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884 volume 31 NumBeR 10 oCToBeR 2013 nature biotechnology
Kevin J. McKernan, Jessica Spangler,
Yvonne Helbert, Lei Zhang and Vasisht
Tadigotla are at Courtagen Life Sciences,
Woburn, Massachusetts, USA.
e-mail: kevin.mckernan@courtagen.com
informing investors on the company’s free-
dom to operate (FTO) are often the gating
item. FTO documents require skilled patent
attorneys to review the relevant claim language
of a set of patents with respect to the meth-
ods that an entrepreneurial company plans
to employ. The cost of an FTO letter written
for a single patent can exceed $20,000. This
is a tractable problem with less than a dozen
patents. When the list balloons to hundreds of
patents, the cost to survey this legal complexity
quickly consumes an unacceptable percentage
of a company’s available funds. The courts’
prolonged ambiguity on the eligibility of gene
patents further underscores the questionable
utility of such legal efforts. Of equal concern,
the thorough report by the Secretary’s Advisory
Committee on Genetics, Health, and Society
demonstrates the challenges in measuring any
positive economic impact provided by gene
patents10. This 392-page review on the eco-
nomics of gene patents states, “[P]atents on
genetic discoveries do not appear to be nec-
essary for either basic genetic research or the
development of available genetic tests.”
Natural versus human-made DNA
Eric Lander’s amicus brief to the Supreme
Court correctly highlights the “demonstrably
incorrect” assumption that fragments of DNA
are exclusively human made11. One example
showcases the evidence of such fragments in
noninvasive prenatal testing techniques12.
The amicus brief implies that only human-
manipulated or synthesized DNA is patent eli-
gible. It also provides the example of cDNAs
as a legitimate human-made DNA molecule,
and the Supreme Court has now affirmed this
position.
If cDNAs are human made, PCR amplifi-
cation of gene panels and exomes would also
constitute human-manipulated isolation of the
DREAMing of a patent-free human genome
for clinical sequencing
Kevin J McKernan, Jessica Spangler, Yvonne Helbert, Lei Zhang & Vasisht Tadigotla
Can methylation be the key to challenging the interpretation of existing gene patent claims? And can a novel PCR
method be used to enable the sequencing of hundreds of genes?
The case for the perils of gene patents and
the negative impact of a profit motive in
scientific endeavors has been made1. Often,
gene patent discussions will conflate economic
and ethical concerns while failing to properly
define the scope of property or the influence of
profit motivations. In an attempt to untangle
these two topics, we review the impact of meth-
ylation on the scope of gene patent property
rights and suggest a simple PCR strategy that
may challenge the interpretation of many pat-
ent claims still in force after the US Supreme
Court’s decision in Association for Molecular
Pathology v. Myriad Genetics2. We also offer
an alternative economic perspective on profit
motivation and its impact on the ethics of gene
patents.
Despite the ruling in Myriad on the patent
ineligibility of natural DNA, cDNAs remain
patentable. In his majority opinion, Justice
Clarence Thomas made the distinction that
although laboratory modification of the DNA
is sufficient as patent subject matter accord-
ing to Section 101 of the Patent Act, that alone
does not guarantee patent eligibility. Because
the modifications required for cDNA isolation
are very similar to those required for making
sequencing libraries, the exact number of
gene patents in force and the scope of their
claims remain the subjects of heated debate3.
Although some convincingly argue that
whole-genome shotgun and single-molecule
sequencers will traverse some of these issues4,
others are less content with the practicality of
this position and rightly point out the lack of
specificity of 15-mer claims currently in
force with the Myriad cDNA-based patents5.
Rosenfeld and Mason’s work5 highlights both
the fact that Myriad is not the only gene patent
to make such broad claims in regard to short
sequences, and that such patents can lay claim
to over 600 other human genes, including
genes from other species.
Does the patent system foster
entrepreneurship?
Graff et al. have made note of the rate of gene
patent abandonment as a sign of potential
freedom to operate in the clinical sequencing
market6. However, for a clinical sequencing
laboratory interested in sequencing diverse
gene panels or entire exomes, the debate over
the existence of 40,000 or 4,000 gene patents
is a bit off target, as even 400 human gene pat-
ents create insurmountable legal costs for most
entrepreneurial enterprises seeking to make
use of the $5,000 genome. To realize the dreams
of personalized or precision medicine, we must
be able to freely read the human genome with-
out paying tolls at thousands of intersections.
This was certainly the intent of the Human
Genome Project and the Bermuda Accord.
The next-generation “sequencing explosion”
has positioned us a decade later for a seismic
shift in translational medicine7. Gene patents
critically obstruct this process and also actively
inhibit solutions that may drastically alter the
health of many patients.
Disruptive technologies like next-generation
sequencing are often first leveraged by start-
ups8. For example, Cambridge, Massachusetts–
based Foundation Medicine offers a test
sequencing hundreds of genes related to can-
cer9, compared to Myriad Genetics’ simple
two-gene sequencing test (for nearly twice the
price). For startups to be funded in the clini-
cal sequencing market, legal opinion letters
paTenTs
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nature biotechnology volume 31 NumBeR 10 oCToBeR 2013 885
Analysis
To further illustrate this point, we demon-
strate a simple PCR process capable of incor-
porating more than the four nucleotides
found to represent most sequence IDs in the
US Patent and Trademark Office. We coined
the term DREAM PCR (decontamination-
ready encoded amplification) to describe a
PCR method that utilizes additional methyl-
ated nucleotides (5me-dCTP) to generate an
amplicon set that is susceptible to methyl-
specific enzymatic digestion. This enzymatic
digestion is analogous to the decontamination
afforded with the use of dUTP in clinical PCR
protocols described in US Patent 5,536,649
(ref. 20). Clinical sequencing laboratories often
segregate pre- and post-PCR processes to avoid
such contamination or alternatively use PCR
products that are digestable. Unlike the pre-
PCR digestion of uracil by UDG, EndoVIII
and PNK, a single methyl-specific enzyme
MspJI can be used to digest background 5me-
dCTP–generated amplicons21. Uracil-based
PCR decontamination methods also suffer
from the fact that most DNA sequencers on
the market today utilize uracil-illiterate poly-
merases to perform emulsion or bridge PCR,
thus making uracilated templates challenging
to amplify and sequence. This has proven to be
challenging for bisulfite sequencing22.
Sequencing methylated DNA. Briefly, we
demonstrate DREAM PCR using the Agilent
Haloplex v2 target enrichment PCR process
to sequence 327 nuclear genes in four human
control samples with varying (0.05–0.2 mM)
molarities of 5me-dCTP added to the PCR
cocktail23,24. One will note that this experi-
ment relies on previously published findings
and off-the-shelf commercial kits. The only
modification is the previously published use
of 5me-dCTP nucleotides in now highly mul-
tiplexed PCR that generates DNA fragments
very similar to unamplified, natively methyl-
ated DNA libraries25. Figure 1 demonstrates
amplification of a Haloplex library with and
without 5me-dCTP (blue and red trace, respec-
tively). Subtle mobility shifts are observed with
this methylated amplification and only the
5me-dCTP library is digestible with 30-min
exposure to 5 units of MspJI (green trace).
Sequencing these Haloplex libraries on an
Illumina MiSeq demonstrates similar but not
identical results (Fig. 2). Subtle coverage dif-
ferences appear, but are less important than
the differences observed using two different
read alignment algorithms26,27. Using GATK,
the control sample discovered 427 unfiltered
variants whereas the 50 mM 5me-dCTP sam-
ple discovered 418 where 99.5% (416/418)
of the overlapping variants were in agree-
advantage of the ambiguity in the wobble
bases of the genetic code by making claims to
any DNA sequence that ‘encodes’ a polypep-
tide sequence. The claimed sequence degen-
eracy thus covers many more off-target DNA
sequences that would not be treated equally by
the cell during transcription and translation.
Successful transcriptional activity is required
for these processes to initiate and transcrip-
tion is not blind to methylation. Thus, meth-
ylation of those sequence IDs exposes claim
sets using encoding language to the fact that
methylated DNA silences transcription16–18.
Cantara also demonstrates an alternative
interpretation of the genetic code induced by
cytidine methylation in tRNAs19, underscor-
ing the lack of specificity in most gene patents
filed before next-generation sequencing and
revealing the many marvels of epigenetics.
claimed DNA sequence. This isolation through
amplification significantly alters the DNA by
failing to faithfully replicate the molecular
epigenetic nature of natural DNA. Myriad has
made the case that isolation of DNA from the
chromatin is analogous to the purification of
adrenalin, which was the subject of the land-
mark case involving Parke-Davis13. Removal
of proteins, chromatin and other biologics
provides utility in reading the DNA’s sequence,
and, therefore, Myriad states that their isola-
tion of DNA qualifies as human manipulation.
Whether or not this argument is valid, it does
not reflect the reality that other epigenetic
modifications to the DNA remain in the form
of methylated nucleotides, and most gene pat-
ents fail to specify methylated sequence IDs. As
a result, the impact of methylation on sequence
function and utility is of paramount impor-
tance in this discussion.
Natural DNA is methylated. Any composition-
of-matter claim to a sequence ID would have
to pass a triple identity test: does methylated
sequence perform substantially the same
function, in the same way, to yield the same
result, as nonmethylated sequence? As the
evidence clearly demonstrates that various
restriction enzymes, cellular processes and
antibodies specifically discern methylated
bases from nonmethylated bases, this argu-
ment of equivalence is becoming harder if
not impossible to make14,15. Modern DNA
sequencers are now capable of differentially
sensing methylated bases; similarly, any oli-
gonucleotide synthesis facility attempting to
replicate such claimed DNA sequence would
require the methyl status of the nucleotides
to be specified. Many gene patents even take
Figure 1 Agilent High Sensitivity chip of Haloplex assays after 15 cycles of PCR with (blue) and
without (red) 5me-dCTP used in the PCR. Green line is the MspJI digestion of the libraries with
5 units of MspJI (New England BioLabs) for 30 min at 37 C.
Figure 2 Illumina MiSeq V2. 2 × 250 bp reads
mapped to hg19 using Bowtie2. Coverage
calculated across the 327 genes related to
epilepsy targets using BEDtools.
Coverage performance for 327 epilepsy genes
% of regions covered
Regions covered at least this many times
at 300X mean coverage
C1 C10 C20 C100
200 μM dCTP CTRL
100
90
80
70
60
50
40
30
20
10
0
150 μM dCTP & 50 μM 5me-dCTP
100 μM dCTP & 100 μM 5me-dCTP
200 μM dCTP & 200 μM 5me-dCTP
PATENTS
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886 volume 31 NumBeR 10 oCToBeR 2013 nature biotechnology
test an economic law: in any noncoerced
economic transaction, both parties profit
in that exchange or the exchange does not
occur. Companies that make a profit do
so only if their customers also desire their
product more than what they exchange it
for. Modern use of currency often obscures
the profit on both sides of every transaction.
Therefore profit is actually a measure of win-
win transactions in the marketplace. Gene
patent–granted monopolies introduce a form
of coercion in these transactions.
Organizations relying on government
funding also leverage coerced funding
through taxation and cannot claim to be
funded through win-win transactions. For
example, in the United States, taxes are mostly
non-negotiable, as the alternatives to paying
them are relocation or prison. Although a
citizen may not always agree with the science
or war funded by his or her government, that
citizen cannot fail to pay taxes or itemize the
use of tax funds. This funding model tends
to generate misuse of resources as there is no
negotiated equilibrium or price mechanism
for goods or services governments offer37.
Despite these economic distinctions, there
are still nonprofit, tax-funded facilities like
the National Institutes of Health that are
among the top ten assignees of ‘in-force’ gene
patent estates6,38. These taxpayer-funded
inventions are then licensed to companies
who defray these licensing costs onto the
patient or insurance company in the form of
a clinical test. The taxpayer is thus paying for
these efforts twice and, in both cases, through
coerced pricing mechanisms. This conflict
CHH (where H is A, T or C) methylation pat-
terns seen in mammalian cells but also intro-
duces non-CpG methylation. The 5me-dCTP
PCR products are even more distant from
the claimed four-nucleotide sequences than
the natively methylated versions of the gene
not described in most gene patents (Fig. 4).
The increasing desire to detect rare alleles in
heteroplasmic or heterogeneous tissues also
demands a PCR decontamination process be
considered34. Thus, the utility of methylated
PCR extends beyond patent claim consider-
ations and may in fact provide a very valuable
clinical PCR decontamination tool.
In summary, methylated nucleotides chal-
lenge the interpretation of composition-
of-matter claims and encoding claims. As
methylated cytosine also alters the melting
temperature of amplicons35, other nonca-
nonical nucleotides may also pose a concern
for other patent claim categories described by
Holman et al.4.
Economics and discussion
With the scope of property rights for some
gene patents challenged by methylation, we
turn our attention to the economic consider-
ations raised by Salzberg1. Not only do gene
patents fail to reflect the reality of nature,
they also fail to reflect the realities of the
marketplace. To address the marketplace
concerns over the negative impact of a profit
motive, we suggest that an Austrian School
of Economics perspective on gene patents be
considered36.
The denouncement of profit motivation
as negatively affecting science may fail to
ment. The two additional variants from the
5me-dCTP sample and 11 additional control
variants were all under called heterozygote
variants where evidence of the alternative allele
exists28. This is perhaps not a surprising result
as methylated nonamplified genomic DNA
libraries have previously been successfully
sequenced with both bridge PCR– and emul-
sion PCR–based sequencing methods29–31.
Additionally, 5me-dCTP has been used in
PCR previously to search for methyl-sensitive
restriction enzymes25. Only recently have methyl-
specific restriction enzymes become available32
to enable decontamination techniques.
As implied by Horton et al.21, a simple
30-min digestion with MspJI of DREAM
PCR libraries shows a 2,000-fold reduction
in amplifiable signal with SYBR green–based
qPCR (Fig. 3). This enables the use of MspJI
to decontaminate PCR reagents of background
DREAM PCR libraries before initiating ampli-
fication of a new patient of interest. The enzy-
matic method we propose provides additional
insurances for clinical sequencing laboratories.
This methylated amplification technique is
very likely to work with any gene enrichment
technique, and is becoming more impor-
tant as the courts are emphasizing a distinc-
tion between human-made ‘synthetic DNA’
and free circulating DNA fragments in the
bloodstream. Methylation is the primary dif-
ference between these two classes of nucleic
acids, as seen in US Patent 8,288,100 (ref. 33).
Amplification products that better reflect the
natural epigenetic code are less likely to be
held to existing patent claims. The technique
described here incorporates CpG, CHG and
Figure 3 Plot of a quantitative PCR assay
run on various DREAM PCR libraries and
controls. Digested DREAM (methylated, blue)
libraries demonstrate significant shifts in
delta Ct compared to digested control libraries
(nonmethylated, green). At 0.3 units of MspJI
with 10 ng of total library, a 2,000-fold reduction
in amplifiable signal is observed with a 30-min,
37 °C digestion. A 65 °C 20-min heat-kill is used
before initiation of qPCR with Kapa Biosciences
Library qPCR kit.
Figure 4 Depiction of natural DNA, conventional four-nucleotide PCR DNA and DREAM PCR DNA.
CpG, CHG and CHH methylation patterns exist in natural DNA and do not exist in conventional PCR
products described in most gene patents. DREAM PCR methylates at CpN sites and thus includes
CpG, CHG and CHH methylation but also contains other methylation patterns not present in natural
DNA. Methylated cytosines are also known to shift the melting temperature of an amplicon 0.5 °C for
every methylated cytosine. (Left-hand side of figure reprinted from Nature 441, 143–145, 2006, with
permission from Macmillan Publishers.)
4-bp PCR DNA (no methylation)
The two main components
of epigenetic code
DNA methylation
Methyl marks added
to certain DNA bases
repress gene activity.
Histone modification
A combination of different
molecules can attach
to the ‘tails’ of proteins
called histones.
These alter the activity
of the DNA wrapped
around them.
Not patentable
Patentable
Not patentable
DREAM PCR DNA (partial CpN)
Natural DNA (CpG, CHG, CHH)
Histones
Histones
Chromosomes
PATENTS
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nature biotechnology volume 31 NumBeR 10 oCToBeR 2013 887
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests:
details are available in the online version of the paper
(doi/10.1038/nbt.2703).
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In conclusion, we disagree with Salzberg’s
assumption that profit motive is counterpro-
ductive to scientific progress, and we further
suggest that gene patents which fail to include
methylated nucleotides should not be granted
ownership over their methylated sequence
IDs. In our opinion, gene patents are immoral
not because of a profit motive but because an
armed authority ultimately enforces them to
protect a piece of imagined property or an
idea. Property is usually defined as some-
thing that consists of matter and is not infi-
nitely replicable (i.e., exhibits scarcity). Ideas
are neither of these. They are not stolen when
shared. DNA has quite elegantly shown the
benefits of sharing ideas rapidly, imperfectly
and without any regard to patent protection
or copyright. If there is one lesson we can
all learn from the code of life, it is to share it
quickly and peacefully while celebrating the
beneficial mutations in the process. Gene pat-
ents fail this test in every respect, and it may
require our respect for the epigenome to fully
appreciate this fact.
ACKNOWLEDGMENTS
We would like to thank C. Gunter, T. Harkins,
S. Grimmond and R. McKernan for valuable editorial
advice. We would like to thank E. Dimalantis for
constructive discussions regarding MspJI.
PATENTS
npg © 2013 Nature America, Inc. All rights reserved.