Analytical Biochemistry 478 (2015) 128130

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Analytical Biochemistry
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Notes & Tips

Comparison of proteases in DNA extraction via quantitative polymerase

chain reaction
Alison M. Eychner, Roberta J. Lebo, Kelly M. Elkins
Department of Chemistry, Towson University, Towson, MD 21252, USA

a r t i c l e i n f o a b s t r a c t

Article history: We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in mul-
Received 26 June 2014 tiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved
Received in revised form 22 August 2014 DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and
Accepted 25 August 2014
immigration, and molecular diagnostic purposes. The Quantiler Kit TaqMan quantitative PCR assay
Available online 4 September 2014
was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax
in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing
Keywords:
gum, and cigarette butts. All methods yielded ampliable DNA from all samples.
Quantitative real-time PCR
DNA extraction
2014 Elsevier Inc. All rights reserved.
QIAamp
Proteinase K
Bromelain
Papain

DNA typing in many eldsbiochemical research, genetic
paternity and immigration, forensic biology, and molecular
diagnosticsrequires the use of extraction methods that efciently
lyse cells from various body uids and recover the highest possible
quantity of DNA suitable for amplication. The rst step in the
DNA typing process is DNA extraction. Along with traditional
methods, such as organic phenolchloroformisoamyl alcohol
DNA extraction, other less time-consuming and safer alternatives
that produce high yields of ampliable DNA are available. Com-
mercially available human DNA extraction kits are faster than tra-
ditional organic and Chelex-100 methods and can be automated.
One of these options is the commercial QIAamp DNA Investigator
Kit (Qiagen) that contains all reagents and consumables, including
a modied proteinase K that is stable at room temperature, for
DNA extraction except ethanol that must be added to one reagent
before use [1,2]. In this study, we compared the modied pro-
teinase K in the Qiagen kit with three other commercially available
proteasesunmodied proteinase K, bromelain, and papainto
determine whether the alternative proteases improve DNA recov-
ery. Proteases aid in the degradation of unwanted proteins and
nucleases that degrade DNA. In a previous study, Li [3] evaluated
clostridiopeptidase and trypsin to develop an improved method
for recovering DNA from bone. We evaluated DNA recovery using
proteinase K, bromelain, and papain due to their success in a study
Corresponding author. Fax: +1 410 704 4265.
E-mail address: kmelkins@towson.edu (K.M. Elkins).
of DNA recovery from ngernail clippings; bromelain exhibited a
1.78-fold higher keratinolytic activity than proteinase K [4]. Both
bromelain and papain outperformed proteinase K [4].
Proteinase K (EC 3.5.21.64), a serine protease enzyme of the
fungus Engyodontium album (Tritirachium album Limber), has a
molecular weight of 18 kDa, has an optimal pH of 7.5 to 9.0 (active
between pH 4.0 and 12.0), and is active between 37 and 60 C
(<65 C) [5,6]. It is one of the most widely studied and used pro-
teases in DNA extraction, is stable in several denaturants, and is
unaffected and stimulated by the denaturation of protein sub-
strates [5,6]. Proteinase K is used for cell and tissue disruption in
combination with a lysis buffer that releases DNA from the nuclei
and mitochondria. Serine proteases share mechanistic features
with the cysteine and threonine proteases [7].
Papain (EC 3.4.22.2) and bromelain (EC 3.4.22.32) are both cys-
teine proteases with broad specicity [4]. They have been studied
for use in biochemistry, pharmacology, and other elds of scientic
research [4]. Both papain, found in the latex of the papaya tree
(Carica papaya), and bromelain, an enzyme found in pineapple
(Ananas comosus), have demonstrated natural activities in protect-
ing plants from invasive herbivorous insects [4,8] and have been
used in DNA extraction methods [4]. In this study, pineapple stem
bromelain (24 kDa), with an optimal pH of 4.5 to 5.5 (active
between pH 4.5 and 9.8) and a temperature range of 40 to 60 C
(<65 C), was employed [4,9]. Bromelain will hydrolyze esters
and amides and will cleave amino acids that are leucine or glycine,
are basic, or exhibit large hydrophobic side chains at the P2

http://dx.doi.org/10.1016/j.ab.2014.08.030
0003-2697/ 2014 Elsevier Inc. All rights reserved.
 Notes & Tips / Anal. Biochem. 478 (2015) 128130
position [10]. The papain (345 aa, 23,406 Da) used in this study is a
member of the peptidase C1 family and has an optimal pH of 6.0 to
7.0 and an optimal temperature of 65 C [10]. Papain destroys anti-
gens on the surface of red blood cells [10]. It is water soluble at
10 mg/ml and stable in the presence of some denaturants [10].
In this study, we compared the effects of substituting different
proteases, including the modied kit proteinase K, unmodied pro-
teinase K, bromelain, and papain, on the extraction of human DNA
from human blood, semen, buccal cells, breastmilk, and earwax
and low-template samples, including diluted samples of blood,
semen, and saliva; computer keyboard swabs; chewing gum; and
cigarette butts. Blood, semen, and saliva are commonly encoun-
tered in forensic casework and used as DNA sources in medical
studies [11]. Because DNA has been recovered from feces and urine
in previous research, we evaluated DNA recovery from cells in
breastmilk and earwax [1114]. Nuclear DNA is located in white
blood cells in blood samples, whereas the epithelial cells found
within semen, saliva, breastmilk, earwax, and ngerprint samples
provide the primary source of DNA in those samples [1114]. With
the exception of the kit proteinase K substituted with the other
proteases, all other buffers, reagents, and procedures were
employed and performed according to the manufacturers protocol.
Simulated forensic samples included 50-ll samples of blood,
semen, or breastmilk pipetted on DNA-free cotton-tipped PurS-
wabs (Puritan, Guilford, ME, USA) in addition to buccal cells and
earwax individually recovered on a PurSwab. Semen was pur-
chased from Lee Biosolutions (St. Louis, MO, USA). Samples were
allowed to dry before use. Diluted samples were prepared as
1:10 and 1:100 dilutions of blood, semen, or saliva, with 50 ll of
each dilution being pipetted onto the cotton swabs. In addition,
low-template simulated forensic evidence samples included swabs
of computer keyboards, chewing gum, and cigarette butts using
moistened swabs. Various keyboards in Smith Hall at Towson
University were sampled using swabs with 50 ll of nuclease-free
water prior to use. Focus was placed on the spacebar, enter key,
backspace key, letters, and numbers. Control gum samples were
provided by anonymous adult donors and processed whole. The
gum was chewed for 30 min at least a half-hour after food or drink
was consumed. Environmental chewing gum and cigarette butts
were collected from the grounds at Towson University and were
processed whole. All samples were performed in triplicate with a
negative control for each set (n = 224 samples). Samples were
obtained from three healthy anonymous donors as approved by
Towson Universitys institutional review board (13-0X22 and
14-X097).
DNA extraction was performed using the QIAamp DNA
Investigator Kit (Qiagen, Valencia, CA, USA) [1] as directed by the
manufacturer with the following modications: kit proteinase K
was used as directed or substituted with lyophilized unmodied
proteinase K (Amresco, Solon, OH, USA), papain liquid
(1640 U/mg protein suspension in 0.05 M sodium acetate, pH
4.5, containing 0.01% thymol, SigmaAldrich, St. Louis, MO, USA),
or lyophilized bromelain (SigmaAldrich). The lyophilized powders
were used to prepare 10-mg/ml stock solutions in nuclease-free
water. Proteinase K was used within 24 h, and the bromelain and
papain were used within 2 h of preparation. In step 10b, 500 ll
of lysate was initially transferred to the spin column and cen-
trifuged. The swab was placed in a spin basket and centrifuged
separately to release trapped lysate. The remaining lysate was
transferred to the spin column and centrifuged again to increase
DNA yield. In step 16, 20 ll of DNA was eluted with nuclease-free
water. Samples were frozen immediately after step 17 of the pro-
cedure. All DNA extractions were performed in a PCR Workstation
using the sterile technique. Negative control sample swabs were
subjected to the DNA extraction protocol and the same quantita-
tion procedure.
129
The quantity of ampliable DNA was determined using the
Quantiler TaqMan assay (Applied Biosystems, Carlsbad, CA,
USA) according to the manufacturers protocol on an ABI Prism
7000 real-time PCR instrument. DNA yields were calculated using
a standard curve created from a dilution series using standard
DNA provided in the kit. The KruskalWallis (H) test was employed
in statistical tests.
PurSwabs are individually packaged, sterile, DNA-free cotton
swabs. Cotton swabs were chosen because many law enforcement
agencies throughout the United States currently use them to col-
lect DNA evidence at crime scenes. Studies suggest that the com-
bination of cotton swab with a spin column extraction method,
such as the QIAamp DNA Investigator Kit used in this research, pro-
duces effective results [15]. The reliability of column-based extrac-
tions and commercial buffers allows for more consistent
preparation of samples for analysis as compared with other meth-
ods. All of the swabs were of the same type and expected to have
the same sample retention rate.
Quantitation of the standards using Quantiler yielded a best t
line with slope of 3.132427 and an R2 value of 0.996477. The
Quantiler quantitation results indicate that all proteases can be
successfully implemented in the kit for DNA extraction from blood,
semen, buccal cells, breastmilk, and earwax (Supplementary
Table 1). For blood, both papain and bromelain gave higher DNA
yields than either proteinase K reagent; extractions using papain
yielded the highest quantity of DNA. On average, 59.1, 40.2, 32.4,
and 27.8 ng of DNA was recovered using papain, bromelain,
unmodied proteinase K, and the kit proteinase K, respectively.
For semen, the proteinase K in the QIAamp kit gave the best DNA
yields. For semen samples, an average of 74.7 ng of DNA was
yielded when extracted with the kit proteinase K, 44.6 ng when
extracted with unmodied proteinase K, 50.6 ng when extracted
with bromelain, and 42.9 ng when extracted with papain. For buc-
cal swabs, both proteinase K reagents gave higher DNA yields than
either of the other proteases. Buccal cell samples extracted with
unmodied proteinase K yielded an average of 56.6 ng of DNA,
60.8 ng with the kit proteinase K, 29.3 ng with bromelain, and
43.9 ng with papain. For breastmilk, the unmodied proteinase K
gave higher DNA yields than the other proteases, although all
results were similar. The unmodied proteinase K yielded an aver-
age of 0.869 ng of DNA from breastmilk samples, the kit proteinase
K yielded an average of 1.67 ng, bromelain yielded an average of
1.09 ng, and papain yielded an average of 1.20 ng. For earwax,
papain gave higher DNA yields than the other proteases, although
all results were similar. An average DNA yield of 0.690 ng was
observed when the earwax samples were extracted with the
unmodied proteinase K, 0.413 ng when extracted with the kit
proteinase K, 0.388 ng when extracted with bromelain, and
0.848 ng when extracted with papain.
DNA yields were quantied for diluted body uid samples of
blood (Supplementary Fig. 1A), semen (Supplementary Fig. 1B),
and saliva (Supplementary Fig. 1C). The average DNA yields for
the 1:10 dilution of blood samples were 1.903, 7.787, 1.079, and
3.477 ng/ll for the kit proteinase K, unmodied proteinase K,
bromelain, and papain, respectively. The blood samples diluted to
1:100 yielded an average of 0.361 ng/ll when extracted with the
kit proteinase K, 0.830 ng/ll when extracted with the unmodied
proteinase K, 0.155 ng/ll when extracted with bromelain, and
0.209 ng/ll when extracted with papain. The average DNA yields
for the 1:10 dilution of semen samples were 6.700, 9.607, 7.530,
and 8.657 ng/ll for the kit proteinase K, unmodied proteinase
K, bromelain, and papain, respectively. Semen samples diluted to
1:100 yielded an average of 0.353 ng/ll when extracted with the
kit proteinase K, 0.571 ng/ll when extracted with the unmodied
proteinase K, 0.149 ng/ll when extracted with bromelain, and
1.281 ng/ll when extracted with papain. The average DNA yields
130 Notes & Tips / Anal. Biochem. 478 (2015) 128130
for the 1:10 dilution of saliva samples were 0.793, 0.601, 0.357, and
0.095 ng/ll for the kit proteinase K, unmodied proteinase K,
bromelain, and papain, respectively. The saliva samples diluted
to 1:100 yielded an average of 0.022 ng/ll when extracted with
the kit proteinase K, 0.007 ng/ll when extracted with the unmodi-
ed proteinase K, 0.018 ng/ll when extracted with bromelain, and
0.009 ng/ll when extracted with papain.
Quantitation results for the keyboard ngerprint swabs, chew-
ing gum, and cigarette butt low-template samples (Supplementary
Figs. 1DG) indicate that all proteases could be implemented into
the kit for DNA extraction. However, the best yields were observed
using bromelain for extracting DNA from control chewing gum
(Supplementary Fig. 1D), papain for extracting DNA from nger-
prints on computer keyboards (Supplementary Fig. 1F) and
environmental chewing gum (Supplementary Fig. 1E), and the kit
proteinase K in extracting DNA from cigarette butts (Supplemen-
tary Fig. 1G). On average, control gum swabs extracted with the
kit proteinase K yielded 0.996 ng/ll DNA, 1.382 ng/ll DNA was
recovered when extracted with the unmodied proteinase K,
1.430 ng/ll DNA was extracted using bromelain, and 1.200 ng/ll
DNA was extracted using papain. On average, the environmental
gum swabs yielded 3.187, 2.470, 3.020, and 15.697 ng/ll for the
kit proteinase K, unmodied proteinase K, bromelain, and papain,
respectively. An average of 0.149, 0.191, 0.185, and 0.220 ng/ll
DNA was recovered when extracted with the kit proteinase K,
unmodied proteinase K, bromelain, and papain, respectively, for
the keyboard swabs from heavily used keyboards in the Depart-
ment of Chemistry computer labs at Towson University. The cigar-
ette butt swabs yielded an average of 4.286, 1.300, 0.469, and
0.135 ng/ll DNA for the kit proteinase K, unmodied proteinase
K, bromelain, and papain, respectively. Several variables likely
played a role in the relatively high standard deviations observed,
including human shedder variation and variation in deposited
DNA on the surfaces [16]. The environmental chewing gum, cigar-
ette butt, and keyboard samples were discarded samples collected
from unknown donors. Because only 1 ng is the optimal quantity
required for forensic STR (short tandem repeats) DNA typing with
commercial kits and many other applications, the DNA yields
recovered from all samples is more than sufcient for this applica-
tion in the 20-ll elution volume. The lower limit of recovery of at
least 1 ng of DNA is estimated to be a 1:10 dilution for saliva, 1:100
dilution for all proteases (and 1:1000 with proteinase K) with
blood, and 1:100 dilution for all proteases (and 1:1000 with pro-
teinase K and papain) with semen. The chewing gum and cigarette
butts yielded sufcient DNA to support partitioning in three (or
more) parts, and the yield from the keyboard supported sampling
only a single area if necessary.
Blood, semen, and buccal cells (or saliva) are commonly found
on submitted forensic evidence samples, and buccal cell swabs
and blood are common sources of biological material for reference
samples in investigations. Although earwax and breastmilk are less
likely to be recovered as forensic evidence, the DNA yields are use-
ful for estimating DNA recovery from those biological materials.
Papain yielded the highest DNA quantity for blood and earwax,
the kit proteinase K included yielded the highest DNA quantity
for semen, and the unmodied proteinase K yielded the highest
DNA quantity for saliva and breastmilk. The papain extractions
were the most consistent overall in terms of lowest overall
standard deviation. Because STR DNA typing is reproducible with
as little as 50 pg of template DNA, sufcient DNA can be extracted
from both sources for DNA typing even in duplicate or triplicate.
Future studies may examine DNA recovery from feces, urine, and
tears using both the aforementioned and additional proteases.
Variation of DNA yield as a result of substituting proteases also
may be studied in other methods to maximize DNA yield.
Acknowledgments
The authors gratefully acknowledge the Department of Chem-
istry at Towson University for funding. The authors disclose that
they have no commercial conicts of interest and that all reagents
were purchased from the stated manufacturers and suppliers. The
funding source had no involvement in the study design; in the col-
lection, analysis, and interpretation of data; in the writing of the
article; or in the decision to submit the article for publication.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ab.2014.08.030.
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