Kerry L. Opel,1M.A.; Erica L. Fleishaker,2B.S.; Janice A. Nicklas,3Ph.D.; Eric Buel,3Ph.D.;
and Bruce R. McCord,1Ph.D.
Evaluation and Quantification of Nuclear DNA
from Human Telogen Hairs*
ABSTRACT: Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individu-
ally extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A
selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by
real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the
extracted DNA. Different quantities of sample (<100pg, 100–500pg, >500pg) were amplified with the Miniplex kits to determine the minimum DNA
template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those sam-
ples containing at least 60 pg using MiniSTRs.
KEYWORDS: forensic science, telogen, hair, nuclear, DNA, rt-PCR, Miniplex, STR
Shed telogen hairs can be an important form of probative foren-
sic evidence. Such samples are often reserved for mtDNA analysis
due to the assumption that little nuclear DNA is present. Unfortu-
nately due to its small size, common matrilineal inheritance, and
haploid nature, mtDNA lacks the probative value of nuclear DNA.
Several studies have been published on the extraction and amplifi-
cation of DNA from hair (1–5). While some success has been
reported for amplification of DNA in hair, accurate quantification
of the extracted DNA has been a problem. Before the use of real
time PCR for quantification, DNA analysts relied on methods
which lacked the sensitivity required to detect the minute amounts
of DNA found in hair. Therefore, information on the actual amount
of recoverable nuclear DNA present in hair is scarce. Recently, a
method has been developed for qPCR which utilizes multilocus
probes and permits quantitation of DNA in the picogram range (6).
This procedure is suitable for quantification of the total DNA pres-
ent in telogen hairs. In order to ensure that the DNA quantified
comes from within the hair, methods which can remove exogenous
DNA must be utilized. The use of a selective extraction step per-
mits removal of epithelial cells from the surface of the hair and
allows for the evaluation of the presence of exogenous DNA (7).
DNA extracted from telogen hair is likely to be degraded due to
the keritinization process which occurs during hair growth. Direct
evaluation of the state of degradation of DNA can normally be
accomplished through agarose gel separation and ethidium bromide
staining (8). Chemically degraded DNA presents as a smear on
the gel, with a range of fragment sizes (1500 to less than 150 base
pairs) (8). However, this method is not applicable when sub-
nanogram amounts of DNA are present (9). In these situations, real
time PCR can be used to determine relative level of degradation of
the template DNA through amplification with different primers.
Comparison of the amounts of large and small amplicons will
indicate if degradation is present in the sample (8).
When DNA is degraded, success of amplification of the DNA using
standard commercial multiplex kits with large amplicons can be lim-
ited. Instead, Miniplex kits specifically designed for use with degraded
DNA can provide an alternative for such samples (10). These Miniplex
kits produce a reduction in amplicon size for STR loci of up to 191
base pairs when compared to commercial kits and have demonstrated
ability to amplify DNA that has been chemically degraded (8,10).
Improved amplification and recovery of dropped alleles has also been
seen with DNA from naturally degraded skeletal samples (11).
This paper presents a large-scale sampling of DNA from telogen
hairs to determine the amount that can be extracted. A method for
assessment of the degradation level of DNA from telogen hairs is
also presented, as well as the effect of degradation on genotyping.
Materials and Methods
Sample Collection and DNA Extraction
Head hair was collected from 60 volunteers using a comb to
remove naturally shed hairs, and subjected to microscopic examina-
tion by the analyst to determine the stage of hair growth. Six to nine
telogen stage hairs from each individual (510 total hairs) were
selected and 3 cm of each hair, including the club end, was sampled.
Comparison DNA samples (to be used to establish full geno-
types) were obtained through buccal swabs and were stored either
on FTA paper or as dried swabs. FTA stored samples were pro-
cessed in accordance with the manufacturer’s protocols (12), and
swab samples were processed in accordance with a previously pub-
lished protocol (13).
1Department of Chemistry and Biochemistry, Florida International Uni-
versity, University Park, 11200 SW 8th Street, Miami, FL 33199.
2Department of Chemistry and Biochemistry, Ohio University, 136 Clipp-
inger Laboratories, Athens, OH 45701.
3Vermont Forensic Laboratory, 103 South Main Street, Waterbury, VT
*This project was supported under award 2002-IJ-CX-K007 from the
National Institute of Justice.
Portions of this paper were presented at the American Academy of Forensic
Sciences Annual Meetings in New Orleans, LA, February 25, 2005 and San
Antonio, TX, February 23, 2007.
Received 26 June 2007; and in revised form 23 Nov. 2007; accepted 1
J Forensic Sci, July 2008, Vol. 53, No. 4
Available online at: www.blackwell-synergy.com
? 2008 American Academy of Forensic Sciences
The individual hairs were incubated at 56?C for 2 h in 300 lL
of differential extraction solution (100 mM NaCl, Spectrum,
Gardena, CA), 10 mM EDTA (Sigma, St. Louis, MO), 0.4% SDS
(Spectrum) (7) with 40 lg of proteinase K (Fermentas, Hanover,
MD) to remove epithelial cells from the outside of the telogen
hairs. The extraction solution was removed and subjected to
organic extraction with 300 lL of 70% phenol⁄chloroform⁄water
(Applied Biosystems, Ventura, CA), and the aqueous layer was
purified in YM-30 Microcon?(Millipore, Inc., Danvers, MA) fil-
ters according to the manufacturer’s protocol (14). The extracted
epithelial DNA was collected in 60 lL of deionized H2O.
The hairs were then rinsed with a 0.9% NaCl solution, then
absolute ethanol to ensure complete removal of the extraction
solution, transferred to a fresh 600 lL tube and incubated at 56?C for
2 h in 300 lL of hair extraction solution (10 mM Tris-HCl [Sigma],
pH 8.0, 100 mM NaCl, 5 mM CaCl2[Sigma], 2% SDS, 39 mM
dithiolthreitol [DTT] [Sigma] ) (modified) with 80 lg of protein-
ase K added to extract the DNA from within the telogen hairs. The
DNA was separated and extracted using 300 lL of 70% phenol/
chloroform⁄water, and the aqueous layer containing the DNA was
purified in YM-30 Microcon?filters according to the manufacturer’s
protocol (aqueous layer centrifuged down to dryness in the filter,
washed with H2O, and centrifuged to dryness again) (14). The
purified DNA was collected in 60 lL of deionized H2O (Sigma).
All human samples were obtained in accordance with the institu-
tional review boards of Ohio University and Florida International
Both epithelial DNA and internal hair DNA extracts were quanti-
fied by real time PCR using the Corbett Rotor-Gene 3000 (Corbett
Research, Sydney, Australia), primers for the 82 base pair Alu
amplicon, and SYBR Green I (Invitrogen, Carlsbad, CA) dye using a
previously published protocol (6). Hairs yielding high concentrations
(>25 pg⁄lL) of template DNA (n = 9) were selected for fragment
size comparison, and were quantified, along with nondegraded
control DNA (Cell line 9948; Applied Biosystems) (with three
replicates of each sample) by primers for an 82 bp amplicon, a
124 bp amplicon, and a 201 bp amplicon in separate reactions.
Primer sequences for these amplicons are shown in Table 1.
Selected samples with the highest concentrations of DNA
(>25 pg⁄lL in 55 lL) were amplified according to published pro-
tocols (10) with all three Miniplex kits (Miniplex 2, Miniplex 4,
and Big Miniplex) (primer sequences listed in Table 2) and the
PowerPlex?16 commercial kit using 5 lL of the sample. Nonacet-
ylated bovine serum albumin (BSA) (Sigma) (0.5 lg) was added to
the reaction mix. Low concentration samples (<25 pg⁄uL) were
concentrated using YM-30 Microcons?(extract was added to the
filter and centrifuged to dryness), collected in a smaller volume
(10 lL) and quantified again. Samples with less than 100 pg total
DNA were amplified with Miniplex 2, samples with 100–550 pg
were amplified with Miniplex 2 and Miniplex 4, and samples with
more than 550 pg were amplified with all three kits. The full vol-
ume of recovered DNA (5–7 lL) was used for amplification. BSA
(0.5 lg) was added to the reaction mix. PCR was performed in the
GeneAmp?PCR System 9700 (Applied Biosystems). The thermal
cycling parameters for the Miniplexes were: 11 min at 94?C soak,
followed by 33 cycles of 95?C for 1 min denaturing, 55?C for
1 min annealing, and 72?C primer extension. The final two soak
steps were at 60?C for 45 min and 25?C forever. Published proto-
cols (15) for Powerplex?16 kits (Powerplex Corporation, Madison,
WI) were used for amplification of the samples analyzed with the
Separation and Analysis
Samples were separated on the ABI Prism?310 genetic analyzer
(Applied Biosystems) using POP-4 polymer and GS ROX?500
(Applied Biosystems) internal standard in a 47 cm 50 lm ID capil-
lary. The samples were injected at 5 kV with 15 kV separation and
a run time of 24 min. The data were analyzed with GeneScan?
and Genotyper?software (Applied Biosystems) using in-house
macros for genotyping.
Results and Discussion
Quantification results for the 82 bp amplicon showed that 30%
of individuals had on average (n = 6–9 hairs per person) less than
100 pg of recoverable DNA per hair, while 41.6% had on average
between 100 and 550 pg of recoverable DNA per hair. Only
28.4% of individuals possessed more than an average of 550 pg of
recoverable DNA (Fig. 1) per shed telogen hair. Quantification of
individual hairs yielded similar results: recovered DNA per hair
ranged from 0 to 9200 pg (n = 510). For all hairs, 33.1% yielded
less than 100 pg of DNA (12.2% of the hairs [62 hairs] yielded no
DNA), 45.6% of hairs yielded 100–550 pg of DNA, and 21.3%
yielded more than 550 pg of DNA. We found no correlation
between the amount of extracted DNA and the age, sex, ancestry,
or hair color of the donor. We did not examine the effect of
TABLE 1—Primer sequences for the three ALU amplicons. All three use
the same forward primer.
82 bp reverse
124 bp reverse
201 bp reverse
TABLE 2—Primer sequences for Miniplex loci.
JOURNAL OF FORENSIC SCIENCES
chemical treatment, which may have some affect on recoverable
DNA. We will be investigating this aspect in the future.
Quantification of the samples which yielded both epithelial and
internal DNA showed varied results. Of the analyzed samples
(n = 20), 45% of the samples contained less DNA on their surface
as they did inside the hair, 25% of the hairs contained more DNA
present on their surface as they did inside the hair, and 30% were
roughly equivalent in DNA concentration between the hairs and
their surface. The small number of hair surface extraction samples
which contained sufficient DNA for amplification (n = 3) were
tested with the Miniplex kits for contamination (Fig. 2). No minor
contributor was evident in any of this small number of samples,
and the STR profile matched the hair donor. Since these samples
were collected in a controlled environment (directly from the
donor), other samples collected in a manner similar to forensic
samples were also tested. Samples from three individuals (n = 9
per person) were collected indirectly (removed from household sur-
faces and placed in sterile containers) and subjected to the same
differential extraction procedure. The surface of the simulated
forensic samples (for which sufficient DNA for amplification was
extracted) also showed no minor contributor for all loci and the
profile matched the donor.
The purpose of this particular study was first to determine if con-
tamination would be present, and second to determine if it would
be advisable to skip the first (‘‘decontaminating’’) extraction step in
order to get more DNA from the hairs. Since there was such a
variation in the amounts found in the epithelial extract, no strong
conclusion could be reached for this question. However, we
recommend using the epithelial extraction step to eliminate the
chance of contamination, particularly since the examiner would
be dealing with low copy DNA.
Degradation of the DNA from the hair was assessed through the
use of real time quantitative PCR (qPCR) and three different size
amplicons (82, 124, and 201 bp). Since the degradation process
breaks the DNA into smaller fragments, the smaller amplicons pro-
duced in real time PCR by one set of primers should be more pre-
valent in the sample than the larger amplicons produced by another
set when compared to nondegraded (control DNA). The control
DNA (9948 cell line) gave consistent quantification results with the
larger two amplicons, while the DNA extracted from telogen hairs
(n = 8) showed a decrease in amplification of the larger size ampli-
cons (Fig. 3). This loss in amplification of larger sized DNA
amplicons can be compared to a similar loss of larger DNA frag-
ments seen when larger quantities of degraded genomic DNA are
separated on a yield gel (8).
Of those individual hair samples which contained sufficient
DNA (greater than 25 pg⁄lL) and were successfully amplified
(peaks detected above the detection threshold of 150 RFUs)
(n = 10), amplification of at least 9 of the 12 Miniplex loci was
observed. The three largest amplicons (FGA, D21S11, D7S820)
most commonly failed to produce amplification. This is likely due
to the effect of degradation (Fig. 4). Amplification of these samples
with the Powerplex?16 kit produced amplification of fewer loci
(2–9 of 12), particularly in the larger sizes, and these results are
consistent with our previous work on degraded DNA from skeletal
remains (11). Because such low concentrations of DNA were pro-
duced from the telogen hair samples, we examined the capability
of the commercial kit to amplify DNA at lower levels, and found
that the concentration of the DNA template did not affect the
amplification success (Fig. 5). This indicates that the problem with
the telogen hair samples is likely an issue with degradation or inhi-
bition and not sensitivity. One must be cognizant that the quantita-
tion is based on the amplification of an 82bp fragment. In the
extreme, if all of the DNAs were in 83bp pieces, the quantitation
would not be affected but no amplification of any STR larger than
82 bp would be possible no matter how much DNA is added.
Amplification was tested on a limited number of low template
samples (n = 30). Prior to these tests, the effect of input quantity of
FIG. 1—Average total DNA in a single hair for the 60 individuals sam-
pled. The errors bars represent two standard deviations from the average
FIG. 2—Amplification of differential extract with the Miniplex 2 kit (D5,
D8, and D16 loci). No discernible minor contributor is present.
FIG. 3—Comparison of quantification of telogen hair extracts using three
different sizes of Alu amplicons: 82 bp, 124 bp, and 201 bp. The reduced
amount of the larger amplicons in the hair samples indicates that the DNA
in hair is degraded.
OPEL ET AL. • NUCLEAR DNA FROM HUMAN TELOGEN HAIR
telogen template hair DNA was tested. A large number of hairs
(n = 35) from a single individual (who had previously yielded large
amounts of DNA from shed hairs) were extracted and the extracts
were combined. The total sample was quantified, and different vol-
umes of the sample corresponding to known amounts of DNA
were tested (n = 3 replicates for each concentration). Miniplex 2
produced full profiles with 120 pg of DNA, and Miniplex 4 pro-
duced full profiles with 150 pg of DNA. Partial profiles (two loci)
for these kits were obtained with 60 pg of DNA. However, with
the Big Miniplex (six loci) kit, only two loci produced results for
all amounts of template tested (30–500 pg). These two loci, TH01
and TPOX, have previously demonstrated lower susceptibility to
PCR inhibition (16), and therefore an inhibition problem was sus-
pected for the tested sample. Two possible methods of relieving
inhibition were tested: addition of BSA and addition of larger
amounts of Taq polymerase. Normally, 0.5 lg of BSA is included
in the amplification mix for noncontrol samples, as this amount has
been shown to remove inhibition for the Miniplex sets (16). A
range of BSA amounts were tested with the control hair sample
(0–5 lg) with no improvement in amplification success. One to
four times the normal amount of Taq (2–8 U) was also tested with
the control sample. One locus (CSF1PO) produced detectable
amplification with 8 U of Taq for some of the samples, but no
improvement was seen in the other three loci (FGA, D21S11, and
D7S820). We are currently testing a variety of methods in further
efforts to reduce this problem.
A limited number of low template samples (n = 30) were re-con-
centrated in smaller volumes and amplified with one or more of
the kits. For these samples, the success of amplification varied. The
samples with less than 100 pg total DNA produced profiles for two
of the three loci in Miniplex 2 in 50% of samples tested (n = 10).
The samples with 100–550 pg of total DNA produced full (six loci
FIG. 5—Amplification of 100 pg of Control DNA with Powerplex?16 kit shows sufficient amplification for typing of all loci. The same amount of DNA
from telogen hairs results in much lower intensity of the larger loci, evidence of degradation and possible inhibition in the DNA sample.
TABLE 3—Relative percent success for each locus for all low
concentration samples. D5S818, D8S1179, and D16S539: n = 30; vWA,
D18S51, and D13S317: n = 20; TH01 – D7S820: n = 10.
FIG. 4—Amplification of hair extract with Miniplex kit (all three kits on
one graph). The larger loci of Big Mini (FGA, D21, and D7, circled) show
much lower intensity than the other loci, evidence of degradation and possi-
ble inhibition in the DNA sample.
JOURNAL OF FORENSIC SCIENCES
Miniplex 2 and Miniplex 4) profiles for 40% of the samples, and Download full-text
at least five loci amplified for 60% of the samples (n = 10). The
samples with greater than 550 pg total DNA produced six loci pro-
files (from Miniplex 2, Miniplex 4, and Big Mini) for 50% of the
samples, but only 30% produced 8–12 loci profiles (n = 10). Some
loci are much more robust, indicated by the percent success for
each locus presented in Table 3.
In our efforts to determine the amount of telogen DNA that can
be extracted from shed telogen hairs of consistent length, we were
able to determine that very low concentrations of DNA can be
extracted, and that the amount extracted varies from person to per-
son. Samples taken of epithelial DNA present on telogen hairs pro-
duced the same type as the hair donor, and the amount also varied
greatly. DNA from telogen hairs shows a high level of degradation,
and this degradation reduced the probability of obtaining a full pro-
file. For samples with recoverable nuclear DNA, the Miniplex kits
with their reduced-size amplicons can provide improved results from
telogen hair over the commercial kit although loss of larger-sized
amplicons is still a problem. Telogen hairs can be an important form
of forensic evidence, both for morphological and DNA studies.
Since the amount of nuclear DNA that can be extracted may be very
low as well as highly degraded, for many samples mtDNA analysis
may provide a better chance of obtaining a profile. However, based
on these results, if sufficient DNA can be recovered from hair
(>60 pg), partial profiles may be produced when using the Miniplex
STR systems. For larger amounts of DNA (>550 pg), more loci can
be obtained; however, full profiles are rarely recovered due to the
extensive degradation that is present. In addition to degradation,
inhibition was found to be a problem for some samples. Additional
BSA and Taq polymerase did not significantly reduce this problem,
which may indicate the source of inhibition in these samples
involvesdamage tothe DNA
Points of view in the document are those of the authors and
do not necessarily represent the official view of the U.S.
Department of Justice. We would like to gratefully acknowledge
Dr. John Butler from the National Institute from Standards and
Technology for his technical support with the Miniplex primer
sets, Wes Garber from Ohio University, Albert Araluce and
Sarah Hughes from Florida International University, and to all
of our volunteer hair donors.
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Additional information and reprint requests:
Bruce R. McCord, Ph.D.
Department of Chemistry and Biochemistry
Florida International University
11200 SW 8th Street
Miami, FL 33199
OPEL ET AL. • NUCLEAR DNA FROM HUMAN TELOGEN HAIR