nij-strbyms.pdf

35

RESULTS AND DISCUSSION OF STR ANALYSISBY MASS SPECTROMETRY

In the course of this work, thou-sands of data points were collectedusing STR markers of forensicinterest verifying that GeneTracesmass spectrometry technology works.During this same time, tens of thou-sands of data points were gatheredacross hundreds of different microsatel-lite markers from corn and soybeanas part of an ongoing plant genomicspartnership with Monsanto Company(St. Louis, MO). Whether the DNAmarkers used come from humans orplants, the characteristics describedbelow apply when analyzing polymor-phic repeat loci.

Marker Selection andFeasibility StudiesWith STR LociPrior to receiving grant funding, feasi-bility work had been completed usingthe STR markers TH01, CSF1PO,FES/FPS, and F13A1 in the summerand fall of 1996 (Becker et al., 1997).At the start of this project, a number

of STR loci were considered as possi-ble candidates to expand upon theinitial four STR markers and to devel-op a set of markers that would workwell in the mass spectrometer andwould be acceptable to the forensicDNA community. Searches were madeof publicly available databases, includ-ing the Cooperative Human LinkageCenter (http://lpg.nci.nih.gov/CHLC),the Genome Database (http://gdbwww.gdb.org), and Weber set 8 of theMarshfield Medical Research Founda-tions Center for Medical Genetics(http://www.marshmed.org/genetics).Literature was also searched for pos-sible tetranucleotide markers withPCR product sizes below 140 bp insize to avoid having to redesign thePCR primers to meet our limited sizerange needs (Hammond et al., 1994;Lindqvist et al., 1996). The desiredcharacteristics also included high het-erozygosity, moderate number of alle-les (

36

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

STR markers used by the PromegaCorporation, Applied Biosystems, andthe Forensic Science Service (FSS),researchers redesigned primer pairsfor each STR locus to produce smallerPCR product sizes. These STR mark-ers included TPOX, D5S818, D7S820,D13S317, D16S539, LPL, F13B, HPRTB,D3S1358, VWA, FGA, CD4, D8S1179,D18S51, and D21S11. The primersfor TH01 and CSF1PO were alsoredesigned to improve PCR efficienciesand to reduce the amplicon sizes.Primers for amelogenin, a commonlyused sex-typing marker, were also test-ed (Sullivan et al., 1993). In addition,two Y-chromosome STRs, DYS19 andDYS391, were examined briefly. Exhibit34 summarizes the STR primer setsthat were developed and tested overthe course of this project. However,

with the announcement of the 13CODIS core loci in the fall of 1997,emphasis switched to CSF1PO, TPOX,TH01, D3S1358, VWA, FGA, D5S818,D7S820, D13S317, D16S539,D8S1179, D18S51, D21S11, and thesex-typing marker amelogenin.

The newly designed GeneTraceprimers produced smaller PCR prod-ucts than those commercially availablefrom Applied Biosystems or Promega(exhibit 2), yet resulted in identicalgenotypes in almost all samples tested.For example, correct genotypes wereobtained on the human cell line K562,a commonly used control for PCRamplification success. Exhibit 35shows the K562 results for CSF1PO,TPOX, TH01, and amelogenin. Theseresults were included as part of a

publication demonstrating that time-of-flight mass spectrometry could per-form accurate genotyping of STRswithout allelic ladders (Butler et al.,1998).

Caveats of STR analysis by mass spectrometryWhile mass spectrometry worked wellfor a majority of the STR markers test-ed, a few limitations excluded someSTRs from working effectively. Twoimportant issues that impact massspectrometry results are DNA sizeand sample salts. Mass spectrometryresolution and sensitivity are dimin-ished when either the DNA size orthe salt content of the sample is toolarge (Ross and Belgrader, 1997; andTaranenko et al., 1998). By designingthe PCR primers to bind close to therepeat region, the STR allele sizes arereduced so that resolution and sensi-tivity of the PCR products are benefit-ed. In addition, the GeneTrace-patentedcleavage step reduces the measuredDNA size even further. When possible,primers are designed to produce ampli-cons that are less than 120 bp, althoughwork is sometimes undertaken withSTR alleles that are as large as 140 bpin size. This limitation in size preventsreliable analysis of STR markers withsamples containing a large numberof repeats, such as most of the FGA,D21S11, and D18S51 alleles (exhibit 2).

To overcome the sample salt problem,researchers used a patented solid-phasepurification procedure that reduced theconcentration of magnesium, potassi-um, and sodium salts in the PCR prod-ucts prior to being introduced to themass spectrometer (Monforte et al.,1997). Without the reduction of thesalts, resolution is diminished by thepresence of adducts. Salt moleculesbind to the DNA during the MALDIionization process and give rise topeaks that have a mass of the DNAmolecule plus the salt molecule.Adducts broaden peaks and thusreduce peak resolution. The sample

HumanChromosome STR Marker

HumanChromosome STR Marker

1 F13B 13 D13S317

2 TPOX 14

3 D3S1358 15 FES/FPS

4 FGA 16 D16S539, D16S2622

5 CSF1PO, D5S818,GATA132B04

17

6 F13A1 18 D18S51

7 D7S820 19

8 LPL, D8S1179 20

D21S11

10 22 D22S445

11 TH01 X HPRTB, Amelogenin

12 VWA, CD4 Y DYS19, DYS391,Amelogenin

Exhibit 34. STR markers examined at GeneTrace during the course of this project as sorted by their chromosomal position. Primers were designed, synthesized, and tested for each of these markers. The most extensive testing was performed with the markers highlighted in green. Amelogenin, which is a gender identification marker rather than an STR, is listed twice because it occurs on both the X and Y chromosomes. The italicized STRs are those not commonly used by the forensic DNA community.

219

37


purification procedure, which wasentirely automated on a 96-tip roboticworkstation, reduced the PCR buffersalts and yielded clean DNA for themass spectrometer. Appropriate caremust be taken to prevent samples frombeing contaminated with salts bothduring and after the sample purifica-tion procedure.

Size reduction methodsThe portion of the DNA product onthe other side of the repeat region fromthe cleavable primer was removed inone of two possible ways: using arestriction enzyme (Monforte et al.,1999) or performing a nested linearamplification with a ddN terminatingnucleotide (Braun et al., 1997a and1997b). Both methods have pros andcons. A restriction enzyme, DpnII,which recognizes the sequence 5...^GATC...3, was used with VWA sam-ples to remove 45 bp from each PCRproduct. For example, the GenBankallele that contains 18 repeat units andis 154 bp following PCR amplificationmay be reduced to 126 nucleotides fol-lowing primer cleavage, but it can beshortened to 81 nucleotides if primercleavage is combined with DpnII diges-tion. At 81 nt or 25,482 Da, the STRproduct size is much more manage-able in the mass spectrometer. Thisapproach works nicely provided therestriction enzyme recognition siteremains unchanged. The DpnII diges-tion of VWA amplicons worked on allsamples tested, including a reamplifi-cation of an allelic ladder from ABI(exhibit 36). However, the cost andtime of analysis are increased with theaddition of a restriction enzyme step.

The second approach for reducing theoverall size of the DNA molecule inthe mass spectrometer involved usinga single ddNTP with three regulardNTPs. A linear amplification exten-sion reaction was performed with theddNTP terminating the reaction onthe opposite side of the repeat fromthe cleavable primer. However, there

Exhibit 35. Mass spectra for CSF1PO, TPOX, TH01, and amelogenin using K562 DNA. Genotypes agree with results reported by the manufacturer (Promega Corporation). The numbers above the peaks represent the allele calls based upon the observed mass. The allele imbalance on the heterozygous samples is because the K562 strain is known to contain an unusual number of chromosomes and some of them are represented more than twice per cell. The TH01 peak is split because it is not fully adenylated (Butler et al., 1998).

15,000 20,000 25,000 30,000

CSF1PO

TPOX 9

8

9

10

9.3TH01

Sign

al

Amelogenin X

Mass (Da)

38


were several limitations with this sin-gle base sequencing approach. First,it only worked if the repeat did notcontain all four nucleotides. For exam-ple, a nucleotide mixture of dideoxycy-tosine (ddC), deoxyadenosine (dA),deoxythymidine (dT), and deoxy-guanosine (dG) will allow extension

through an AATG repeat (as occurs inthe bottom strand of TH01) but willterminate at the first C nucleotide in aTCAT repeat (the top strand of TH01).Thus one is limited with the DNAstrand that can be used for a givencombination of dideoxynucleotide andcorresponding deoxynucleotides. In

addition, primer position and STRsequence content are important. If addC mix is used, the DNA sample can-not contain any C nucleotides prior tothe repeat region or within the repeat,or the extension will prematurely haltand the information content of the fullrepeat will not be accurately captured.In most cases, this requires the exten-sion primer to be immediately adja-cent to the STR repeat, a situation thatis not universally available due to theflanking sequences around the repeatregion. For example, this approachwill work with TH01 (AATG) but notVWA, which has three different repeatstructures: AGAT, AGAC, and AGGT.Thus with VWA, a ddC would extendthrough the AGAT repeat but wouldbe prematurely terminated at the C inthe AGAC repeat, and valuable poly-morphic information would be lost.

The use of a terminating nucleotidealso provides a sharper peak for anamplified allele compared with the splitpeaks or wider peaks (if resolution ispoor) that can result from partiallyadenylated amplicons (i.e., -A/+A).Exhibit 37 illustrates the advantage ofa ddG termination on a D8S1179 het-erozygous sample containing 11 and13 TATC repeats. In the bottom panel,23 nt were removed compared withthe top panel, which corresponds toa mass reduction of almost 8,000 Da.The peaks are sharper in the lowerpanel, as the products are blunt ended.Identical genotypes were obtainedwith both approaches, illustrating thatthe ddG termination is occurring atthe same point on the two differentsized alleles.

To summarize, STR sample sizes werereduced using primers that have beendesigned to bind close to the repeatregion or even partially on the repeatitself. A cleavable primer was incorpo-rated into the PCR product to allowpost-PCR chemical cleavage and subsequent mass reduction. Two additional post-PCR methods werealso explored to further reduce themeasured DNA size. These methods

20,000 22,000 24,000 26,000 28,000 30,000 32,000

14,000 16,000 18,000 20,000 22,000 24,000

14,000 16,000 18,000 20,000

16,000 18,000 20,000 22,000 24,000 26,000 28,000 30,000

Exhibit 36. Mass spectra of STR allelic ladders from CSF1PO, TPOX, TH01, and VWA. The numbers above each peak designate the allele name (number of repeats). Peak widths vary among samples based on DNA size and salt content. Smaller sizes (e.g., TH01) give sharper peaks than larger sizes (e.g., CSF1PO). On a mass scale as shown here, each nucleotide is approximately 300 Daltons (Da). The VWA ladder was digested with DpnII restriction enzyme following PCR to reduce the overall size of the amplicons.

CSF1PO

TPOX

TH01

VWA

67

8 9

10

11

1213

1514

6 7 8 910 11 12

13

6 78 9 10

5 9.3

11 12 1516 17 18

1314

19 20 21

Mass (Da)

39


included restriction enzyme digestionin the flanking region on the otherside of the repeat region from thecleavable primer and a primer exten-sion through the repeat region with asingle dideoxynucleotide terminator(single base sequencing approach).

To illustrate the advantages of theseapproaches to reduce the overallDNA product mass, researchers exam-ined the STR locus TPOX. Using a

conventional primer set, a samplecontaining 11 repeats measured 232bp or ~66,000 Da. By redesigning theprimers to anneal close to the repeatregion, a PCR product of 89 bp wasobtained. With the cleavable primer,the size was reduced to 69 nt or21,351 Da. By incorporating a ddCtermination reaction, another 20 ntwere removed leaving only 49 nt or~12,000 Da (primarily only the repeatregion). The repeat region contained

44 nt (4 nt x 11 repeats) or ~10,500Da. The ddC termination was alsoused in multiplex STR analysis toproduce a CSF1PO-TPOX-TH01triplex (exhibits 4 and 5). The repeatsequences used for these STR loci wereAGAT for CSF1PO, AATG for TPOX,and AATG for TH01. The level ofsequence clipping by ddC was as follows: CSF1PO (-14 nt), TPOX (-20 nt), and TH01 (-4 nt).

-A/+A

Mass reduced by 7,477 Da

Exhibit 37. Mass spectra of a D8S1179 sample illustrating the benefit of a dideoxynucleotide termination approach. The top panel displays a result from a regular PCR product; the bottom panel contains the same sample treated with a linear amplification mix containing a ddG terminator with dA, dT, and dC deoxynucleotides. With the ddG approach, the problem of incomplete adenylation (both A and +A forms of a PCR product) is eliminated, and the amplicons are smaller, which improves their sensitivity and resolution in the mass spectrometer. The red oval highlights the broader -A/+A peaks present in the regular PCR product. Note: The genotype (i.e., 11 and 13 repeats) is identical between the two approaches, even though almost 8,000 Da are removed with the ddG termination.

12,000 14,000 16,000 18,000 20,000 22,000 24,000 26,000 28,000 30,000

1113

(not fully adenylated)

Sign

al

Mass (Da)

1113

ddG approach

regular PCR products

40


Multiplex STR WorkDue to the limited size range of DNAmolecules that may be analyzed bythis technique, a new approach tomultiplexing was developed thatinvolved interleaving alleles fromdifferent loci rather than producingnonoverlapping multiplexes. If theamplicons could be kept under~25,000 Da, a high degree of massaccuracy and resolution could be usedto distinguish alleles from multipleloci that may differ by only a fractionof a single nucleotide (exhibit 10).Allelic ladders are useful to demon-strate that all alleles in a multiplexare distinguishable (exhibit 9).

The expected masses for a triplexinvolving the STR loci CSF1PO,TPOX, and TH01 (commonly referredto as a CTT multiplex) are schemati-cally displayed in exhibit 4. All knownalleles for these STR loci, as defined bySTRBase (Ruitberg et al., 2001), arefully resolvable and far enough apartto be accurately determined. Forexample, TH01 alleles 9.3 and 10 fallbetween CSF1PO alleles 10 and 11.For all three STR systems in this CTTmultiplex, the AATG repeat strand ismeasured, which means that the alle-les within the same STR system differby 1,260 Da. The smallest spreadbetween alleles across multiple STRsystems in this particular multiplexexists between the TPOX and TH01alleles, where the expected mass dif-ference is 285 Da. TPOX and CSF1POalleles differ by 314 Da, while TH01and CSF1PO alleles differ by 599 Da.By using the same repeat strand in themultiplex, the allele masses betweenSTR systems all stay the same distanceapart. Each STR has a unique flankingregion and it is these sequence differ-ences between STR systems that per-mit multiplexing in such a fashion asdescribed here. An actual result withthis CTT multiplex is shown in exhib-it 5. This particular sample is homozy-gous for both TPOX (8,8) and CSF1PO(12,12) and heterozygous at the TH01locus (6,9.3).

It is also worth noting that this partic-ular CTT multiplex was designed toaccount for possible, unexpectedmicrovariants. For example, a CSF1POallele 10.3 that appears to be a singlebase shorter than CSF1PO allele 11was recently reported (Lazaruk et al.,1998). With the CTT multiplex primerset described here, a CSF1PO 10.3allele would have an expected mass of21,402 Da, which would be fully dis-tinguishable from the nearest possibleallele (i.e., TH01 allele 10) becausethese alleles would be 286 Da apart.Using a mass window of 100 Da asdefined by previous precision studies(Butler et al., 1998), all possible allelesincluding microvariants should befully distinguishable. STR multiplexesare designed so that expected allelemasses between STR systems are offsetin a manner that possible microvari-ants, which are most commonly inser-tions or deletions of a partial repeatunit, may be distinguished from allother possible alleles. The larger thealleles mass range, the more difficult itbecomes to maintain a high degree ofmass accuracy. For example, exhibit 8shows the observed mass for TH01allele 9.3 is -52 Da from its expectedmass, while TPOX allele 9 is only 3Da from its expected mass. In this par-ticular case, the mass calibrants usedwere 4,507 Da and 10,998 Da. Thus,the TPOX alleles mass measurementwas more accurate and closer to thecalibration standard. The ability todesign multiplexes that have a rela-tively compact mass range is importantto maintaining the high level of massaccuracy needed for closely spacedalleles from different, overlapping STRloci. The mass calibration standardsshould also span the entire region ofexpected measurement to guaranteethe highest degree of mass accuracy.

Two possible multiplexing strategiesfor STR genotyping are illustratedin exhibit 38. Starting with a singlepunch of blood stained FTA paper, itis possible to perform a multiplex PCR(simultaneously amplifying all STRs

of interest) followed by another PCRwith primer sets that are closer to therepeat region. With this approach,single or multiplexed STR productscan be produced that are smallenough for mass spectrometry analy-sis. Alternatively, multiple punchescould be made from a single blood-stain on the FTA paper followed bysingleplex or multiplex PCR with massspectrometry primers. After the geno-type is determined for each STR locusin a sample, the information would becombined to form a single samplegenotype for inclusion in CODIS orsome other DNA database. This multi-plexing approach permits flexibilityfor adding new STR loci or only pro-cessing a few STR markers across alarge number of samples at a lowercost than processing extensive andinflexible STR multiplexes.

Comparison TestsBetween ABI 310 andMass SpectrometryResultsA plate of 88 samples from the CDOJDNA Laboratory was tested with 10different STR markers and comparedwith results obtained using the ABI310 Genetic Analyzer and commer-cially available STR kits. The sampleswere supplied as a 200 L aliquot ofextracted genomic DNA in a 96-welltray with each sample at a concentra-tion of 1 ng/L. A 5 L aliquot wasused for each PCR reaction, or 5 ngtotal per reaction. Since each markerwas amplified and examined individu-ally, approximately 35 ng of extractedgenomic DNA was required to obtaingenotypes on the same 7 markers aswere amplified in a single AmpF1STR

COfilerTM STR multiplex. Only 2 ng ofgenomic DNA were used per reactionwith the AmpF1STR COfilerTM kit.Thus, a multiplex PCR reaction is muchbetter suited for situations where thequantity of DNA is limited (e.g., crimescene sample). However, in most cases

41


involving high-throughput DNA typ-ing (e.g., offender database work),hundreds of nanograms of extractedDNA would be easily available.

A major advantage of the mass spec-trometry approach is speed of thetechnique and the high-throughput

capabilities when combined withrobotic sample preparation. The datacollection times required for the 88CDOJ samples using the ABI 310Genetic Analyzer and GeneTracesmass spectrometry method are com-pared in exhibit 11. While it took the

ABI 310 almost 3 days to collect thedata for the 88 samples, the samegenotypes were obtained on the massspectrometer in less than 2 hours.Even the ability to analyze multipleSTR loci simultaneously with differentfluorescent tags on the ABI 310 couldnot match the speed of GeneTracesmass spectrometry data collection witheach marker run individually.

To verify that the mass spectrometryapproach produces accurate results,comparison studies were performed onthe genotypes obtained from the twodifferent methods across 8 differentSTR loci. Exhibits 1219 contain adirect comparison with 1,408 possibledata points (2 methods 88 samples 8 loci). With a few minor excep-tions, there was almost a 100% corre-lation between the two methods. Inaddition to the data obtained on the8 loci from both the ABI 310 and themass spectrometer, two additionalmarkers (D8S1179 and DYS391) weremeasured by mass spectrometry acrossthese same 88 samples (exhibits3940). Both the D8S1179 and theDYS391 primer sets worked extre-mely well in the mass spectrometer(exhibit 41). Thus, it is likely that ifresults were made available on thesesame samples with fluorescent STRprimer sets (e.g., D8S1179 is in theAmpF1STR Profiler PlusTM kit), therewould also be a further correlationbetween the two methods.

PCR Issues

Null allelesWhen making comparisons betweentwo methods that use different PCRprimer sets, the issue is whether or nota different primer set for a given STRlocus will result in different allele callsthrough possible sequence polymor-phisms in the primer binding sites. Inother words, do primers used for massspectrometry that are closer to therepeat region than those primers used

STEPS INPROCESS

Blood samples

DNA FTA paper with blood spotExtraction

Single punch Multiple punchesfrom the same spot

DNA (PCR ) Multiplex PCR with STR- Singleplex, duplex, or triplexAmplification

locus-specific primers PCR with GeneTrace (14 loci simultaneously) primers optimized for MS

SPLIT SAMPLESecond-round (nested PCR)

with GeneTrace primersto probe 1-3 STRs per assay

Purification 384-well purification on robotic workstation

Mass Spec Automated mass spec detectionDetection

Data Analysis/ Automated genotyping at each locusGenotyping

Databasing Data combined to form single sample genotype

Exhibit 38. Multiplexing strategies for STR genotyping using FTA paperTM

42


Exhibit 39. CDOJ D8S1179 STR results with the mass spectrometry method

Position Mass Spec Allele 1 (Da) Allele 2 (Da) Position Mass Spec Allele 1 (Da) Allele 2 (Da)A1 12,15 24,693 28,228 A7 13,13 25,791B1 14,15 27,159 28,369 B7 13,14 25,845 26,954C1 11,14 23,342 26,933 C7 12,15 24,540 28,101D1 15,15 28,199 D7 13,14 25,772 26,931E1 11,14 23,421 26,988 E7 14,14 26,960F1 13,17 25,822 30,529 F7 13,16 25,785 29,381G1 11,14 23,449 27,093 G7 14,14 27,097H1 14,14 27,067 H7 14,14 27,039A2 13,13 25,777 A8 13,15 25,770 28,204B2 12,14 24,597 27,007 B8 11,13 23,383 25,816C2 11,11 23,361 C8 14,15 27,033 28,043D2 11,13 23,399 25,768 D8 14,14 27,076E2 13,14 25,904 27,012 E8 14,15 26,984 28,221F2 14,14 27,125 F8 13,14 25,781 26,946G2 No data G8 10,12 22,183 24,581H2 11,15 23,344 28,151 H8 12,15 24,587 28,197A3 15,15 28,160 A9 15,16 28,265 29,390B3 13,15 25,904 28,256 B9 13,14 25,835 26,963C3 12,14 24,640 27,022 C9 11,14 23,447 27,084D3 11,15 23,407 28,201 D9 11,14 23,391 26,988E3 11,14 23,461 27,093 E9 11,14 23,361 26,931F3 14,15 27,086 28,186 F9 14,15 27,031 28,225G3 10,15 22,218 28,230 G9 14,14 26,978H3 13,16 25,849 29,355 H9 11,13 23,451 26,008A4 14,15 27,018 28,112 A10 13,14 25,789 26,841B4 12,14 24,605 27,018 B10 15,15 28,206C4 13,13 25,797 C10 13,13 25,791D4 No data D10 14,15 26,999 28,123E4 14,14 27,108 E10 13,14 25,895 26,980F4 14,15 27,080 28,215 F10 13,15 25,777 28,197G4 12,13 24,311 25,519 G10 14,15 26,733 27,909H4 13,14 25,824 27,044 H10 12,14 24,688 27,093A5 12,16 24,548 29,321 A11 12,15 24,574 28,230B5 13,14 25,893 26,997 B11 13,14 25,822 27,054C5 12,13 24,727 25,885 C11 14,16 26,963 29,324D5 No data D11 13,14 25,812 26,950E5 14,14 26,960 E11 12,15 24,617 28,232F5 13,14 25,916 27,014 F11 14,14 26,982G5 14,15 27,009 28,106 G11 14,14 27,005H5 13,14 25,804 26,708 H11 12,13 24,631 25,818A6 14,14 26,988B6 14,16 26,982 29,364C6 15,15 28,249D6 14,14 26,716E6 13,16 25,933 29,584F6 8,14 19,884 27,041G6 15,15 28,394H6 14,14 27,009

43


in fluorescent STR typing yield thesame genotype?

Differences between primer sets arepossible if there are sequence differ-ences outside the repeat region thatoccur in the primer binding region ofeither set of primers (exhibit 42). Thisphenomenon produces what is knownas a null allele, or in other words, theDNA template exists for a particularallele but fails to amplify during PCRdue to primer hybridization problems.In all cases except the STR locusD7S820, there was excellent correla-tion in genotype calls between the twomethods (where mass spectrometryand CE results were obtained), signify-ing that the mass spectrometry primersdid not produce any null alleles.

For the STR locus D7S820, 17 of 88samples did not agree with the twomethods (exhibit 18). The bottom twopanels in exhibit 43 illustrate moremicroheterogeneity at this locus thanpreviously reported. On the lower leftplot, only the allele 10 peak can beseen; allele 8, which was seen withPCR amplification using a fluorescentprimer set, is missing (see position ofred arrow in exhibit 43). On the lowerright plot, both allele 8 and allele 10are amplified and detected in the massspectrometer, confirming that the prob-lem is with the PCR amplification andnot the mass spectrometry data collec-tion. In this particular case, there is adifference between those two alleles 8,meaning that the mass spectrometerprimer set identified a new, previouslyunreported allele. When using fluores-cent primer sets that anneal 50100bases or more from the repeat region,a single-base change (e.g., T to C) outof a 300 bp PCR product is difficult todetect. Upon comparing the results ofmass spectrometer data where therewere missing alleles with the resultsfrom the ABI 310, it was noted thatthe situation occurred only with someallele 8s, 9s, and 10s (see underlinedalleles in the ABI 310 column ofexhibit 18). Thus, these null alleleswere variants of alleles with 8, 9, or

Exhibit 40. CDOJ DYS391 STR results with the mass spectrometry method

Position Mass Spec Allele 1 (Da) Position Mass Spec Allele 1 (Da)A1 10 24,489 A7 10 24,416B1 11 25,672 B7 10 24,406C1 10 24,487 C7 10 24,353D1 10 24,455 D7 8 21,905E1 10 24,471 E7 11 25,662F1 11 25,641 F7 10 24,353G1 11 25,637 G7 10 24,422H1 10 24,436 H7 10 24,451A2 10 24,459 A8 10 24,455B2 10 24,465 B8 11 25,529C2 10 24,440 C8 10 24,410D2 10 24,455 D8 10 24,473E2 10 24,359 E8 12 26,841F2 11 25,837 F8 10 24,438G2 10 24,444 G8 10 24,359H2 10 24,451 H8 12 26,805A3 11 25,639 A9 10 24,367B3 10 24,414 B9 10 24,416C3 11 25,654 C9 10 24,463D3 11 25,591 D9 10 24,343E3 10 24,463 E9 10 24,471F3 11 25,648 F9 10 24,457G3 10 24,475 G9 10 24,465H3 10 24,436 H9 10 24,479A4 10 24,408 A10 11 25,631B4 10 24,475 B10 10 24,617C4 11 25,625 C10 10 24,560D4 No data D10 10 24,444E4 11 25,581 E10 11 25,650F4 10 24,396 F10 10 24,414G4 11 25,562 G10 10 24,453H4 10 24,463 H10 11 25,866A5 10 24,390 A11 11 25,662B5 10 24,446 B11 10 24,432C5 10 24,599 C11 10 24,400D5 11 25,652 D11 10 24,463E5 10 24,451 E11 10 24,420F5 10 24,436 F11 9 23,175G5 10 24,380 G11 10 24,459H5 12 26,775 H11 11 25,585A6 11 25,550B6 10 24,428C6 11 25,583D6 10 24,457E6 11 25,658F6 10 24,436G6 10 24,473H6 10 24,463

44


10 repeats. Most likely, a sequencemicrovariant occurs within the repeatregion near the 3-end of the reverseprimer, which anneals to two fullrepeats. Unfortunately, time con-straints restricted the gathering ofsequence information for these sam-ples to confirm the observed variation.Interestingly enough, the D7S820locus has been reported to cause simi-lar null allele problems with otherprimer sets (Schumm et al., 1997).

MicrovariantsSequence variation between allelescan take the form of insertions, dele-tions, or nucleotide changes. Allelescontaining some form of sequencevariation compared with more com-monly observed alleles are oftenreferred to as microvariants becausethey are slightly different from full

repeat alleles. For example, the STRlocus TH01 contains a 9.3 allele, whichhas 9 full repeats (AATG) and a par-tial repeat of 3 bases (ATG). In thisparticular example, the 9.3 allele dif-fers from the 10 allele by a single basedeletion of adenine. Microvariantsexist for most STR loci and are beingidentified in greater numbers as moresamples are being examined aroundthe world. In this study, three previ-ously unreported STR microvariants(exhibit 44) were discovered duringthe analysis of 38 genomic DNA sam-ples from a male population data setprovided by Dr. Oefner (exhibit 45).These microvariants occurred in thethree most polymorphic STR loci thatpossess the largest and most complexrepeat structures: FGA, D21S11, andD18S51.

The ability to make accurate massmeasurements with mass spectrome-try is a potential advantage whenlocating new microvariants. If themass precision is good, then anypeaks that have large offsets from theexpected full repeat alleles could besuspect microvariants in the form ofinsertions or deletions because theirmasses would fall outside the expectedvariance due to instrument variation.This possibility is especially true whenworking with heterozygous samples.Microvariants can be detected by usingthe mass difference between the twoalleles and comparing this value withthe expected value for full repeats orwith the allele peak mass offsets. Ifthe peak mass offsets shift together,then both alleles are full repeats, butif one of the peak mass offsets is sig-nificantly different (e.g., ~300 Da), apossible insertion or deletion existsin one of the alleles. Exhibit 46 illus-trates this concept by plotting the massoffset (from a calculated allele mass)of allele 1 verses the mass offset (froma calculated allele mass) of allele 2.Note that the 9.3 microvariant (i.e.,partial) repeat alleles for TH01 clus-ter away from the comparison of fullrepeat versus full repeat allele. On the other hand, results for the otherthree STR loci, which have no knownmicrovariants in this data set, havemass offsets that shift together for theheterozygous alleles. Exhibit 47 com-pares the peak mass offsets for theamelogenin X allele with the Y alleleand demonstrates that full repeatsshift together during mass spectrome-try measurements.

Nontemplate additionDNA polymerases, particularly theTaq polymerase used in PCR, oftenadd an extra nucleotide to the 3-endof a PCR product as template strandsare copied. This nontemplate addi-tionwhich is most often an adenine,hence, the term adenylationcan be favored by adding a final incuba-tion step at 60 oC or 72 oC after the

15,000

20,000

25,000

30,000

35,000

40,000

45,000

50,000

55,000

0 96 192 288 384

Mas

s (Da

)

FGA D8S1179 DYS391

9 repeats

20 repeats16 repeats

30 repeats

8 repeats

15 repeats

8 repeats

12 repeats

CDOJ Samples Plate 970805A

Sample number

D3S1358

Exhibit 41. Plot of measured masses versus sample number from four different STR loci. FGA, which has a high degree of scatter, is the most polymorphic marker and has the largest mass alleles. The highest and lowest alleles observed for each STR locus in this study are shown on the plot.

45


temperature cycling steps in PCR(Clark, 1988, and Kimpton et al.,1993). However, the degree of adeny-lation is dependent on the sequenceof the template strand, which in thecase of PCR results from the 5-end of the reverse primer. Thus, every

locus will have different adenylationproperties because the primersequences are different. From a meas-urement standpoint, it is better to haveall molecules of a PCR product as sim-ilar as possible for a particular allele.Partial adenylation, where some of the

PCR products do not have the extraadenine (i.e., -A peaks) and some do(i.e., +A peaks), can contribute to peakbroadness if the separation systemsresolution is poor (see top panel ofexhibit 37). Sharper peaks improve thelikelihood that a systems genotypingsoftware can make accurate calls.Variation in the adenylation status ofan allele across multiple samples canhave an impact on accurate sizing andgenotyping potential microvariants.For example, a nonadenylated TH0110 allele would look the same as afully adenylated TH01 9.3 allele inthe mass spectrometer because theirmasses are identical. Therefore, it isbeneficial if all PCR products for aparticular amplification are either +Aor -A rather than a mixture (e.g., A).By using the temperature soak at theend of thermal cycling, most of theSTR loci were fully adenylated, withthe notable exception of TPOX, whichwas typically nonadenylated, andTH01, which under some PCR condi-tions produced partially adenylatedamplicons. For making correct geno-type calls, the STR mass ladder file(exhibit 30) was altered according tothe empirically determined adenyla-tion status.

During the course of this project,Platinum GenoTYPETM Tsp DNA poly-merase (Life Technologies, Rockville,MD) became available that exhibitslittle to no nontemplate nucleotideaddition. This new DNA polymerasewas tested with STR loci that had beenshown to produce partial adenylationto see if the +A peak could be elimi-nated. Exhibit 48 compares massspectrometry results obtained usingAmpliTaq Gold (commonly used)polymerase with the new Tsp poly-merase. The Tsp polymerase producedamplicons with only the -A peaks,while TaqGold showed partial adenyla-tion with these TH01 primers. Thus,this new polymerase has the potentialto produce sharper peaks (i.e., nopartial adenylation) and allele masses that can be more easily predicted

*

*

*(A)

(B)

(C)

Exhibit 42. Effects of sequence variation on PCR amplification in or around STR repeat regions. The asterisk symbolizes a DNA difference (base change, insertion, or deletion of a nucleotide) from a typical allele for a STR locus. In situation (A), the variation occurs within the repeat region (depicted in green) and should have no impact on the primer binding and the subsequent PCR amplification, although the overall amplicon size may vary slightly. In situation (B), the sequence variation occurs just outside the repeat in the flanking region but interior to the primer annealing sites. Again, PCR should not be affected, although the size of the PCR product may vary slightly. However, in situation (C), the PCR can fail due to a disruption in annealing a primer because the primer no longer perfectly matches the DNA template sequence. Therefore, if sequence variation occurs in the flanking region for a particular locus, one set of primers may work while another may fail to amplify the template. The template would therefore be a null allele.

46


(i.e., all PCR products would be nonadenylated).

Stutter productsDuring PCR amplification of STR loci,repeat slippage can occur and result inthe loss of a repeat unit as DNA strandsynthesis occurs through a repeated

sequence. These stutter products aretypically 4 bases, or one tetranucleotiderepeat, shorter than the true allele PCRproduct. The amount of stutter productcompared to the allele product variesdepending on the STR locus and thelength of the repeat, but typically stut-ter peaks are 210% of the allele peakheight (Walsh et al., 1996). Forensic

DNA scientists are concerned aboutstutter products because their presencecan interfere in the interpretation ofDNA mixture profiles.

When reviewing plots of GeneTracesmass spectrometry results for STR loci,forensic scientists have commented onthe reduced level of stutter product

Exhibit 43. Mass spectra of CDOJ samples amplified with D7S820 primers. From left side, top-to-bottom followed by right side, top-to-bottom, sample genotypes are (10,11), (9,10), (10,10), (null allele 8, 10), (8,11), (11,13), (10,12), and (8,10). The arrow indicates the position where allele 8 should be present in the sample but is missing due to a primer annealing binding site sequence polymorphism that results in a null allele. The mass range shown here is 12,00025,000 Da.

1.5

x 104

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)2 2.5 1.5 2 2.5

1.5 2 2.5

x 104 x 104

Inte

nsity

(arbit

rary u

nits)

Inte

nsity

(arbit

rary u

nits)

Inte

nsity

(arbit

rary u

nits)

Inte

nsity

(arbit

rary u

nits)

Inte

nsity

(arbit

rary u

nits)

Inte

nsity

(arbit

rary u

nits)

Inte

nsity

(arbit

rary u

nits)

Inte

nsity

(arbit

rary u

nits)10,000

0

2

0

15,000

1.5 2 2.5 1.5 2 2.5

10,000

0

5,000

1.5 2 2.5

1.5 2 2.5 1.5 2 2.5

10,000

0

5,000 10,000

5,000

0

2

0

1

00

x 104

10,000

10,000

47


detection (exhibit 35). There are twopossibilities for this reduction:

Since the primers are closer to therepeat region, smaller PCR productsare amplified, which means that theDNA polymerase does not have tohold on to the extending strand as

long for synthesis purposes. It ispossible that the polymerase readsthrough the repeat region fasterand, therefore, the template strandsdo not have as much of an oppor-tunity to slip and reanneal out ofregister on the repeat region. Forexample, Taq polymerase has a pro-

cessivity rate of ~60 bases before itfalls off the extending DNA strand;therefore, the closer the PCR prod-uct size is to 60 bases, the better theextension portion of the PCR cycle.GeneTraces PCR product sizes,which are typically less than 100bp, are much smaller than the

1,500

Exhibit 44. Electropherograms of ABI 310 results for new STR microvariants seen in the Stanford male population samples. The D18S51 16.2 allele, D21S11 30.3 allele, and FGA 28.1 allele have not been reported previously in the literature. These plots are views from Genotyper 2.0 with results overlaid on shaded allele bins. The base pair size range is indicated at the top of each plot. Note: The microvariant alleles (indicated by the red arrows) fall between the shaded bins, but the other alleles in the heterozygote set contain complete repeats and fall directly on the shaded (expected) allele bin.

2,000

1,000500

2,0003,000

1,000

3,0004,000

2,0001,000

48


Nam

eSa

mpl

eA

mel

- A

1A

mel

- A

2D

8-

A1

D8- A2

D21

-A1

D21

- A

2D

18-

A1

D18

-A

2D

3-

A1

D3-

A

2VW

A-

A1

VWA

-A2

FGA

- A

1FG

A-

A2

D5-

A

1D

5-

A2

D13

- A

1D

13

-A2

D7-

A

1D

7-

A2

Aus

21X

Y8

1229

31.2

1218

1517

1417

1822

1212

1112

911

Aus

28X

Y14

1529

31.2

1519

1617

1517

1920

1212

1012

810

Ber

g15

XY

1028

,30.

231

.212

,17

1415

,17

18,1

916

17,1

820

,21

2411

13w

8,14

117w

10B

erg1

9X

Y14

1432

.232

.212

1816

1614

1822

2212

1312

129

13B

I12

XY

1212

3031

.217

1716

1817

1924

2712

1211

119

11B

sk09

2X

Y14

1429

33.2

1316

1717

1717

2323

1112

1212

1111

Bsk

111

XY

1213

3032

.216

1914

1416

1618

1912

128

108

9B

sk11

8X

Y13

1430

.230

.211

1416

1617

1723

2510

128

108

8C

H17

XY

1214

32.2

34.2

1423

1515

1618

2026

912

812

1112

CH

23X

Y12

1329

3116

1615

1514

1724

2411

129

1210

12C

H42

XY

1214

28.2

31.2

1315

1616

1717

2122

1212

1012

1111

F18

XY

1414

3132

.219

1915

1514

1621

2313

1311

128

12F2

1X

Y14

1527

3114

1915

1517

1822

2612

1311

138

8J1

3X

Y14

1432

.233

.217

1915

1714

1620

2210

118

1110

10J3

XY

1314

3030

1315

1616

1717

2224

1014

811

1010

J37

XY

1013

3131

.213

2015

1516

1822

2510

128

1110

10

J39

XY

1314

3031

.213

1315

1817

1723

2411

1312

138

12

JK29

21X

Y10

1029

31.2

1415

1516

1419

w19

2210

1111

119

9JK

2979

XY

1012

32.2

33.2

1414

1516

1418

2124

.211

1411

1211

11M

DK

204

XY

1417

2830

.212

1914

1516

1623

2512

1311

128

10

Mel

12X

Y13

1430

32.2

1720

1516

1718

2122

1314

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1112

1212

Mel

15X

Y14

1528

31.2

14,1

522

1618

1618

2324

1010

1213

1111

Mel

18X

Y15

1528

32.2

1415

1516

1718

2424

1011

1213

1011

NG83

XY

1313

3031

13

1815

161

51

723

2411

118

88

10N

G85

XY

1515

2931

.215

2116

1717

1923

2613

138

811

12N

G88

XY

1215

33.2

38.2

1317

1515

1616

2528

.111

118

118

10O

M13

5X

Y12

1328

3915

1616

1616

1821

2512

1212

1211

11P1

03G

XX

1217

2930

1920

1717

1519

2024

1313

1012

88

P109

XY

1114

2834

1721

1516

1519

2227

1112

1212

910

P205

XY

1115

2836

1515

1415

1516

2124

810

1212

910

P240

XY

1414

30.3

3416

1616

1715

2122

22.2

1011

813

1112

P33

XY

1314

2930

16.2

1814

1716

1720

248

1011

1210

10P3

7GX

Y14

1529

32.2

1420

1617

1718

2424

1213

912

811

P37G

?X

Y14

1529

32.2

1420

1617

1718

2424

1213

912

811

P73

XY

1415

2930

1416

1516

1419

22.2

2412

1211

1310

11PG

1162

XY

814

31.2

32.2

1415

1516

1618

1823

1112

911

810

PKH

062

XY

1215

2931

1316

1618

1618

2122

1111

913

1012

SDH

053

XY

1012

3033

.216

1618

1817

1721

2511

128

88

8

Exhibit 45. ProfilerPlusTM results from Stanford male population samples 12

,13

Not

e:Th

e ST

R lo

ci ar

e co

lor c

oded

to in

dica

te th

eir f

luor

esce

nt d

ye la

bel c

olor

;the

sha

ded

boxe

s in

the

body

of t

he e

xhib

it re

fer

to th

e m

icro

varia

nt a

llele

s (ex

hibi

t 44),

3-ba

nded

patte

rns, or

an u

nex

pect

ed x

,x

am

elo

geni

n.

49


fluorescently labeled primer setsused by most forensic DNA labora-tories (exhibit 2). However, thisneeds to be studied more extensive-ly with multiple primer sets on aparticular STR locus that generatesvarious sized amplicons. For exam-ple, the primer sets described inexhibit 24 could be fluorescentlylabeled and analyzed on the ABI310, where the stutter product peakheights could be quantitativelycompared to the allele peak heights.

The more likely reason that lessstutter is observed by mass spec-trometry is that the signal-to-noiseratio is much lower in mass spec-trometry than in fluorescencemeasurements. Fluorescence tech-niques have a much lower back-ground and are more sensitive forthe detection of DNA than massspectrometry. Thus, stutter may bepresent at similar ratios comparedwith those observed in fluorescencemeasurements, but because stutteris part of the baseline noise of massspectrometry data, it may not beseen in the mass spectrum. Thislatter explanation is probably morelikely, as indicated in very strongstutter peaks for some dinucleotiderepeat markers (exhibit 49).

Whether stutter products are presentor not, GeneTraces current STR geno-typing software has been designedto recognize them and not call themas alleles.

Primer sequence determinations from commercial STR kitsPrimarily, two commercial manufac-turers supply STR kits to the forensicDNA community: Promega Corpora-tion and Applied Biosystems. Thesekits come with PCR primer sequencesthat permit simultaneous multiplexPCR amplification of up to 16 STRloci. One of the primers for each STRlocus is labeled with a fluorescent

-800

-600

-400

-200

0

200

400

600

800

0 200 400 600

Mas

s of

fset

2 (D

a)

300 Da window(1 base)

100 Da window

Microvariants

-600 -400 -200

Mass offset 1 (Da)

Exhibit 46. Plot of allele mass offsets (allele 1 versus allele 2) for heterozygous samples from four different loci. These are 88 CDOJ samples for the STR loci TH01, TPOX, CSF1PO, and D16S539.

Exhibit 47. Plot of X allele mass offset versus Y allele mass offset for 88 amelogenin samples. The red box shows 100 Da around the expected values and the green box shows 300 Da. The blue line is the ideal situation where heterozygous peaks would shift in unison compared with the expected masses.

-600

0

600

600

Mas

s of

fset

Y a

llele

(Da) 300

300

-300

-300-300

Mass offset X allele (Da)

0

50


dye to permit fluorescent detection ofthe labeled PCR products. Since theprimer sequences are not disclosedby the manufacturers, mass spectrome-try was used to determine where theyannealed to the STR sequences com-pared with GeneTrace primers (seeprevious discussion on null alleles).

First, the primer mixtures were spot-ted and analyzed to determine eachprimers mass (top panel of exhibit50). Then a 53 exonuclease wasadded to the primer mix and heated to37 oC for several minutes to digest theprimer one base at a time. An aliquotwas removed every 510 minutes toobtain a time course on the digestionreaction. Each aliquot was spotted in3-hydroxypicolinic acid matrix solu-tion (Wu et al., 1993), allowed to dry,and analyzed in the mass spectrometer.

A digestion reaction produces a seriesof products that differ by one nucleo-tide. By measuring the mass differencebetween each peak, the original primersequence may be determined (bottompanel of exhibit 50). Only the unla-beled primers will be digested becausethe covalently attached fluorescent dyeblocks the 5-end of the dye-labeledprimer. Using only a few bases ofsequence (e.g., 45 bases), it is possibleto make a match on the appropriateSTR sequence obtained from GenBankto determine the 5-end of the primerwithout the fluorescent label.

With the full-length primer massobtained from the first experiment, the remainder of the unlabeled primercan be identified. The position of the5-end of the other primer can be deter-mined using the GenBank sequenceand the PCR product length for theappropriate STR allele listed in GenBank(exhibit 2). The sequence of the labeledprimer can be ascertained by using theappropriate primer mass determinedfrom the first experiment and subtract-ing the mass of the fluorescent dye.The primer mass is then used to obtainthe correct length of the primer on theGenBank sequence and the primers

Exhibit 48. Mass spectra comparing an STR sample amplified with TaqGold polymerase and Tsp polymerase. The Tsp polymerase favors production of the nonadenylated form of PCR products, which results in a single peak for each allele (bottom panel). TaqGold produces a mixture of A and +A peaks, which leads to two peaks for each allele (top panel). The peak masses in Daltons are indicated next to each peak. Mass difference measurements of 308 Da and 305 Da between the +A and A peaks reveal that a T is added by TaqGold instead of the expected A (expected masses: T = 304 Da andA = 313 Da). The samples genotype was TH01 6,8.

14,895

15,203

17,706

17,421

14,907

Tsp polymerase (only -A)

TaqGold polymerase (only A)

12,000 14,000 16,000 18,000 20,000

17,401

Mass (Da)

Sign

al

Exhibit 49. Mass spectrum demonstrating detection of stutter products from a particularly stutter-prone dinucleotide repeat locus. The mass differences between the stutter product peaks and the allele peaks can be used to determine the repeat sequence that is present on the measured DNA strand. Note: The amount of stutter is larger in the longer repeat allele than in the shorter allele.

14,000 16,000 18,000 20,000 22,000Mass (Da)

Sign

al

CT repeat = 593 Da

13 repeats

18 repeatsm = 592

m = 587

m = 590

m = 590

m = 587

51


sequence. Finally, an entire STRmultiplex primer set can be measuredtogether in the mass spectrometer toobserve the primer balance (exhibit51). High-performance liquid chro-matography fraction collection can beused to pull primers apart from com-plex, multiplex mixtures, and eachprimer can be identified as previouslydescribed. The primer sequences fromboth Promega and Applied BiosystemsSTR loci TH01 (exhibit 52), TPOX(exhibit 53), and CSF1PO (exhibit 54)were identified using this procedure.A comparison of the primer sequencesfrom the two manufacturers found thatthey were very similar. The 3-endsof the primer setsthe most criticalportions for annealing duringwerealmost identical between the differentkits. The ABI primers were typicallyshorter at the 5-end and, therefore,produced PCR products that were ~10bases shorter than those produced bythe corresponding Promega primers.In all three STR loci, the primersannealed further away from the repeatregion than the GeneTrace primer sets.

Analytical Capabilitiesof This Mass Spectro-metry MethodUsing the current primer design strate-gy, most STR alleles ranged in sizefrom ~10,000 Da to ~40,000 Da. Inmass spectrometry, the smaller themolecule, the easier it is to ionize anddetect (all other things being equal).Resolution, sensitivity, and accuracyare usually better the smaller the DNAmolecule being measured. Because thepossible STR alleles are relatively farapart, reliable genotyping is readilyattainable even with DNA moleculesat the higher mass region of the spec-trum. For example, neighboring full-length alleles for a tetranucleotiderepeat, such as AATG, differ in massby 1,260 Da.

5,000 6,000 7,000 8,000 9,000

Dye-labeledprimer

Original primer pair(not desalted)

Use of mass spectrometryto determine PCR primersof unknown sequence

Following 6 min digestion ofprimers with 5 3 exonuclease

Exhibit 50. Primer sequence determination with exonuclease digestion and mass difference measurements. This example is a D5S818 primer pair purchased from Promega Corporation and used in its PowerPlexTM STR kit. The top panel shows a mass spectrum of the original primer pair prior to digestion. The bottom panel is the mass spectrum of the same primers following a 6 minute digestion at 37 oC with calf spleen phosphodiesterase, which is a 53 exonuclease. The dye-labeled primer is not digested because the dye protects the 5-end of the primer. Mass difference measurements between the digestion peaks leads to the sequence determination of the 5-end of the unlabeled primer (see underlined portion of forward sequence). The determined sequences are 5-GGTGATTTTCCTCTTTGGTATCC-3 (forward) and 5-fluorescein dyeTTTACAACATTTGTATCTATATCTGT-3 (reverse).

GGG TT TT

A

Sign

al

Mass (Da)

CSF-R

TH01-F

TPOX-R

TPOX-F

CSF-FTH01-R

AMEL-R

AMEL-F

8,0007,0006,0005,000

Exhibit 51. Mass spectrum of AmpF1STR Green I primer mix. Each peak has been identified with its corresponding primer. Peaks containing the fluorescent dye (JOE) are underlined.

Mass (Da)

Sign

al

52


AmpFlSTR Green I Kit Reverse primer is labeled with JOE dye (fluorescein derivative)

PCR product =184 bp (9 repeats) PowerPlexTM Kit

Forward primer is labeled with TMR dye (tetramethylrhodamine)

PCR product = 195 bp (9 repeats)

Exhibit 52. TH01 STR primer positions for commercially available primers highlighted on the GenBank sequence. The forward primer is shown in blue with the reverse primer in brown. The repeat is highlighted in green on the strand that contains the fluorescent dye. The 3-positions of the forward primers are identical but differ by a single base for the reverse primers. Promega primers are longer at the 5-end, which produces a larger PCR product by 11 bp (6 bases on forward and 5 bases on reverse).

53


AmpFlSTR Green I KitForward primer is labeled with JOE dye (fluorescein derivative)

PCR product = 237 bp (11 repeats) PowerPlexTM Kit

Reverse primer is labeled with TMR dye (tetramethylrhodamine)


Exhibit 53. TPOX STR primer positions for commercially available primers highlighted on the GenBank sequence. The forward primer is shown in blue with the reverse primer in brown. The repeat is highlighted in green on the strand that contains the fluorescent dye. The 3-positions of the reverse primers are identical but differ by a single base for the forward primers. Promega primers are longer at the 5-end, which produces a larger PCR product by 7 bp (4 bases on forward and 3 bases on reverse).

54


AmpFlSTR Green I KitForward primer is labeled with JOE dye (fluorescein derivative)

PCR product = 304 bp (12 repeats) PowerPlexTM Kit

Forward primer is labeled with TMR dye (tetramethylrhodamine)


Exhibit 54. CSF1PO STR primer positions for commercially available primers highlighted on the GenBank sequence. The forward primer is shown in blue with the reverse primer in brown. The repeat is highlighted in green on the strand that contains the fluorescent dye. The 3-positions of the reverse primers are identical but differ by a single base for the forward primers. Promega primers are longer at the 5-end, which produces a larger PCR product by 11 bp (5 bases on forward and 6 bases on reverse).

55


ResolutionDinucleotide repeats, such as CArepeats, require a resolution of at least2 bp in order to resolve stutter prod-ucts from the true allele or heterozy-gotes that differ by a single repeat.Trinucleotide and tetranucleotiderepeats, with their larger repeat struc-ture, are more easily resolved becausethere is a larger mass differencebetween adjacent alleles. However,the overall mass of the PCR productincreases more rapidly with tri- ortetranucleotide repeats. For example,the repeat region for 40 GA repeatsis 25,680 Da, while the mass of therepeat region quickly increases to37,200 Da for 40 AAT repeats and50,400 Da for 40 AATG repeats.

GeneTrace has demonstrated that aresolution of a single dinucleotiderepeat (~600 Da) may be obtainedfor DNA molecules up to a mass of~35,000 Da. This reduced resolutionat higher mass presents a problem forpolymorphic STR loci such as D18S51,D21S11, and FGA because single baseresolution is often required to accu-rately call closely spaced alleles or todistinguish a microvariant containing apartial repeat from a full-length allele.These three STR loci also contain long alleles. For example, D21S11 hasreported alleles of up to 38 repeats(mixture of TCTA and TCTG) inlength, D18S51 up to 27 AGAArepeats, and FGA up to 50 repeats(mixture of CTTT and CTTC).Heterozygous FGA alleles that differedby only a single repeat were more dif-ficult to genotype accurately thansmaller sized STR loci due to poorresolution at masses greater than~35,000 Da (see samples marked inred in exhibit 19).

The analysis of STR allelic laddersdemonstrates that all alleles can beresolved for an STR locus. Allelic lad-ders from commercial kits were typi-cally diluted 1:1000 with deionizedwater and then reamplified with the

Exhibit 55. Mass spectrum of a TH01 allelic ladder reamplified from AmpF1STR Green I allelic ladders. The PCR product size of allele 10 is only 83 bp with a measured mass of 20,280 Da and separation time of 204 s. The allele 9.3 and allele 10 peaks, which are only a single nucleotide apart, differ by only 1.5 s on a separation timescale and can be fully resolved with this method.

14,000 16,000 18,000 20,000

m/z

5 6 7

89

9.3

10

Sign

al

Exhibit 56. Mass spectra of D5S818 allelic ladders from two manufacturers. The Applied Biosystems D5S818 ladder contains 10 alleles (top panel); the Promega D5S818 ladder contains only 8 alleles (bottom panel). The GeneTrace primers bind internally to commercially available multiplex primers, and all alleles in the commercial allelic ladders are therefore amplified, demonstrating that the GeneTrace primers can amplify all common alleles for this particular STR locus.

14,000 16,000 18,000 20,000 22,000 24,000 26,000 28,000

Sign

al

Mass (Da)

D5S818 Allelic Ladderfrom Promega

D5S818 Allelic Ladder from Applied Biosystems

7

8 9

10

1112 13 14

15

16

7

8 910 11

12 1314

56


GeneTrace primers that bound closerto the repeat region than the primersfrom the commercial kits. This ream-plification provided PCR products fordemonstrating that the needed level ofresolution (i.e., distinguishing adjacentalleles) is capable at the appropriatemass range in the mass spectrometeras well as demonstrating that theGeneTrace primers amplify all alleles(i.e., no allele dropout from a nullallele). A number of STR allelic ladderswere tested in this fashion, includingTH01 (exhibit 55); CSF1PO, TPOX,and VWA (exhibit 36); and D5S818(exhibit 56). All tetranucleotide repeatalleles were resolvable in these exam-ples, demonstrating 4 bp resolution,and TH01 single base pair resolutionwas seen between alleles 9.3 and 10.

SensitivityTo determine the sensitivity of Gene-Traces STR typing assay, TPOXprimers were tested with a dilutionseries of K562 genomic DNA (20 ng,10 ng, 5 ng, 2 ng, 1 ng, 0.5 ng, 0.2 ng,and 0 ng). Promegas Taq polymeraseand STR buffer were used with 35PCR cycles as described in the scopeand methodology section. Peaks forthe correct genotype (heterozygote8,9) could be seen down to the lowestlevel tested (0.2 ng or 200 picograms),while the negative control was blank.Exhibit 57 contains a plot with themass spectra for 20 ng, 5 ng, 0.5 ng,and 0 ng. While each PCR primer paircan exhibit a slightly different efficien-cy, human DNA down to a level of ~1ng can be reliably PCR amplified anddetected using mass spectrometry.GeneTraces most recent protocolinvolved 40-cycle PCR and the use ofTaqGoldTM DNA polymerase, whichshould improve overall yield for STRamplicons. All of the samples testedfrom CDOJ were amplified with only5 ng of DNA template and yieldedexcellent results (exhibits 1219, 31,39, 40, 43, 58, and 59). In terms ofabsolute sensitivity in the mass spec-

trometer, several hundred femtomolesof relatively salt-free DNA moleculeswere typically found necessary fordetection. GeneTraces PCR amplifica-tions normally produced several pico-moles of PCR product, approximatelyan order of magnitude more materialthan is actually needed for detection.

Mass accuracy and precisionMass accuracy is an important issuefor this mass spectrometry approach toSTR genotyping, as a measured massfor a particular allele is compared withan ideal mass for that allele. Due to

Exhibit 57. Mass spectra of TPOX PCR products from various amounts of K562 DNA template material. This sensitivity test demonstrates that DNA templates in the quantity range of 0.520 ng may be effectively amplified and detected by mass spectrometry.

14,000 16,000 18,000 20,000 22,000 24,000

9

Sign

al

8

98

20 ng K562

5 ng K562

98

0.5 ng K562primerdimer

0 ng (negative control)

Mass (Da)

57


the excellent accuracy of mass spec-trometry, internal standards are notrequired to obtain accurate DNA siz-ing results as in gel or CE measure-ments (Butler et al., 1998). To makean inaccurate genotype call for atetranucleotide repeat, the mass offsetfrom an expected allele mass wouldhave to be larger than 600 Da (half the mass of a ~1,200 Da repeat).

GeneTrace has observed mass accura-cies on the order of 0.01 nucleotides(

58


Precision is important for STR allelemeasurements in mass spectrometrybecause no internal standards arebeing run with each sample to makeadjustments for slight variations ininstrument conditions between runs.To demonstrate the excellent repro-ducibility of mass spectrometry, 15mass spectra of a TPOX allelic ladderwere collected. A table of the obtained

masses for alleles 6, 7, 8, 9, 10, 11, 12,and 13 shows that all alleles wereeasily segregated and distinguishable(exhibit 61). Statistical analysis of thedata found that the standard deviationabout the mean for each allele rangedfrom 20 to 27 Da, or approximately0.1% relative standard deviation (RSD).The mass between alleles is equal tothe repeat unit, which in the case of

TPOX is 1,260 Da for an AATG repeat(exhibit 62). Thus, each allele is easilydistinguishable.

Measurements were made of the sameDNA samples over a fairly wide time-span, revealing that masses can beremarkably similar, even when datapoints are recollected months later.Exhibit 60 compares 57 allele meas-

Exhibit 59. Mass spectra of CDOJ samples amplified with TH01 primers. From left side, top-to-bottom followed by right side, top-to-bottom, sample genotypes are (6,7), (6,8), (6,9.3), (6,10), (7,7), (7,7), (7,8), and (7,9.3). The split peaks for each allele result from partial adenylationi.e., both A peaks are present. The mass range shown here is 12,00022,000 Da.

1.2

x 104

x 104

x 104

Mass (Da)

Inte

nsity

(arbit

rary u

nits)

Mass (Da)

Inte

nsity

(arbit

rary u

nits)

Mass (Da)

Mass (Da)

Inte

nsity

(arbit

rary u

nits)

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(arbit

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(arbit

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(arbit

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nits)

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(arbit

rary u

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(arbit

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Mass (Da)1.4

1

x 104

x 104

x 104

x 104

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x 104

0

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x 104 x 104

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1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

1.2 1.4 1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

1.2 1.4 1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

1.2 1.4 1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

0

1

2

2

59


urements from 6 different TPOX alle-les collected 6 months apart. The firstdata set was collected on October 1,1998, and the second data set onMarch 26, 1999. Amazingly enough,some of the alleles had identical meas-ured masses, even though differentmass calibration constants (and evendifferent instruments) were used.

The bottom line is whether or not acorrect genotype can be obtained usingthis new technology. Exhibit 63 com-pares the genotypes obtained using aconventional CE separation methodand this mass spectrometry techniqueacross 3 STR markers (D16S539,D8S1179, and CSF1PO) and indicatesan excellent agreement between the

methods. With the CDOJ samples test-ed, there was complete agreement onall observed genotypes for the STR lociCSF1PO, TH01, and D3S1358 as wellas the sex-typing marker amelogenin(exhibits 12, 1416). Some gas-phasedimers and trimers fell into the allelemass range and confused the callingfor TPOX (exhibit 13) and D16S539(exhibit 17) on several samples. Gas-phase dimers and trimers are assayartifacts that result from multipleexcess primer molecules colliding inthe gas phase and being ionized dur-ing the MALDI process. A mass offsetplot like that shown in exhibit 46 canbe used to detect these assay artifactsas they fall outside the tight grouping

and inside the 300 Da window. Withthe CDOJ samples, D7S820 exhibitednull alleles (exhibit 18) and FGA hadsome unique challenges due to its larg-er size, such as problems with resolu-tion of closely spaced heterozygotesand poorer mass calibration since themeasured alleles were further awayfrom the calibration standards (exhibit19). Thus, when the PCR situationssuch as null alleles are accounted forand smaller loci are used, this massspectrometry method produces resultscomparable to traditional methods ofSTR genotyping.

Data collection speedThe tremendous speed advantage ofmass spectrometry can be seen inexhibit 11. Over the course of thisproject, data collection speed increasedby a factor of 10 from ~50 seconds/sample to less than 5 seconds/sample.This speed increase resulted fromimproved software and hardware onthe automated mass spectrometersand from improved sample quality(i.e., better PCR conditions that yield-ed more product and improved samplecleanup that in turn yielded cleanerDNA). With data collection time around5 seconds per sample, achieving sam-ple throughputs of almost 1,000 sam-ples per hour is possible, and 3,0004,000 samples per system per day isreasonable when operating at fullcapacity. Sample backlogs could beerased rather rapidly with this kind ofthroughput. By way of comparison, ittakes an average of 5 minutes to obtaineach genotype (assuming a multiplexlevel of 6 or 7 STRs) using conven-tional CE methods (exhibit 11).Thus, the mass spectrometry methoddescribed in this study is two orders ofmagnitude faster in sample processingtime than conventional techniques.

24,000

15,000 16,000 17,000 18,000 19,000 20,000 21,000 22,000 23,000 24,000

Obs

erve

d m

ass

(Da)

(Mar.

26, 1

999)

23,000

22,000

21,000

20,000

19,000

18,000

17,000

16,000

15,000

Observed mass (Da) (Oct. 1, 1998)

Exhibit 60. Comparison of allele masses collected 6 months apart. This plot compares 57 allele measurements of 6 different TPOX alleles. The ideal line is shown on the same plot to demonstrate how reproducible the masses are over time. The average standard deviation of allele mass measurements between these 2 data sets was 47 Da. This result further confirms that no allelic ladders or other internal DNA standards are needed to obtain accurate measurements with mass spectrometry.

60


Allele 6 Allele 7 Allele 8 Allele 9 Allele 10 Allele 11 Allele 12 Allele 13Expectedmass (Da) 15,345 16,605 17,865 19,125 20,385 21,644 22,904 24,164

1 15,346 16,623 17,903 19,130 20,388 21,623 22,860 24,0742 15,387 16,667 17,901 19,129 20,387 21,639 22,893 24,1143 15,372 16,629 17,887 19,143 20,400 21,615 22,877 24,0914 15,385 16,653 17,903 19,163 20,384 21,642 22,903 24,0765 15,388 16,642 17,898 19,155 20,337 21,654 22,870 24,1116 15,336 16,600 17,857 19,105 20,362 21,599 22,832 24,0647 15,388 16,637 17,894 19,131 20,383 21,635 22,904 24,1108 15,363 16,618 17,872 19,129 20,368 21,604 22,853 24,0879 15,365 16,628 17,891 19,150 20,385 21,620 22,892 24,087

10 15,373 16,638 17,892 19,136 20,394 21,631 22,878 24,08511 15,383 16,640 17,896 19,152 20,387 21,621 22,884 24,12912 15,388 16,648 17,912 19,172 20,388 21,623 22,850 24,14913 15,407 16,660 17,941 19,208 20,425 21,674 22,944 24,14814 15,410 16,659 17,930 19,174 20,425 21,666 22,893 24,13215 15,390 16,648 17,915 19,157 20,423 21,636 22,897 24,126

Averagemass 15,379 16,639 17,899 19,149 20,389 21,632 22,882 24,106

Std. dev. 20.2 17.8 20.6 24.7 23.7 21.0 27.2 27.3%RSD 0.13 0.11 0.12 0.13 0.12 0.10 0.12 0.11

% error 0.22 0.21 0.19 0.13 0.02 -0.06 -0.10 -0.24Obs-exp 33.7 34.3 34.5 23.9 4.1 -11.9 -22.0 -58.5

Exhibit 61. Fifteen replicate analyses of a TPOX allelic ladder to measure mass precision and accuracy. The precision was less than 30 Da for a single standard deviation, which corresponds to less than 0.1 nucleotide. The measured mass accuracy from the calculated expected allele masses averaged ~30 Da. Across the 8 alleles in the ladder, 120 data points are used to make this determination. All numbers are in Daltons (Da). Percentage error was calculated as (observed-expected)/ expected. This same data is also presented in histogram format; see figure 1 in Butler et al., 1998.

Samplenumber

Upper Strand Expected (Da) Observed (Da)Allele 56 1,211 1,210Allele 67 1,211 1,211Allele 78 1,211 1,215Allele 89 1,211 1,215Allele 99.3 907 915Allele 9.310 304 306Allele 910 1,211 1,221

Repeat = TCAT = 1,210.8 Da Repeat = AATG = 1,259.8 Da= --CAT = 906.6 Da = --ATG = 946.6 Da

Exhibit 62. Upper strand (TCAT repeat) and lower strand (AATG repeat) mass differences for the TH01 allelic ladder. The upper strand was discernible from the lower strand due to the different sequence contents of the repeats. The STR repeat structure and nucleotide content can be seen using mass spectrometry. Note: The upper strand mass difference between alleles 9.3 and 10 is 306 Da, or a T, and the lower strand mass difference between these same two alleles is 315 Da, or an A. For more details, see Butler et al., 1998b.

Allele 56 1,260 1,259Allele 67 1,260 1,262Allele 78 1,260 1,269Allele 89 1,260 1,267Allele 99.3 947 948Allele 9.310 313 315Allele 910 1,260 1,263

Upper Strand Expected (Da) Observed (Da)

61


D81179

02468

1012141618

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le c

all (r

epea

t #)

ABI 310Mass Spec

D16S539

02468

101214

0 20 40 60 80 100 120 140 160 180Number of Alleles

Alle

le c

alls

(repe

at #)

CSF1PO

02468

101214

Alle

le c

all (r

epea

t #)

Exhibit 63. Comparison of ABI 310 and mass spectrometry allele calls for 90 CEPH/diversity samples. Out of 1,080 possible allele calls with these 3 STR loci, there were 100 with no data collected (indicated as a 0 on the allele call axis), and only 12 calls differed between the two methods, or ~98% correlation.

Number of Alleles

Number of Alleles

0 20 40 60 80 100 120 40 160 180

0 20 40 60 80 100 120 40 160 180

ABI 310Mass Spec

ABI 310Mass Spec

63

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

Work began on the develop-ment of multiplexed SNPassays in the summer of1998 after notice that a second NIJgrant, Development of MultiplexedSingle Nucleotide PolymorphismAssays from Mitochondrial and Y-Chromosome DNA for Human IdentityTesting Using Time-of-Flight MassSpectrometry, had been funded.Excellent progress was made towardthe milestones on this grant, but thework not finished because this grantwas prematurely terminated on thepart of GeneTrace in the spring of1999. The completed work focused on two areas: the development of a10-plex SNP assay from the mtDNAcontrol region using a single ampliconand the development of a multiplexPCR assay from Y-chromosome SNPmarkers that involved as many as 18loci amplified simultaneously. Thissection describes the design aspects of multiplex PCR and SNP assaysalong with the progress made towardthe goal of producing assays thatwould be useful for high-throughputscreening of mitochondrial and Y-chromosome SNP markers.

The approach to SNP determinationdescribed here has essentially threesteps: (1) PCR amplification, (2)phosphatase digestion, and (3) SNPprimer extension. Either strand ofDNA may be probed simultaneouslyin this SNP primer extension assay.PCR primers are designed to generatean amplicon that includes one ormore SNP sites. The initial PCR reac-tion is performed with standard (unla-beled) primers. A phosphatase is thenadded following PCR to remove allremaining dNTPs so that they will notinterfere with the single base exten-sion reaction involving ddNTPs.These reactions can all be performedin the same tube or well in a sampletray. A portion of the phosphatase-

treated PCR product is then used forthe primer extension assay.

In the SNP primer extension assay, aspecial primer containing a biotin moi-ety at the 5-end permits solid-phasecapture for sample purification priorto mass spectrometry analysis. Thisprimer hybridizes upstream of the SNPsite with the 3-end immediately adja-cent to the SNP polymorphic site. Acleavable nucleotide near the 3-endallows the 3-end of the primer to bereleased from the immobilized portionand reduces the overall mass of themeasured DNA molecule (exhibit 21)(Li et al., 1999). The complementarynucleotide(s) to the nucleotide(s) pres-ent at the SNP site is inserted duringthe extension reaction. In the case of aheterozygote, two extension productsresult. Only a single base is added to

the primer during this process becauseonly ddNTPs are used and the dNTPsleft over from PCR are hydrolyzed withthe phosphatase digestion step. If theextension reaction is not driven tocompletion (where the primer wouldbe totally consumed), then both primerand extension product (i.e., primerplus single nucleotide) are presentafter the primer extension reaction.The mass difference between these twoDNA oligomers is used to determinethe nucleotide present at the SNP site.In the primer extension SNP assay, theprimer acts as an internal standard andhelps make the measurement moreprecise. A histogram of mass differencemeasurements across 200 samples (50per nucleotide) is shown in exhibit 64.The ddT and ddA differ by only 9 Daand are the most difficult to resolve asheterozygotes or distinguish from one

Exhibit 64. Histogram of mass difference measurements for 200 samples (50 for each ddN). Expected masses for the dideoxynucleotides are 273.2 Da for ddC, 288.2 Da for ddT, 297.2 Da for ddA, and 313.2 Da for ddG. The overall mass precision with this set of samples was less than 2 Da. For more details, see Li et al., 1999.

Num

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40

30

40

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0270 280 290 300 310 320

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ddC273.2 Da

ddT

288.2 Da

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297.2 Da313.2 Da

ddG

64


another in terms of mass. As reportedin a recently published paper (Li et al.,1999), this approach has been used toreliably determine all four possibleSNP homozygotes and all six possibleheterozygotes.

Mitochondrial DNA WorkThe control region of mtDNA, com-monly referred to as the D-loop, ishighly polymorphic and contains anumber of possible SNP sites foranalysis. MITOMAP, an internet data-base containing fairly comprehensiveinformation on mtDNA, lists 408 poly-morphisms over 1,121 nucleotides ofthe control region (positions 16020576) that have been reported in litera-ture (MITOMAP, 1999). However,many of these polymorphisms are rareand population specific. The presentstudy focused on marker sets from afew dozen well-studied potential SNPsites. Special Agent Mark Wilson fromthe FBI Laboratory in Washington,D.C., who has been analyzing mtDNAfor more than 7 years, recommended aset of 27 SNPs that would give a rea-sonable degree of discrimination andmake the assay about half as informa-tive as full sequencing. His recom-mended mtDNA sites were positions16069, 16114, 16126, 16129, 16189,16223, 16224, 16278, 16290, 16294,16296, 16304, 16309, 16311, 16319,16362, 73, 146, 150, 152, 182, 185,189, 195, 198, 247, and 309. Theunderlined sites are those reported in a minisequencing assay developed bythe Forensic Science Service (Tully etal., 1996). GeneTraces multiplex SNPtyping efforts began with 10 of theSNPs used in the FSS minisequencingassay, since those primer sequenceshad already been reported and studiedtogether.

L15997

H00401

1021 bp PCR Product

H16069 G or AH16129 C or TH16189 A or GH16224 A or GH16311 A or G

H00073 T or CL00146 T or CH00152 A or GL00195 T or CH00247 C or T

HV1 HV2

* * * * * * * *

* *

Exhibit 65. Schematic representation of the mtDNA control region 10-plex SNP assay. The asterisks represent the relative positions of the SNP sites and the strand that is probed.

Exhibit 66. SNP ions impacting multiplex design. Doubly charged and triply charged ions from higher mass primers can interfere with singly charged ions of smaller mass primers if the multiplex is not well designed.

2,000 3,000 4,000 5,000 6,000 7,000

Extensionproduct

SNP primer

Primerimpurity

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3+ ions

1+ ions

Na adduct

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65


The reported FSS sequences weremodified slightly by removing thepoly(T) tail and converting the degen-erate bases into the most commonsequence variant (identified by exami-nation of MITOMAP information atthe appropriate mtDNA position). Acleavable base was also incorporated atvarying positions in different primersso that the cleaved primers would beresolvable on a mass scale. Exhibit 26lists the final primer set chosen for a10-plex SNP reaction. Eight of theprimers detected SNPs on the heavyGC-rich strand and two of them iden-tified SNPs on the light AT-richstrand of the mtDNA control region(exhibit 65). Five of the primersannealed within hypervariable region I(HV1) and five annealed within hyper-variable region II (HV2). All of the 10chosen SNP sites were transitions ofeither A to G (purine-to-purine) orC to T (pyrimidine-to-pyrimidine)rather than transversions (purine-to-pyrimidine).

Besides primer compatibility (i.e., lackof primer dimer formation or hair-pins), another important aspect ofmultiplex SNP primer design is theavoidance of multiple-charged ions.Doubly charged and triply chargedions of larger mass primers can fallwithin the mass-to-charge range ofsmaller primers. Depending on thelaser energy used and matrix crystal-lization, the multiple-charged ions canbe significantly abundant (exhibit 66).Primer impurities, such as n-1 failureproducts, can also impact how closetogether primers can be squeezed ona mass scale. These primer synthesisfailure products will be ~300 Da small-er in mass than the full-length primer.Since an extension product rangesfrom 273 (ddC) to 313 Da (ddG) larg-er than the primer itself, a minimumof 650700 Da is needed betweenadjacent primers (postcleavage mass)if primer synthesis failures exist to

avoid any confusion in making thecorrect SNP genotype call.

Primer synthesis failure products wereobserved to become more prevalentfor larger mass primers. Because reso-lution and sensitivity in the mass spec-trometer decrease at higher masses,it is advantageous to keep the multi-plexed primers in a fairly narrow masswindow and as small as possible. Theprimers in this study ranged from1,580 to 6,500 Da. Exhibit 23 displaysthe expected primer masses for themtDNA SNP 10-plex along with theirdoubly and triply charged ions. Thesmallest four primers, in the massrange of 1,5803,179 Da, had primerand extension masses that were simi-lar to multiple-charged ions of largerprimers. For example, in the bottompanel of exhibit 6, which shows the10-plex primers, the doubly chargedion from MT4e (3,250 Da), whichprobes site L00195, fell very closeto the singly charged ion from MT3(3,179 Da), which probes site H16189.The impact of primer impurity prod-ucts can also be seen in exhibit 6. Anexamination of the extension productregion from primer MT7/H00073(~6,200 Da) shows two peaks whereonly one was expected (top panel).The lower mass peak in the doubletis labeled as +ddC (6,192 Da), butthe larger peak in the doublet was aprimer impurity of MT4e/L00195(6,232 Da). The mass differencebetween these two peaks was 40 Daor exactly what one would expect fora C/G heterozygote extension of primerMT7/H00073. Thus, to avoid a falsepositive, it was important to run the10 primers alone as a negative controlto verify any primer impurities.

To aid development of this multiplexSNP assay, large quantities of PCRproduct were produced from K562genomic DNA (enough for ~320 reac-tions) and were pooled together sothat multiple experiments would have

the same starting material. With theK562 amplicon pool, the impact ofprimer concentration variation wasexamined without worrying about theDNA template as a variable. The K562amplicon pool was generated usingthe PCR primers noted in exhibit 26,which produced a 1,021 bp PCR prod-uct that spanned the entire D-loopregion (Wilson et al., 1995). Thus, all10 SNP sites could be examined froma single DNA template.

Using ABIs standard sequencing proce-dures and dRhodamine dye-terminatorsequencing kit, this PCR pool wassequenced to verify the identity of thenucleotide at each SNP site in the 10-plex. The sequencing primers werethe same as those reported previously(Wilson et al., 1995). Identical resultswere obtained between the sequencingand the mass spectrometer, which ver-ified the method (exhibit 6).

A variety of primer combinations andprimer concentrations were tested onthe way to obtaining results with the10-plex. For example, a 4-plex and a 6-plex were developed first withprimers that were further apart interms of mass and, therefore, could bemore easily distinguished. An early 6-plex was published in Electrophoresis(Li et al., 1999). Primer concentra-tions were balanced empirically byfirst running all primers at 10 pmoland then raising or lowering theamount of primer in the next set toobtain a good balance between thosein the multiplex primer mix. In gener-al, a higher amount of primer wasrequired for primers of higher mass.However, this trend did not alwayshold true, probably because ionizationefficiencies in MALDI mass spectrome-try differ depending on DNA sequencecontent. The primer concentrations inthe final optimized 10-plex rangedfrom 10 pmol for MT3 (3,179 Da) to35 pmol for MT7 (5,891 Da). Primerextension efficiencies also varied

66


between primers, making optimizationof these multiplexes rather challenging.

Originally, this study set out to exam-ine 100 samples, but due to the earlytermination of the project, researcherswere unable to run this multiplex SNPassay across a panel of samples to veri-fy that it worked with more than onesample. Future work could includeexamination of a set of population sam-ples and correlation to DNA sequenc-ing results. Examination of the impactof SNPs that are close to the one beingtested and that might impact primerannealing also needs to be done. Inaddition, more SNP sites can be devel-oped and the multiplex could beexpanded to include a larger numberof loci.

Y-Chromosome WorkWhile the mtDNA work produced anopportunity to examine the mass spec-trometry factors in developing an SNPmultiplex, this work involved only asingle DNA template with multipleSNP probes. A more common situa-tion for multiplex SNP developmentis multiple DNA templates with oneor more SNP per template. SNP sitesmay not be closely spaced along thegenome and could require uniqueprimer pairs to amplify each sectionof DNA. To test this multiplex SNPsituation, researchers investigated mul-tiple SNPs scattered across the Y chro-mosome. Through a collaboration withDr. Oefner and Dr. Underhill, 20 Y-chromosome SNP markers were exam-ined in this study. Dr. Oefner and Dr.Underhill have identified almost 150SNP loci on the Y chromosome, someof which have been reported in the lit-erature (Underhill et al., 1997). Byexamining an initial set of 20 Y SNPsand adding additional markers as need-ed, researchers attempted to developa final multiplex set based on ~50 YSNP loci. The collaboration provided

detailed sequence information on basesaround the SNP sites (typically severalhundred bases on either side of theSNP site), which is important for mul-tiplex PCR primer design. Dr. Oefneralso provided a set of 38 male genomicDNA samples from various populationsaround the world for testing purposes.

The sequences were provided in twobatches of 10 sequences each. In thefirst two sets, primer designs wereattempted for a 9-plex PCR and a 17-plex PCR, respectively. Due to primerincompatibilities, it was impossible to

incorporate all SNPs into each multi-plex set. However, with a larger set ofsequences to choose from, it is con-ceivable that much larger PCR multi-plexes can be developed. According toDr. Underhills nomenclature, the firstset of Y SNP markers included the fol-lowing loci: M9 (CG), M17 (1 bpdeletion, 4Gs3Gs), M35 (GC),M42 (AT), M45 (G>A), M89(CT), M96 (GC), M122 (TC),M130 (CT), and M145 (GA).The second set of Y SNP markers con-tained these loci: M119 (AC), M60(1 bp insertion, a T), M55 (TC),

PCR Mix: 5 mM MgCl2, 1X PCR buffer II, 250 M dNTPs, 2 U TaqGold, 40 pmol univ-F primer, 40 pmol univ-R primer, and 0.4 pmole each locus-specific primer pair in 20 L volume Thermal Cycling: 95 oC for 10 min; 50 cycles at 94 oC for 30 sec, 55 oC for 30 sec, 68 oC for 60 sec, 72 oC for 5 min, and 4 oC forever

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