Use of Alternative Primers for Gender Discrimination in - Defense

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Office of Aerospace Medicine
Washington, DC 20591

Use of Alternative Primers for Gender
Discrimination in Human Forensic

Doris M. Kupfer
Marita Jenkins
Dennis Burian
Dennis V. Canfield
Civil Aerospace Medical Institute
Oklahoma City, OK 73125

April 2008

Final Report

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                                                   Technical Report Documentation Page
 1. Report No.                                      2. Government Accession No.                             3. Recipient's Catalog No.
 4. Title and Subtitle                                                                                      5. Report Date
 Use of Alternative Primers for Gender Discrimination in                                                    April 2008
 Human Forensic Genotyping                                                                                  6. Performing Organization Code

 7. Author(s)                                                                                               8. Performing Organization Report No.

 Kupfer DM, Jenkins M, Burian D, Canfield DV
 9. Performing Organization Name and Address                                                                10. Work Unit No. (TRAIS)

 FAA Civil Aerospace Medical Institute
 P.O. Box 25082                                                                                             11. Contract or Grant No.

 Oklahoma City, OK 73125

 12. Sponsoring Agency name and Address                                                                     13. Type of Report and Period Covered

 Office of Aerospace Medicine
 Federal Aviation Administration
 800 Independence Ave., S.W.
 Washington, DC 20591                                                                                       14. Sponsoring Agency Code

 15. Supplemental Notes

 Work was accomplished under approved task AM-TOXLAB
 16. Abstract

 An assay using the Federal Bureau of Investigation’s human Combined DNA Identity System (CODIS) primers
 has been developed for polymerase chain reaction (PCR)-based human identity testing. Recent forensic literature
 has identified several human populations that carry a deletion mutation in the Y-chromosome copy of the
 amelogenin locus. This is the standard locus used for gender determination in CODIS. Additionally, the
 amelogenin male PCR products are very close in size requiring manual annotation of PCR electrophoresis results
 for this locus. This study was designed to test several gender-specific primers which are to loci outside the
 amelogenin region, have well-separated PCR products, and could serve as additions or replacements to
 amelogenin in our human identity testing assay.

 17. Key Words                                                                        18. Distribution Statement
 PCR, Genotyping, Gender Determination, Electrophoresis                               Document is available to the public through the
                                                                                      Defense Technical Information Center, Ft. Belvior, VA
                                                                                      22060; and the National Technical Information
                                                                                      Service, Springfield, VA 22161
 19. Security Classif. (of this report)        20. Security Classif. (of this page)                      21. No. of Pages                22. Price
                 Unclassified                                     Unclassified                                       10
Form DOT F 1700.7 (8-72)                                                                                  Reproduction of completed page authorized


  We thank Dr. Brandt Cassdy, DNA Solutons, Inc., for hs helpful dscussons and for
provdng the ntal ZFX/ZFY prmer mxture for gender determnaton.


INtroDuCtIoN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
MAterIAlS AND MethoDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
       Sample sets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
       Genomc DNA extracton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
       PCr prmers and amplficaton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
       electrophoress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
reSultS AND DISCuSSIoN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
reFereNCeS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

               Use of AlternAtive Primers for Gender discriminAtion
                          in HUmAn forensic GenotyPinG

               INTrOduCTION                                          Addtonal genotypng assays have been developed
                                                                 for a seres of Y-specfic markers, whch nclude a seres
   Polymerase chan reacton-based human dentty test-          of short tandem repeat regons n the DYS locus found
ng has been used successfully for genotypng of forensc        on the q-arm of the Y chromosome (see, for example,
samples. the Federal Bureau of Investgaton developed           studes done by Butler10 and rutberg11). Glson reported
a standardzed set of loc and prmers for use wth PCr          the use of a mxture of prmers for two loc that can be
for genotypng. the Combned DNA Identty System                 used n a wde varety of mammals ncludng humans
(CoDIS) contans a core set of Str markers for hu-               for sex determnaton.12 one prmer par s desgned to
man dentty testng1 that are dscrmnatory over a wde        a regon of the Y-specfic gene SrY,12,13 and the second
range of ethnctes. An addtonal marker, amelogenn,2         s to the homologous znc finger proten genes of the X
has been ncluded n the CoDIS seres for gender ds-            and Y chromosomes, ZFX/ZFY.14 the successful use of
crmnaton.                                                     these prmers n humans has been reported.15
   the amelogenn prmers are specfic for a locus wthn            the current study took advantage of the avalablty of
the X-Y homologous regon. Gender determnaton s               human forensc samples to test alternatve gender deter-
based on the presence of an X copy and the presence or           mnng prmers. We tested Y-specfic short tandem repeat
absence of a 6 base-par shorter Y copy.2 In 1998, Santos        prmer sets for loc DYS390,10 DYS438, and DYS439,16
reported that two Sr lankan ndvduals from 350 males          all q-arm loc, and the prmers for p-arm loc ZFX/ZFY
from varous ethnc groups carred a p-arm deleton of           and SrY. the results were compared to amelogenn for
the Y chromosome whch ncluded the amelogenn re-               confirmaton of gender.
gon.3 thangaraj, n a 2002 study of 270 Indan males,
found five carryng a smlar deleton.4 In a 2003 study                mATErIAls ANd mEThOds
of 338 ndvduals of Malay, Chnese, and Indan eth-
ncty Chang reported that four Indans and one Malay            Sample sets
showed a deleton of the Y copy of the amelogenn locus.5           Four samples were used for characterzaton stud-
there are addtonal reports of amelogenn Y nulls n an         es. two male samples, 04010722-1 from lver, and
Australan Caucasan,6 a Moroccan father-son par,7 and          03010577-11 from blood, were forensc n orgn. Samples
sx Austrans.8 Chang concludes that there appears to be         23A and 23B were blood from female volunteers, col-
an amelogenn falure as hgh as 3.6% n the Malaysan           lected under a local Internal revew Board protocol for
Indan populaton and a lower but notceable defict of          developmental work. After ntal characterzaton of
0.02% n the Caucasan populaton.5                              the prmers, 12 addtonal forensc samples were used to
   recently, we reported the development of a genotypng         valdate the selected condtons across a varety of tssue
protocol based on the use of prmers from the CoDIS set,         types and DNA qualty (table 1).
ncludng the gender determnng marker, amelogenn,9
usng mcrofludcs rather than capllary electrophoress        genomic dNA extraction
for amplcon detecton. Based on the low but sgnficant            We stored and processed samples as descrbed n a
probablty of msdentfyng samples, we consdered t          prevous report.9 Brefly, tssue and blood were stored
advantageous to our genotypng protocol to develop an            at -20° C. Approxmately 25 mg of tssue was mnced
addtonal gender determnaton assay for use n suspected       and processed wth the ChargeSwtch gDNA Mn ts-
Y nulls. Addtonally, we were nterested n developng an       sue kt, #CS11204, usng the manufacturer’s drectons
assay that could potentally replace or be an addendum           (Invtrogen; Carlsbad, CA). DNA from whole blood was
to the amelogenn assay snce the very close sze dffer-        extracted from 200ul whole blood usng the QIAamp
ence between the X and Y allele products was sometmes           DNA Mn Kt #51304 (Qagen, Valenca, CA).
dfficult for mcrofludcs nstruments to automatcally
detect, thus requrng manual annotaton.

PCr primers and amplification                                       An ntal 95° C 11 mn, 96° C 1 mn; then 94° C
   the sequences for and expected product sze of the sx        30sec, ramp 68 sec to 60° C, hold for 30 sec, ramp 50 sec
prmer pars used n the study are shown n table 2. All         to 70° C, hold for 45 sec, for 10 cycles. then 90° C 30
prmers were syntheszed commercally (Integrated DNA            sec, ramp 50 sec to 70° C, hold for 45 sec, for 30 cycles.
technologes; Coralvlle, IA). A mx of ZFX/ZFY and              then 60° C for 30 mn, 4° soak.
SrY prmers for ntal testng was provded by Brandt
Cassdy, DNA Solutons; oklahoma Cty, oK.                       Electrophoresis
   reacton setup was for 25ul final volume. each reacton          Detecton of a 1ul alquot from a 1:3 dluton n dstlled
contaned 5ng template, 400-800nM prmers, 2.5mM                 water of each amplficaton product was performed on a
dNtPs, 0.1% trtonX-100, 1x Ampltaq Gold buffer, and            2100 Boanalyzer usng Aglent expert software verson
2.25unts Ampltaq gold. Amplficaton was as prevously         B.02.03.SI307 and DNA 1000 Seres II labchp kts
reported.9 thermal cyclng was performed on a GeneAmp            #5067-1504 (Aglent technologes; Palo Alto, CA).
9600 thermal cycler (Perknelmer; Wellesley, MA) usng
the followng cyclng profile for 40 cycles:                             rEsulTs ANd dIsCussION

                                                                    PCr was performed wth the four characterzaton
          Table 1. Additional forensic                           samples usng DYS prmers at 800nM and mxture prmers
          samples used in the study.
                                                                 at 400nM under standard PCr condtons (see Materals
          Sample ID               Source                         and Methods secton). DYS390 and DYS439 gave the
                                                                 expected product for the two males and were negatve
          03010577-1              blood
                                                                 for female samples. DYS438 showed the expected sngle
          03010577-7              liver
                                                                 peak wth the male samples but showed three products
          03010577-11             blood                          wth the female. Due to the presence of these spurous
          08010522-1              blood                          products, DYS438 was elmnated from further testng.
          08010522-3              heart                          A mxture of ZFX/ZFY and SrY prmers receved from
          08010522-5              brain                          Brandt Cassdy was tested at 400nM wth the four samples
          01010622-1              liver                          and gave the expected product results, a sngle peak for
          01010622-3              kidney                         females and two peaks for males (see table 2 for expected
          04010622-4              kidney                         products and szes).
          08010622-1              blood                             the crtera for a relable gender determnaton test
          04010722-1              kidney                         were the ablty to detect both males and females wth
          04010722-2              skin                           a dfferental sgnal for each, as seen wth amelogenn.

   Table 2. Primers used in study.

                                                                           Product Size or
         Locus        Primer Sequence                                        Range (bp)          Gender      Reference
      Amelogenin      ACC TCA TCC TGG GCA CCC TGG TT                      212(Y) or 218(X)      X and Y      Mannucci (2)
                      AGG CTT GAG GCC AAC CAT CAG
       DYS390         TAT ATT TTA CAC ATT TTT GGG CC                          189-233          Y-specific    Butler (10)
                      GTG ACA GTA AAA TGA AAA CAT TGC
       DYS438         TGG GGA ATA GTT GAA CGG TAA                             202-242          Y-specific    Ayub (16)
                      GTG GCA GAC GCC TAT AAT CC
       DYS439         TCC TGA ATG GTA CTT CCT AGG TTT                         236-256          Y-specific    Ayub (16)
                      GCC TGG CTT GGA ATT CTT TT
         SRY          CCC ATG AAC GCA TTC ATT GTG TGG                            224           Y-specific    Gilson (12)
                      ATT TTA GCC TTC CGA CGA GGT CGA TA
       ZFX/ZFY        GCA CTT CTT TGG TAT CTG AGA AAG T                          445            X and Y      Aasen (14)
                      ATA ATC ACA TGG AGA GCC ACA AGC T

ths ruled out the use of the DYS or SrY prmers, alone,
snce they are Y-specfic. therefore, three prmer mxes,              A.
each contanng the ZFX/ZFY prmer par, whch gves a
characterstc 445 bp product wth a female sample, and                                      Ameloginin

one male-specfic prmer par were tested. the results were                   female
compared to amelogenn, whch gves a sngle peak for a                                                                         MW Marker
female template, a doublet peak wth a male. the mxes,                        male
all at 800nM for each prmer, were DYS390 + ZFX/ZFY,                   MW Marker
DYS439 + ZFX/ZFY, and SrY + ZFX/ZFY. the last                                                         Secondary
mx, SrY+ZFX/ZFY, s smlar to the one receved from
Brandt Cassdy but at 800nM for each prmer, rather
than 400nM (Fgure 1).
    From Fgure 1A, t s clear that the small dfference n
                                                                                                    female                      High
sze of the X versus Y chromosome amelogenn product                                                                          MW Marker
results n a doublet rather than the well-separated peaks                                   SRY
as seen n Fgure 1B-D for male samples wth the three                 MW Marker
prmer mxes. Note that the ZFX/ZFY products seen
for the female sample have a hgher sgnal than the male                           male              male
snce there are two copes of the X chromosome present.                                                            products

Also note that secondary products are present for the
male samples for all Y-specfic prmers tested, nclud-
ng amelogenn. All three mxes gave dstnct peaks for                                                      ZFX/ZFY

male samples. there was, however, a dfferental level                 C.
of sgnal between the three Y-specfic prmer products                                                                          High
                                                                                                                              MW Marker
and ZFX/ZFY. ZFX/ZFY showed a fluorescent unt
(Fu) measure on the Boanalyzer of roughly 100 Fu at                     Low
                                                                       MW Marker
800nM wth the male samples. But the Y-specfic prm-
ers ranged from a hgh of 100 Fu wth SrY to 40 Fu                                                   male
wth DYS390 to a low of less than 40 Fu wth DYS 439                               male                            products

(Fgure 1B-D). the ampltude of secondary products
wth DYS and SrY prmers were all n the range of 5-15
Fu. the SrY + ZFX/ZFY mx was determned to have
optmal performance of the three prmer mxes showng                  D.                                    ZFX/ZFY

the hghest overall sgnal ntenstes of products, the most                                      female
closely equvalent product sgnal ntenstes, and lowest                                                                 MW Marker
secondary product sgnal strengths.
    We attempted to reduce the secondary products                      MW Marker
presence by reducng the prmer concentraton n the
SrY + ZFX/ZFY mx. A range of 400, 600, and 800nM                                           DYS439                Secondary
was examned for product and secondary product peak                                 male
strength wth the same four samples. No secondary
products were detected at 400nM, but sgnal ntensty
was reduced to 15 Fu. Increasng SrY concentraton
to 600 and 800nM, respectvely, showed the presence                Figure 1. Gender primer mix PCR products. Shown
of the secondary product doublet at less than 5 Fu for             are overlays of bioanalyzer results of the PCR
                                                                   products from the three mixes and amelogenin
600nM and 5-8 Fu 800nM. the SrY amplcon peak                      (see Results and Discussion section). Male
heght at 600nM averaged 50 Fu and 70 Fu at 800nM                  sample 04010722-1 and female sample 23A (taller
(Fgure 2). the relatvely low level of secondary prod-            peaks in Amelogenin and ZFX/ZFY overlays) are
uct and hghest product Fu at 800nM suggested that                 compared. A. Amelogenin; insert is a close-up of the
                                                                   characteristic male doublet. B. SRY+ZFX/SFY C.
ths was the optmal concentraton. the dfference n              DYS390+ ZFX/SFY D. DYS439+ ZFX/SFY.

sgnal ntensty seen n the two SrY PCr seres usng               In summary, the avalablty of addtonal gender
800nM prmers wth the same templates suggests that             determnaton loc wll expand our repertore of robust
the varatons between experments may result n sgnals        prmers for use n forensc sample dentficaton. ths s
rangng from 70-100 Fu for the SrY products and 5-              mportant because of the null-Y mutaton that results n
15 for the secondary product doublet. these levels are          a deleton of the amelogenn gene located at 6.79Mbp on
acceptable for detecton on the Boanalyzer n the case         the Y chromosome.3 4 5 the mutaton occurs especally
of the product and reman below the default cutoff for          wthn certan Indan populatons but has also been found
the secondary products.                                         n Caucasan populatons.6 7 8 the result of ths deleton
    twelve forensc samples were tested wth the mx at         s the ncorrect dentficaton of the ndvdual as female
800nM to assure consstent results across a varety of          when usng amelogenn as the gender-determnng locus.
samples. these samples had been prevously tested by            the extent of the mutaton has been estmated. the MSY1
PCr wth amelogenn. All the samples appeared male,             locus, a Y-specfic mn-satellte marker on the same p-arm
based on amelogenn results, except 04010722-2, whch           as amelogenn, but less than a Mbp away, was negatve
gave a negatve amelogenn result (data not shown). Four        wth an amelogenn deleton group.5 Subjects carryng
samples were from blood, and eght were from a varety          the null-Y marker had a Y-chromosome deleton of about
of tssues (table 1). eleven of the samples showed the          1Mbp.4 Fnally, the tSPYP1 locus, located at 6.19Mbp,
expected peaks for SrY and ZFX/ZFY at sgnal strengths          was found to be a proxmal endpont of the deleton.3 the
rangng from 50-100 Fu. Sample 04010722-2 showed a              StY and ZFY genes are on the p-arm of the Y chromo-
very weak sgnal, a sngle peak for SrY wth 10 Fu but          some at 2.56Mbp and 2.86 Mbp respectvely. Both are
no ZFX/ZFY peak. ths was not surprsng snce ths             sgnficant dstances from the amelogenn gene deleton
sample had been negatve wth amelogenn. the absence           regon and therefore are good canddates for alternatve
of the ZFX/ZFY peak, whch represents an amplcon of            gender determnaton loc.
445bp compared to the weak 245bp SrY amplcon, sug-                 Certanly, n human forensc cases where there s a
gests sample degradaton. Furthermore, the presence of a        possblty of an amelogenn deleton, t would be neces-
peak from the SrY locus unambguously determnes that           sary to use an alternatve locus. however, t also wll be
the sample s from a male, regardless of the presence or        very helpful to use the ZFX/ZFY and SrY prmer mx to
absence of a sgnal from the X-determnant locus. over-         facltate automatc annotaton of the Boanalyzer results
all, the gender mx performs satsfactorly wth forensc       because of the excellent separaton and sgnal strength
samples, gvng expected results wth DNA solated from         of the products.
both tssue and blood.


                                600nM                                                   600nM



                                                                                              Secondary Products

Figure 2. Comparison of PCR product signal strengths with SRY +ZFX/ZFY primer mixes. The product peaks
are from male sample 04010722-1. The concentration of primers in the mix is shown.
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