mass spectral information.These new variables are then used for samplecomparisons and derivation of statisticalsignificance for the analysis.


The developed analysis protocols for glassanalysis have been used for casework with the

Vancouver, WA Police Department. Laserablation-ICP-MS s a powerful and versatiletechnique for determining the elementalcomposition of a variety of different types ofsamples. The Materials Analysis Unit of theFBI laboratory has previously worked with theAmes Laboratory research group to transfermultivariate analysis methods developed forlaser ablation-ICP-MS analysis of plain carbonsteel. The analysis protocols weresuccessfully implemented n a round robin

orqanized by the Illinois State Police to

determine source attribution of glass fragmentsby different analytical techniques. The analysisprotocols are currently being used in acollaborative study with Lawrence BerkleyNational Laboratory to investigate fractionationissues in nuclear proliferation materials. Amanuscript outlining the experimental andanalytical protocols for glass analysis is plannedfor submission to a forensic journal.


Baldwin, D.P., R.S. Houk, S.J. Bajic,“Laser Ablation-Inductively CoupledPlasma-Mass Spectrometry of ForensicGlass Samples,”Midwest ForensicsResource Center/Ames Laboratory FinalReport. Ames, IA: Ames Laboratory,2004, IS-5163.

Bajic, S.J., D.P. Baldwin, D.B.Aeschliman, and R.S.Houk, “LaserAblation Inductively Coupled PlasmaMass Spectrometry of Forensic GlassSamples,”Oral Presentation at AmericanAcademy of Forensic Sciences, 56thAnnual Meeting, 2004, Dallas, TX, PaperB164.

Aeschliman, D.B., S.J.Bajic, D.P.

Baldwin, and R.S.Houk, ‘MultivariatePattern Matching of Trace Elements inSolids by Laser Ablation InductivelyCoupled Plasma - Mass Spectrometry:Source Attribution and Diagnosis ofFractionation.”Analytical Chemistry 76,

(2004): 3119-3125.


WORK

This project determined that a minimum glasssample size of approximately 750 ng is requiredfor an analysis by these methods. This amountof material would be completely consumed in ananalysis. Work continues on understandingmechanisms that lead to fractionation andmethods for addressing these issues. This workcontinues under US Department of Energyfunding in collaboration with Lawrence BerkleyNational Laboratory. One area of investigationincludes the use of ultrafast lasers to eliminate

fractionation.26

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Group D Score Plot

A I I I IA

1 .2 ~1 0 ~

A 3

51035

si033

si009

1. I-1.2xi0Q

I I I I-1 .6~10 '~ -1.4~10'~ -1.2x10'~ -1 O X i O ' ~

Principle Component 1 (99.39%)

This plot illustrates the Principle Component Analysis (PCA) of the mass

spectra acquired by LA-ICP-MS from three glass fragments that are not

differentiable based on their refractive indices or density. The entire massspectra (elemental signatures of the samples) are used in the analysis. PCA

reduces the mass spectra to a few variables, which are used for sample

comparisons. When these variables are plotted, the repetitions of the

samples cluster together, but the samples occupy different areas on the plot

indicating that the glass samples are distinguishable based on their elemen-

tal compositions.

CONTACTS

Dr. David P. BaldwinAmes Laboratory Ames Laboratory Ames Laboratory130 Spedding Hall 827 Spedding Hall 9 Spedding HallAmes, IA 50011 Ames, IA 50011 Arnes, IA 50011

[email protected] rshouk@ astate.edu [email protected]

Dr. R. Sam Houk Dr. Stanley J. Bajic

(515) 294-2069 fax: -4748 (515) 294-9462 f a : -5233 (515) 294-2086 fax: -4748


http://iastate.edu/

http://iastate.edu/



http://iastate.edu/


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FORENSIC ANALYSIS OF TRACE EXPLOSIVES


The rise in terrorist bombings has made thechemical identification of explosives a priorityfor the forensic scientist. The volume ofevidence to be analyzed and the importance ofsuch testing for preventative and investigativepurposes have increased the need fordetection methods specific for explosives thatare reliable, fast, and cost effective. In thisproject, work was performed, as acollaboration between the Chemistry

Department at the University of Nebraska-Lincoln and the Nebraska State Patrol CrimeLaboratory, to develop new analytical methodsfor the forensic analysis of trace explosives bycombining immunoextraction and capillaryelectrophoresis (CE).


The analysis method created in this project fortrace explosives makes use of antibody-basedextraction (Le., immunoextraction) for theconcentration and selective isolation of desiredexplosive agents, followed by the separationand analysis of the isolated agents by CE(Figure 1). The immunoextraction sample pre-treatment and CE portions were developedseparately and then merged to create atandem analytical system.

High-performance liquid chromatography(HPLC) and gas chromatography (GC) are two

common methods employed in analyzingexplosives. CE is an alternative that has beenexplored for detecting explosives. Some of theadvantages of CE versus HPLC includesmaller sample volume requirements andgreater efficiency, allowing CE to separatemore compounds per run. CE, in contrast toGC, can work directly with liquid samples anduses lower separation temperatures, which isimportant when dealing with thermally unstableexplosive compounds. Both HPLC and GC

are used for moderate to large amounts of

sample. The CE method developed here utilize5nanoliter amounts of sample, unlike the millilitersample amounts required in HPLC, and in anondestructive manner (unlike GC). In addition,this CE method requires only nanoliter amountsof other reagents.

TECHNOLOGY BENEFITS

The CE method developed in this project hasbeen developed to separate fourteen target

organic compounds including HMX, RDX, TNT,PETN, and Tetryl. The current CE protocol hasbrought the following benefits:

Nanoliter sample requirements,translating into lower consumption ofevidence15-minute total analysis time per sampleSmaller reagent requirements thancurrently employed methods lowerspurchasing and waste disposal cost

In addition, immunoextraction columns havebeen successfully developed and tested for bothHMX and TNT-related compounds. The bindingand elution properties of these columns havebeen determined, as well as some of the cross-reactivity with related agents. Based on pastwork that has been performed with similarcolumns for herbicides and pesticide analysis inenvironmental samples, the availability of suchcolumns should greatly reduce the amount of

time and effort that is required by forensicscientists to process explosives samples prior toanalysis.


The Nebraska State Patrol CriminalisticsLaboratory has provided valuable guidance inthe development of these methods. This hashelped ensure that the protocols andinstrumentation hat have been developed are

28

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F l y r e 1. Separation &Analy sis ofSeveral Ep l mi ve A ge n e & Related Compounds by CE

~~

practical and relevant to the forensic sciencefield. Dissemination of this research has beenconducted through presentations that havebeen made at the 2004 Joint Meeting of theMidwest, Mid-Atlantic, Southern, and Canadian

Societies of Forensic Scientists. An invitedpresentation was also made on this work at aspecial meeting that was held in Fall 2004 bythe U.S. Army Corps of Engineers onBiosensing Methods for Explosives. Inaddition, the Pl’s laboratory is planning to

submit a paper on this topic for publication inthe Journal of Forensic Sciencesby May 2005.

The Pl’s laboratory is continuing to focus onthis project with work being done to furtheroptimize the CE separation andimmunoextraction. Future work will focus onminiaturizing the analytical system used in thisproject for the purpose of developing a portableexplosives detection system for screeningevidence at the site of an explosion.

The Army Corps of Engineers has contactedthe PI with an interest in funding field studieswith this approach, which are scheduled to takeplace in the summer of 2005.

Publications and PresentationsHage, D.S., “Forensic Analysis of

Trace Explosives,” Midwest ForensicsResource Center/Ames LaboratoryFinal Report,Ames, IA: AmesLaboratory, 2005, IS-5168.

Burks,R. “Forensic Analysis of Trace

Explosives,” Presentation at MidwestForensics Resource Center AnnualMeeting, July 15, 2004, Ames, IA,50014.

CONTACTS

PrinciDal Investiaator:David S. HageChemistry Dept., Univ. Nebraska608 Hamilton Hall, Lincoln NE 68588

[email protected]

Technoloav User:Dr. John DietrichNebraska State Patrol Crime Laboratory1223Arapahoe, Lincoln, NE 68502

jdietrica nsp.state.ne.us

(402)-472-2744 fax: -9402

(402)-471-8950



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