research paper str genotyping of exogenous hair shaft dna

Australian Journal of Forensic SciencesVol. 39, No. 2, December 2007, 107–122

RESEARCH PAPER

STR genotyping of exogenous hair shaft DNA

Kate S. Robertsona*, Dennis McNevinb and James Robertsona

aForensic and Technical, Australian Federal Police bForensic Studies,School of Health Sciences, University of Canberra, Australia

Most hairs found at crime scenes yield low quality and/or low quantities of nuclearDNA. This DNA is further depleted when stringent hair cleaning procedures are appliedin the laboratory, suggesting that detectable DNA exists exogenously. The phenomenonof exogenous hair DNA is the subject of this study. DNA was extracted from washed andunwashed hairs and the resulting ProfilerTM Plus STR genotypes were compared withthose of reference (buccal) swabs from the hair donors. The DNA extraction procedureinvolved no prior cleaning of the hair sample and no dissolution of the hair duringdigestion, in contrast to standard procedures. The STR genotyping success was measuredby recording the two dominant alleles at each locus and comparing them with thereference DNA profile. The effect of hair cleanliness was examined by leaving donors’hair unwashed for periods of 1, 3 and 7 days before sampling. It was found that thegenotyping success for unwashed hair was significantly higher than that for freshlywashed hair, with the majority of clean hair samples producing little or no DNA.Genotyping success was also lower for donors with cosmetically treated hair comparedwith those having untreated hair. Although the quality of STR profiles (i.e. alleledropout, differential amplification) from hair shafts or telogen hair clubs is reducedcompared with those from other biological sources, the genotypes obtained in this studymay be usable and are certainly discriminating if alternative interpretational methods areapplied.

Keywords: Hair; shaft; telogen; extraction; short tandem repeat (STR); low copynumber (LCN)

1. Introduction

Hair is a biological tissue that can be very useful as forensic trace evidence in criminalinvestigations9,8,34. It can be used as evidence to exclude or associate individuals with acrime scene or object. Until recently microscopic examination of hair morphologicalcharacteristics has been the principal method of hair analysis34. A major limitation of hairmicroscopy includes its highly subjective nature, which makes it difficult for the hairexaminer to place a statistical value on a proposed hair ‘match’. These issues have led to agreater focus on DNA analysis, which can potentially individualise DNA evidence with avery high statistical certainty. With the advent of the polymerase chain reaction (PCR)29,highly sensitive nuclear DNA (nuDNA) genotyping systems exist today, allowingidentification of short tandem repeat (STR) microsatellite alleles from minute amountsof biological material40,42.

*Corresponding author. Email: [email protected]

ISSN 0045-0618 print/ISSN 1834-562X online

� 2007 Australian Academy of Forensic Sciences

DOI: 10.1080/00450610701650096

http://www.informaworld.com

Currently, nuclear DNA analysis is predominantly conducted only on the roots ofhairs in the active growth (anagen) phase rather than on hair shaft or hairs in the resting(telogen) phase. While anagen phase hair has metabolically and mitotically active root andfollicle material that are amenable to DNA typing, hair shaft and telogen hair clubs arefully keratinised containing only very small amounts of DNA that are thought to be of avery degraded nature25,36,39. This is problematic for forensic scientists as it is the fullykeratinised telogen phase hairs that are naturally shed and comprise the majority ofevidentiary hairs found at crime scenes4,11.

As a result there has been considerable focus on mitochondrial DNA (mtDNA)analysis, which has been more successful than nuDNA analysis20,21,23,28,31 due to mtDNAexisting in much higher copy numbers within a cell.43 However, mtDNA analysis hasseveral limitations26, a significant one being that it is maternally inherited and thusmtDNA profiles cannot individuate between maternal relatives. Hence the establishmentof a successful and reliable technique for the typing of nuDNA from keratinised hairwould be of great value to the forensic community.

Recent research into the improvement of STR typing of DNA from keratinised hairhas mainly focused on post-extraction procedures, including techniques such as reducedvolume PCR (RV-PCR), extended PCR cycles, nested PCR and the use of redesigned PCRprimers that generate shortened PCR products7,12,13,15,16,18. However, it is not clearwhether nuDNA actually persists endogenously within the hair shaft or the telogen hairclub26. McNevin et al. 27 found that there was a greater chance of obtaining STR allelesthat corresponded with donors’ buccal swabs (‘consensus’ alleles) from single hair shaftsnot cleaned prior to extraction rather than from cleaned hair shafts, where cleaning hadinvolved sequential washes with SDS, dH2O and alcohol. They also showed that the mosteffective procedure for obtaining consensus alleles from hair shaft involved only rinseswith water and a Tris–HCl buffer which did not dissolve the hair27. A better result was notobtained by dissolution of the hair in a lysis buffer containing commonly used detergents,proteinase K and reducing agent. The observations that the degradation of the keratin hairstructure has little effect on nuDNA quantity and quality, and that pre-cleaning the hairdecreases the chance of obtaining nuDNA, provides evidence that recoverable nuDNAresides towards the hair exterior or is exogenous. Indeed, there is considerable evidencethat nuDNA exists in the cuticle layer of the hair shaft19,24,30,35.

Using an extraction method involving simple leaching of exogenous nuDNA fromhair, McNevin et al. 27 found that STR genotyping success was highly dependent on thedonor. This is consistent with the proposition that exogenous DNA is more vulnerable tothe environmental influences on the hair. From a forensic perspective, if the majority ofrecoverable nuDNA is indeed exogenous, a better understanding of the factors orconditions that can affect the presence of nuDNA on hair, and thus the success of STRgenotyping, is critical.

The aims of this study were to identify factors that may affect the presence ofexogenous DNA on hair shaft, with a major focus on hair cleanliness. The effect of hairwashing on the ability to obtain a STR profile from hair shaft is investigated bycomparison of DNA profiles produced from clean hair and dirty hair from a number ofdonors. It is hypothesised that if hair shaft DNA is exogenous, and therefore morevulnerable to environmental influence, then hair washing will remove the recoverableexogenous DNA and consequently there will be a marked difference between the ability toobtain a profile from freshly washed hair and dirty hair. An alternative STR profileinterpretation method is recommended for this type of low copy number (LCN)genotyping.

108 K. S. Robertson et al.

2. Materials and methods

2.1 Sampling procedures

A total of 29 volunteers were recruited to the study in accordance with the University ofCanberra Human Ethics Manual (2007)22. The participants consisted of 8 males and 21females and ranged in age from 22 to 53 years. They each collected a minimum of 15individual hairs directly after washing their hair with supplied shampoo (De Lorenzo�,Hair and Cosmetic Research Pty. Ltd., Silverwater, Australia) and then another minimumof 15 hairs a number of days later without further shampoo washing (washing with wateronly was permitted). A clean comb was provided each time hair was sampled. Themajority of participants fell into three groups, those that left their hair unwashed for 1 day(6 participants), 3 days (12), or 7 days (11). Hairs were self-sampled by participants eitherby running a comb or gloved hands through the hair. Hair samples were stored at roomtemperature in folded paper inside sealable plastic bags until examined.

To assist in identification of other factors that may influence genotyping success, eachparticipant completed a questionnaire, which provided information on the features,condition and recent history of their hair and how it was treated during the period betweencollection of the clean and dirty hair samples (factors identified in the Questionnaire areincluded in Table 1).

A buccal (cheek) swab (Medical Wire & Equipment Co. (Bath) Ltd., Corsham, UK)was obtained from each participant in order to produce a reference DNA profile. Thebuccal swab was stored in a paper envelope at 4�C until required for DNA extraction.

2.2 Hair sample preparation

Eight hairs were used from each clean and dirty hair sample supplied by the participants.The hairs were examined under a stereomicroscope (40� resolution) to identify the hairroot, then for each hair approximately 1.5 cm was cut from the proximal end anddiscarded. A further 2.5 (�0.3) cm segment from each of the eight hairs was then cut fromthe proximal end and placed in a sterile 1.5ml tube, yielding an equivalent total of 20 cm ofhair in each sample tube. Hair sample preparation was conducted in a separate room toreference swabs. All equipment used was washed with 20% bleach and 70% ethanolsolutions before processing each hair sample to minimise the risk of contamination.

2.3 Sample processing

As a precaution against extraneous contamination, DNA extraction and DNAamplification were performed in different rooms. All samples were handled in apresterilised biological safety (laminar flow) cabinet with latex gloves, hair net, andfacemask. Hair samples were processed separately from reference samples withapproximately 1 month between reference and hair sample processing. The laminar flowcabinet and all equipment were decontaminated between the processing of each samplebatch. All reagents and chemicals used were of Analytical Reagent (AR) grade ormolecular biology grade as appropriate.

2.4 DNA extraction

DNA from the clean and dirty hair samples was extracted using a phenol-chloroformextraction method optimised for the extraction of exogenous hair DNA, as described byMcNevin et al. 27 This protocol involved no cleaning of the hair samples prior to extractionand no dissolution of the hair during the extraction process. Each sample was soaked

Australian Journal of Forensic Sciences 109

Table 1. Demographic information for the 29 participants obtained from thequestionnaire.

SexMales 8Females 21

Age18–35 years 1836–53 years 11

Ethnic OriginNW European 24S European 2NW European, S European 1NW European, African 1NW European, Oceanian (Maori) 1

Hair TypeWavy hair 10Straight hair 17Curly hair 2

Hair LengthShort hair 15Short-medium 3Medium 7Long 4

Treated and Natural Hair (Hair used in experiments)Treated hair 9Permanent dye and/or bleached (6)Permanent foils (1)Semi-permanent (1)Temporary dye rinse (1)

Natural hair 20Blonde (4)Blonde-Brown (1)Brown (11)Black (1)Red (0)Grey (3)

Use of Supplied ConditionerConditioner used 27No conditioner used 2

Hair Products used during ExperimentNo hair product used 16Hair product used 13Conditioning & smoothing products (2)Hairspray, wax, putty, gel & curling creme (11)

Conditions Hair is Exposed toNo specific conditions 25Specific conditions 4Chorinated water regularly, during experiment (2)Chorinated water regularly, but not duringexperiment

(1)

Paint fumes and dust, everyday (1)

(continued)


overnight in a simple digestion buffer containing 10mM Tris–HCl, 10mM EDTA, 2%Tween�20 and 100mM NaCl. After extraction with phenol:chloroform:isoamyl alcohol(25:24:1) and chloroform each sample was subjected to centrifugal ultrafiltration usingMicrocon� YM-30 centrifugal filter devices (Millipore Corporation, Bedford, USA). Theresulting 100ml extracts were stored at 4�C until quantitation.

DNA from the buccal swabs was extracted using Chelex� 100 Resin (Bio-rad,Hercules, CA) according to standard procedures41.

2.5 DNA quantitation

The amount of extracted DNA was estimated via real-time PCR as described byMcNevin et al.27

2.6 DNA amplification

DNA extracts from buccal swabs and hair samples were genotyped against AmpF‘STR�

Profiler PlusTM STR loci (Applied Biosystems, Foster City, CA). Extracted DNA wasamplified in a GeneAmp� 9600 thermal cycler (Applied Biosystems) in a 50 ml volumeaccording to the AmpF‘STR� Profiler PlusTM protocol5 except that, for hair samples, 34PCR cycles (rather than the standard 28 cycles) were employed and a 100� dilution ofAmpF‘STR� Control DNA 9947A (to make final concentration of 1 pg/ml) was used as apositive control because of the extra 6 PCR cycles. PCR product (1.5ml) was then addedto 14.4ml of Hi-DiTM deionised formamide (Applied Biosystems) and 0.6ml ofGeneScan�-500 [ROX]TM internal standard (Applied Biosystems) and then separatedinto STR alleles via capillary electrophoresis on an ABI Prism� 3100 Genetic Analyser(Applied Biosystems) under the following conditions: 36 cm array; POP-4TM sievingpolymer (Applied Biosystems); filter set F; 10 s, 5 kV injections; 60�C, 15 kV runs.Electropherograms were generated using GeneScan� Analysis Software2. STR genotypeswere determined in the Genotyper� Software1 environment.

2.7 Interpretation of STR profiles

Allele peaks (homozygous and heterozygous) that were less than 100 RFU (relativefluorescence units) were not recorded, in accordance with standard Australian FederalPolice procedures3. Stutter artefacts were interpreted according to the AmpF‘STR�

Table 1. Continued.

Oiliness of hairDry 4Normal 16Oily 7Combination 2

Usual Regularity of Hair WashingEvery 1 day 11Every 2 days 8Every 3 days 5Every 4 to 7 days 4Greater than every 7 days 1


Profiler PlusTM PCR Amplification User’s Manual (2000)5 and, if present below themaximum expected stutter percentage for the locus in question, they were ignored.

Due to a relatively high incidence of PCR artefacts and possible contaminating DNA(‘non-consensus’ alleles) in the hair extract profiles, only the two dominant alleles (twohighest peaks as measured by RFU) at each locus were recorded. The profile resultingfrom the application of this rule was termed a ‘primary profile’.

The genotypes for the hair extracts were compared with their corresponding referencegenotypes obtained from buccal swabs. The STR genotyping success was determined asthe number of dominant alleles that matched the reference genotype (a maximum of 20). Ifone of the two dominant peaks at a locus in a sample DNA profile matched a homozygousreference allele for that particular locus, then both homozygous alleles were considered tobe present.

2.8 Statistical analysis

Non-parametric statistical analyses were conducted on the data produced due to veryskewed data distributions. All statistical analysis was performed using SPSS� AnalyticalSoftware for Windows� Version 11.537. Only the primary profiles were considered in thestatistical calculations.

3. Results

The demographic and hair information obtained from the questionnaires issued toparticipants is depicted in Table 1. The participants ranged in age from 22 to 53 years, withonly 27.6% (8) being male and the majority being of a North Western European ethnicorigin (82.8%). Two-thirds of the participants had naturally coloured hair (69.0%), whichranged in colour from blonde to black. Treated hair consisted of temporarily dyed (colourrinses), bleached, permanently dyed (including foils) and semi-permanently dyed hair.

The quantities of DNA in the hair shaft extracts, as estimated by real-time PCR, werevery low. Only six samples contained detectable DNA, ranging from 1.6 pg ml�1 to5.1 pg ml�1. The capacity to measure the DNA concentration was not indicative of theability to obtain a DNA profile, as several samples that produced a full profile did notproduce a measurable DNA concentration by real-time PCR. The quantitation method,based on the Alu transposon in the human genome27, may not be appropriate for theanalysis of very low levels of DNA.

Contaminating alleles appeared in two of the extraction controls and one of thenegative controls. The average number of contaminating alleles in all negative andextraction controls analysed was 0.33 alleles per control. There was no consistency as towhich contaminating alleles were present in the controls and these alleles were not presentin hair profiles unless they corresponded with reference profiles. When genotyping of thecontrols was repeated, contaminating alleles disappeared. These observations suggest thatthe contamination of the controls was a random event and not due to contamination of thereagents used.

Only 42 of the 58 hair extracts produced DNA profiles (72.4%), 15 of the clean hairextracts and 27 of the dirty hair extracts. A total of 87 and 289 consensus alleles wereobserved in the clean hair and dirty hair primary profiles, respectively. Analysis of only thedominant alleles (primary profile) did not affect participants’ genotyping success except inone case where it was reduced by one, dropping the total consensus allele count for alldirty hair profiles from 290 to 289. However, it did reduce the total number of


non-consensus alleles in the profiles dramatically, from a total of 30 when all observable

peaks were considered to a total of 16 when only the dominant alleles (primary profile)

were considered. Although the number of non-consensus alleles was higher in the dirty

hair primary profiles (12) than the clean hair primary profiles (4), the difference was not

significant (Sign test, p¼ 0.114).Figure 1 shows the distribution of the number of consensus alleles in the clean and

dirty hair profiles. The number of consensus alleles in the clean hair profiles varied from

0 to 19 with just under half the participants producing no profile at all (48.3%). There were

two outliers that produced 85% and 95% of their full consensus profile in their clean hair

sample (17 and 19 alleles, respectively). In contrast, the dirty hair data had a much less

skewed distribution with 5 (17.2%) samples producing full profiles, 22 (75.9%) producing

partial profiles and only 2 (6.9%) producing no profile. A sign test was used to compare

the median number of consensus alleles in the clean and dirty hair profiles. The clean hair

(a)

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2

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16

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Number of consensus alleles in clean hair primary profile

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Number of consensus alleles in clean hair primary profile

Nu

mb

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Nu

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Figure 1. The distribution of the number of consensus alleles in the (a) clean hair primary profilesand (b) dirty hair primary profiles. The arrows highlight the two participants who produced 85%and 95% of their full profile.


median (1.0) and the dirty hair median (9.0) were found to be significantly different(p¼ 0.000).

Table 2 shows the number of consensus alleles observed in the clean and dirty profilesof each group. For the 3-day and 7-day group data, the dirty hair median was significantlyhigher than the clean hair median (Sign test, p¼ 0.000 and p¼ 0.006, respectively);however, for the 1-day group the clean and dirty hair median were not significantlydifferent (Sign test, p¼ 0.063). Although there was no significant difference between thedirty hair data of the 3-day and 7-day groups (p¼ 0.440), the number of consensus allelesdetected in the participants dirty hair profiles varied greatly, ranging from 1 to 20 alleles(data not shown). Hair treatment was investigated as a possible contributing factor to thisvariation in genotyping success. The data of the participants in the 3-day and 7-day groupswere pooled into one set as they were not significantly different, which allowed for agreater population sample to be studied (23 participants), and then separated according towhether they had natural hair or treated hair (Figure 2). Treated hair was considered to behair that had been dyed (permanent, semi-permanent or rinse treatments), bleached orcontained foils (see Table 1). To take into account the presence of re-growth, participantswho stated that their hair had been treated 4 months ago or longer were classified as‘natural’. This limit was based on the estimation that hair grows approximately 1 cm every

Treated Natural

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Figure 2. Consensus allele counts for the 23 participants’ dirty hair profiles (�3 days dirty),grouped according to whether the profiles were produced from natural or treated hair. Thegrey boxes represent the middle 50% of values and each whisker represents 25% of the values toeither side. The black lines represent the medians.

Table 2. The total number of consensus alleles observed in the clean and dirty profiles for eachgroup and the median number of consensus alleles in the clean and dirty hair primary profiles foreach group.

Clean Hairc Dirty Hair

Groupa Nb Total Alleles Median Median Total Alleles Median Median

1-day 6 4 0 29 23-days 12 37 1 142 117-days 11 46 2 118 9

aParticipants grouped according to the day they collected their dirty hair sample.bNumber of participants in each group.cRepresents the sample taken at day 0 for each group.


month17 and the hair samples used for DNA analysis in this study were obtained fromwithin the first 4 cm of the proximal end of each hair. A Mann–Whitney Test was used tocompare the two independent data sets and showed that the two groups were significantlydifferent (p¼ 0.011). Similar analyses were conducted with regard to the use of hairproduct and participant hair type (curly/straight/wavy), however no significant differenceswere found between these groups and the number of consensus alleles in participant dirtyhair profiles (data not shown).

There are many other factors that may have some influence on the STR genotypingsuccess of hair shaft DNA. Table 1 contains information on several characteristics thatmay be potential contributing factors. For example, a participant’s ethnic origin, hair type,oiliness of the hair or the use of hair products may affect the ability to obtain DNA fromtheir hair shaft. The number of participants involved in this study does not provide enoughstatistical power to measure many of these factors.

It should be noted that the influence of extraneous variables during hair samplingmight interfere with drawing clear conclusions from the data obtained. Steps taken toreduce error variance due to these variables included treatment of subjects as similarly aspossible, consistency in presentation of instructions to each participant and use ofstandard sampling and laboratory protocols. Even so, subjects and their livingenvironments differ from one another in innumerable and unidentifiable ways, and thepossibility that participants deviated from the instructions provided cannot be discounted.

Several features were observed that affected the quality of the hair extract profiles.Differential amplification of heterozygote alleles resulting in unbalanced peak heights waspresent in most of the profiles and was also present in the 100�diluted positive control(Figure 3a). Another common observation was that homozygous and heterozygous alleleswere not present in their usual proportions, with homozygote peaks sometimes beingsmaller than heterozygote peaks (Figure 3b and c).

Allelic dropout was also a very common observation in the partial profiles obtained(Figure 3c). Figure 3d is a further example of allelic dropout, although this time with anon-consensus contaminating allele present as well, another common feature observed.

Contaminating ‘non-consensus’ alleles were present in both the clean and dirty hairsamples. There was no consistency except possibly for allele 14 of the D8S1179 locus andallele 8 of the D1S317 locus, both of which appeared in two different profiles (but not inthe controls). The number of non-consensus alleles did not increase with the number ofconsensus alleles in the participants’ dirty hair profiles (rs¼�0.082, p¼ 0.661).

Although most of the peaks corresponding to non-consensus alleles were smaller thanthe peaks of consensus alleles, as in Figure 3d, in two cases the contaminating allele had ahigher RFU value than the consensus allele (Figure 3e and f). In every hair profile but one,all consensus alleles present at a locus were included in the two dominant peaks of theprimary profile. In the one exception (Figure 3f), the contaminating allele was bigger thanone of the consensus heterozygous alleles, which excluded that heterozygous allele fromthe primary profile.

The measure of STR genotyping success so far mentioned does not indicate theaccuracy of the profile with respect to the presence of the non-consensus alleles. The degreeof reliability of a genotype is defined here as the ratio of the number of non-consensusalleles to the number of consensus alleles present in the DNA profile. Table 3 shows thegenotype reliability for the clean hair and dirty hair primary profiles for each of theparticipants. Even though the total ratio value is lower for clean hair (1.72), indicating thatobtaining a genotype from clean hair is more reliable, the difference between the total ratiovalues of clean and dirty hair is not significant (Sign test, p¼ 0.194). In addition, there is


no significant difference between the non-consensus-to-consensus ratios for the dirty hairsamples of the 3- and 7-day groups (Mann–Whitney U test, p¼ 0.651). The 1-day groupwas excluded from this analysis due to the small sample size.

Increased amplification of stutter artefacts beyond the expected maximum loci stutterpeak percentages as expressed in the AmpF‘STR� Profiler PlusTM PCR AmplificationUsers Manual (2000)5 may explain the presence of some non-consensus alleles: one

Figure 3. Profiler PlusTM electropherograms of several STR loci depicting cases of reduced profilequality. All were obtained from dirty hair except 3 c. (a) Electropherogram of the D21S11 locus,showing differential amplification of heterozygous alleles. (b) Electropherogram of the D13S317 andD7S820 loci, showing disproportional peak heights of heterozygote and homozygote loci, in whichthe homozygous peak is approximately the same as the heterozygous peaks. (c) Electropherogram ofthe D3S1358 and vWA loci, illustrating another case of disproportional peak heights of heterozygoteand homozygote loci in addition to allele drop out. The peak at the vWA locus corresponds to aheterozygote locus although only one heterozygous allele was detected. The arrow indicates that thehomozygote peak at the D3S1358 locus is approximately half the size of the heterozygous peak.(d) Electropherogram of locus D3S1358 for a heterozygote individual in which allele dropout hasoccurred resulting in detection of only one heterozygous allele (allele 14). The arrow highlights thepresence of a smaller contaminating allele (allele 15). (e) Electropherogram of the D21S11 locus for aheterozygote individual in which allele dropout has occurred resulting in detection of only oneheterozygous allele (allele 30). The arrow highlights the presence of a larger contaminating allele(allele 29). (f) Electropherogram of the D5S818 locus for a heterozygote individual, in which acontaminating peak (allele 14) has been detected in greater amount than one of the heterozygouspeaks. (g) Electropherogram of the D5S818 locus for a homozygote individual, in which acontaminating allele (allele 11) appears alongside the taller homozygote peak. (h) Electropherogramof the D5S818 locus showing base pair ladder and allele designations. The arrow indicates thepresence of a short non-consensus peak that is four base pairs smaller in size than the heterozygousallele 11, which may be the result of over-amplification of a stutter artefact.


example is observed in Figure 3h. The presence of these potentially over-amplified stutterpeaks did not have any effect on the respective primary profiles obtained.

4. Discussion

Our results show that it is possible to obtain discriminating STR profiles from hair shaftthat does not undergo a cleaning step prior to extraction and is soaked in a simple digestbuffer, which does not dissolve the hair. Genotyping success of hair shaft DNA wasdependent on the hair donor, as found by McNevin et al. 27 The genotyping success of the(dirty) hair extracts varied extremely between individuals, ranging from 100% success with

Table 3. Ratio of the number of non-consensus alleles to the number ofconsensus alleles in the clean hair and dirty hair primary profiles for eachparticipant.

Days N-C/Cb

Participant Code Dirtya Clean Hair Dirty Hair

1 Bx 1 – 02 Ex 1 – 03 Ix 1 – –4 Nx 1 – –5 Aax 1 – 0.506 Afx 1 0 0

7 Ax 3 0 08 Fx 3 0 0.149 Hx 3 – 010 Kx 3 – 0.1711 Ox 3 – 012 Qx 3 0 013 Rx 3 – 014 Sx 3 0 015 Vx 3 – 0.4416 Wx 3 0 017 Xx 3 0 018 Zx 3 1.00 0

19 Cx 7 – 020 Dx 7 – 0.1321 Jx 7 0.17 0.0722 Lx 7 0 023 Px 7 0 0.1324 Tx 7 0 025 Yx 7 – 0.3326 Abx 7 0.50 027 Adx 7 0.05 028 Aex 7 0 0.0629 Agx 7 – 0

Total N/A 1.72 1.97Median N/A 0 0

aIndicates the number of days after washing and before sampling.bThe ratio of the number of non-consensus alleles to consensus alleles in a profile.A dash indicates that no profile was obtained. A value of ‘0’ indicates there wereno non-consensus alleles observed (most reliable genotype).


all of the possible 20 alleles present (17.2% of participants) to no success with no alleles

present (6.9% of participants).A comparison of the DNA profiles produced from each participant’s clean and dirty

hair samples showed that there was a significant increase in the genotyping success for

dirty hair. The increase in the DNA detected indicates that the hair has acquired DNA

during the period of time between shampooing and sampling of the dirty hair, which

suggests that the additional DNA was most likely deposited onto the hair and exists

exogenously. The significantly lower success in STR genotyping of freshly washed hair

would indicate that hair washing removes some of the detectable DNA that exists

exogenously on the hair shaft. This has been shown in several other studies30,35. Schreiber

et al. 35 found that washing hair with an aqueous solution of the detergent SDS drastically

reduced the DNA yield obtained from the hair. Heywood et al. 19 showed that washing

hairs with a diluted alkaline shampoo (12% sodium lauryl ethyl sulfate, 2% tegobetaine)

resulted in detectable DNA in the wash solution after 20 sequential washes.Although significantly lower, consensus alleles were still detected in several of the clean

hair profiles (51.7% of participants). Indeed, two individuals produced 85% and 95%

(17 and 19 alleles, respectively) of their full consensus profile in their clean hair sample.

This only serves to illustrate the donor-variability of genotyping success. If endogenous

DNA exists at all in hair shaft, it is most likely to be found in the cuticle cells19,24,30 and

thus may be accessible to a digestion buffer.According to our results, if (telogen phase) hair evidence found at a crime scene was

freshly washed or had been unwashed for only one day, it most likely would not yield a

discriminating STR profile. The chance of obtaining a useful DNA profile increases if the

hair has been unwashed for 3–7 days. However, although all samples produced DNA in

these two groups, the majority of profiles produced at 3 and 7 days were only partial

profiles (82.6% or 19 participants). Only 17.4% of the profiles (4 participants) had the

maximum 20 consensus alleles providing the highest discriminatory power possible using

the Profiler PlusTM genotyping system. Due to the inability to obtain a full DNA profile

from the majority of participants, it would be desirable to conduct further studies to

determine if the genotyping success for hair would increase further if the hair were left

dirty for longer than 7 days. From a forensic investigative perspective, this may be

pertinent as hair evidence often presents in a relatively dirty state33.In contrast to the above results, one participant whose hair was sampled one day after

washing produced a full DNA profile (the median number of alleles for similar samples

was 1.5). This hair sample collected as much exogenous DNA as hairs that were left

unwashed for 3 and 7 days. A feature that may be relevant in distinguishing this

participant from others is the participant’s use of a ‘hair putty’ product.The genotyping success for untreated hair was significantly higher than that for treated

hair. Other studies have reported similar findings. Heywood et al. 19 showed that the

amount of extractable DNA was significantly reduced after permanent colour treatment of

hair (with commercial hair colour treatment), while McNevin et al. 27 found that bleaching

hair (with commercial hair bleach) similarly reduced the amount of recoverable DNA. The

correlation between hair treatment and recoverable DNA may be due to alteration of the

hair surface (physical or chemical), which may either damage the cuticle cells or decrease

the ability of DNA to adhere to them. It has been widely documented that treatment of

hair with cosmetic products can induce limited temporary surface changes or more

extensive, permanent whole-fibre changes to the basic morphology and chemistry of

the hair10,32.


Low copy number (LCN) PCR inevitably results in a reduction in the quality of thehair extract profiles obtained and these have important implications for forensic casework.Increased stutter artefacts, allele dropout and unbalanced heterozygosity were regularlyobserved and are thought to be the result of stochastic variation when pipetting very diluteDNA extracts14,38. Standard interpretation methods, such as use of peak size andproportion, for allocation of genotypes to DNA profiles are probably not appropriatewhen very low quantities of DNA are extracted from hair. The amplification oflaboratory-based random contaminating alleles, otherwise known as ‘allele drop-in’, wasdetected in two extraction controls and a negative control and may possibly be responsiblefor several non-consensus alleles in the hair profiles. This is a common feature of LCNDNA amplification where 34 PCR cycles (or more) are used. Theoretically, this method issensitive enough to detect the equivalent of a single copy of DNA15. A sterile workplaceand the use of DNA-free reagents and instruments are essential for the analysis of DNAfrom keratinised hair.

We have applied an alternative STR profiling strategy for assigning genotypes to LCNDNA which involves the examination of a ‘primary profile’ comprising the two dominantalleles at each locus. There will always be a danger here that a contaminating allele willexclude an allele derived from the hair donor. This occurred in one case in our results(Figure 3f). However, we have demonstrated that with use of this method, the effect of lowlevels of random contaminating alleles and enhanced stutter peaks can be reduced to aminimum.

Several areas of further improvement were identified that must first be addressedbefore advocating this method for operational use. It is highly recommended that replicateanalyses are conducted with LCN DNA to identify random contaminating or ‘drop-in’alleles and achieve unambiguous genotypes15,38. It may be necessary to quantify theprobability of random contamination by performing numerous extraction and negativecontrols. Identification of non-reproducible random ‘drop-in’ contamination would helpto prevent misinterpretation of an individual’s primary profile.

In this study, 8 � 2.5 cm proximal hair segments (20 cm in total) were combined in eachsample so that differences between long and short hair were minimised. Obviously,casework will provide a range of hair lengths, as well as single hairs, and therefore it wouldbe ideal to conduct additional tests using more realistic scenarios. Also, because buccalswabs were available from hair donors, homozygous genotypes could be identified herewhere this would not be possible in casework. Identification of homozygosity is alimitation of this and other approaches to LCN DNA genotyping. However, there aremethods available for interpretation of these profiles14,15,38.

At present, this and other LCN strategies may not be tenable in court. However, theyare very useful in focusing forensic investigations and their power for inclusion andexclusion of suspects should not be discounted.

5. Conclusion

This study has provided further evidence for the existence of exogenous hair shaft DNAand has shown that it is quite possible to obtain highly discriminating STR profiles fromhair shafts. Factors affecting the ability to obtain a profile have also been identified. Thechance of obtaining a native STR profile is much greater when hair has not been washedfor 3 days or more. Given the variability between individuals in obtaining an STR profileand the issues concerning poor profile quality, the STR genotyping of keratinised hair


remains problematic. In this study, an alternative strategy for the assignment of genotypeswhen using LCN techniques has been proposed, while the limitations of this strategy havebeen identified.

Analysing nuDNA from hairs continues to attract interest from several groups ofresearchers. Most recently Anslinger et al.6 reported on the analysis of 96 hairs taken fromroutine casework, using two short amplicon or mini STR kits and real-time PCR as ascreening method for hair selection. Sixty-five of the hairs tested gave no detectable DNA.All of these were telogen hairs. Several of the successful hairs had visible cellular (sheath)material present and the root type in others was unknown. Even with these hairs theoverall success rate was just over 20%.

Hence, it is clear that success in obtaining nuDNA from teleogen hairs remainstechnologically challenging. We believe that the hair should be viewed as a substrate forexogenous DNA and that any protocol for the selection of hairs should be based on aknowledge of the donor history where possible. nuDNA testing of telogen hairs cannot yetbe considered a routine technique but it is within our grasp. We predict limitedapplications of this approach in the next few years after further study of variables whichmight affect potential successful typing and further advances in short amplicon analyticalapproaches.

Acknowledgments

The authors wish to thank Jennelle Kyd for valuable advice on experimental design forthis study.

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