BACKGROUND
[0001] This application relates generally to collection and amplification of
circulating nucleic acids (CNAs) from a biological sample. More particularly, the
application relates to separation, collection, amplification and further detection of
circulating nucleic acids from the biological sample.
[0002] Circulating nucleic acids are released from a variety of tissues and are
accumulated in bodily fluids. A variety of intact and/or fragmented nucleic acids
have been identified in CNAs, including mRNA, miRNA, mitochondrial DNA,
genomic DNA, and retrotransposons. CNAs are ideally suited for early detection of
diseases as well as prognostic and theranostic applications. The diagnostic potential
of CNAs has been demonstrated over a wide spectrum of diseases, including
tumorigenesis, inflammation, myocardial infarction, autoimmune disorders and
pregnancy-associated complications.
[0003] Circulating nucleic acids may be detected using minimaly invasive
methods that sample bodily fluids. However, CNAs are present in very low
abundance in the bodily fluids. Hence, analysis of CNAs generally often requires
collection and processing of large volumes (milliliters or liters) of bodily fluids.
However, many times, only very small amounts of bodily fluid sample (microliters)
may be available for analysis, especially in the fields of in vitro diagnostics,
pathology, and forensics. Moreover, large-volume sample collection often leads to
significant set-up costs, transportation/handling costs and sample artifacts.
Additionally, since CNAs are present outside of cells in bodily fluids, this circulating
pool of nucleic acids can be gradually swamped out by intra-cellular DNAs or RNAs
that are released through lysis of resident cells in bodily fluids. This swamping out or
contamination may be a multi-parameter function of time, temperature, type of
treatment for stabilization, and separation forces used for isolation of bodily fluids.
These pre-analytical variables can produce undesirable genomic contamination from
the resident cells that are present in the bodily fluid. For example, in whole blood
samples, DNAs or RNAs may be released into plasma or serum from blood cells
during storage and processing. This may interfere with the analysis of extra-cellular,
circulating nucleic acids that are present in the plasma or serum. Genomic
contamination of circulating nucleic acid pools may be reduced by maintaining the
blood sample at 4°C and processing the sample within 2 hours. However, such
conditions are often not feasible and/or cost-effective for many applications.
[0004] Whole-genome amplification may be used expand the natural pool of
circulating nucleic acids. However, prior attempts at whole-genome amplification of
CNAs using multiple displacement amplification (MDA) techniques have highlighted
unique challenges that are associated with the poor quality and low quantity of CNAs
in the bodily fluids. Generally, by nature, CNAs are highly fragmented due to their
origion from apoptotic/necrotic cells. The nucleic acid fragmentation pattern of
CNAs is not ideal for conventional whole-genome amplification and thus leads to
allelic drop-out and/or sequence-biased amplification patterns. Additionally, many of
the conventional whole-genome amplification techniques require nanogram quantities
of input nucleic acids. Hence, CNAs must be purified from large volumes of noncellular
fraction to meet these template concentration demands. In view of the above,
there is a critical need for technologies that streamline the separation, collection,
stabilization and/or amplification of circulating nucleic acids from a biological
sample, particularly when analyzing small sample volumes containing picogram
quantities of CNAs.
BRIEF DESCRIPTION OF THE INVENTION
[0005] The present invention is directed at collection and subsequent
amplification of CNAs from a biological sample.
[0006] One aspect of the invention relates to a method for amplification of
circulating nucleic acids that are present in a non-cellular fraction of a biological
sample. The method includes the steps of filtering the biological sample to separate
the non-cellular fraction from intact cells, collecting the separated, non-cellular
fraction onto a dry solid matrix, and extracting CNAs from the collected, non-cellular
fraction. The method further includes the steps of circularizing the extracted CNAs to
form single-stranded nucleic acid circles, and amplifying the single-stranded nucleic
acid circles by random-primed rolling circular amplification to form an amplified,
CNA product. If CNAs are in double-stranded form, the method also includes the
step of denaturing the double-stranded CNAs to a single-stranded form prior to the
intra-molecular ligation reaction for making single-stranded nucleic acid circles. The
circularization of linear single-stranded CNAs may be achieved by a ligase that is
capable of intra-molecular ligation of single-stranded nucleic acids.
[0007] Another aspect of the invention relates to a method for processing
whole blood at a point-of-collection to collect plasma or serum that contains
circulating nucleic acids. The method includes the steps of filtering the whole blood
to separate the plasma or serum from the whole blood at the point-of-collection,
collecting the separated plasma or serum on to a dry solid matrix and drying the
collected plasma on to the solid matrix. The solid matrix is devoid of any detergent.
[0008] Another aspect of the invention relates to a method for detection of
CNAs from a dried sample of plasma or serum. The method includes steps of
extracting the CNAs from the dried plasma or serum, performing a whole genome
amplification of the extracted circulating nucleic acids to form an amplified,
circulating nucleic acid (CNA) product, and detecting a specific circulating nucleic
acid sequence in the amplified, CNA product. The whole genome amplification is
performed by first circularizing the extracted CNAs using a ligase that is capable of
intra-molecular ligation of single-stranded nucleic acids to form single-stranded
nucleic acid circles, and amplifying the single-stranded nucleic acid circles by rolling
circular amplification employing random primers. If the CNAs are in double-stranded
forms, the method also includes the step of denaturing the double-stranded CNAs to
its single-stranded form prior to the intra-molecular ligation reaction.
[0009] Another aspect of the invention relates to a device for collecting a noncellular
fraction of a biological sample that comprises circulating nucleic acids. The
device comprises a filtration membrane configured to separate the non-cellular
fraction of the biological sample from intact cells, and a dry solid matrix configured
to collect the separated, non-cellular fraction. The filtration membrane and the solid
matrix are configured to establish a direct contact between them. Further, the solid
matrix is devoid of any detergent.
DRAWINGS
[0010] These and other features, aspects, and advantages of the described
invention will become better understood when the following detailed description is
read with reference to the accompanying drawings in which like characters represent
like parts throughout the drawings, wherein:
[0011] FIG. 1 depicts a flow diagram illustrating an embodiment of the
method of the invention.
[0012] FIG. 2 depicts a schematic representation of a lateral flow device for
the separation and collection of non-cellular fraction of a biological sample.
[0013] FIG. 3 depicts a schematic representation of an embodiment for
making the lateral flow device for the separation and collection of non-cellular
fraction of a biological sample.
[0014] FIG. 4 depicts a schematic representation of a vertical flow device for
the separation and collection of non-cellular fraction of a biological sample.
[0015] FIG. 5 depicts plasma DNA collection on to a dry solid matrix
following lateral or vertical separation of human whole blood.
[0016] FIG. 6 depicts ligase-assisted whole genome amplification that enables
detection of four different chromosomal loci from plasma DNA (i.e., circulating DNA
extracted from plasma) separated from whole blood by lateral or vertical flow.
[0017] FIG. 7 illustrates a schematic representation of an embodiment of a
ligase-assisted whole-genome amplification of a linear double stranded DNA.
[0018] FIG. 8 illustrates the size profiles of circulating DNA isolated from
blood plasma of healthy individuals.
[0019] FIG. 9A illustrates a ligase-assisted whole-genome amplification of
circulating DNA extracted from the non-cellular fraction of whole blood, using
CircLigase™ II.
[0020] FIG. 9B illustrates a ligase-assisted whole-genome amplification of
circulating DNA extracted from the non-cellular fraction of whole blood, using T4
DNA ligase.
[0021] FIG. 9C illustrates a ligase-assisted whole-genome amplification of
circulating DNA extracted from the non-cellular fraction of whole blood, using E.
Coli ligase.
[0022] FIG. 10 illustrates the effectiveness of ligase-assisted whole-genome
amplification for sensitive and balanced DNA amplification of four different CODIS
loci.
[0023] FIG. 11 illustrates the effectiveness of ligase-assisted whole-genome
amplification for sensitive and balanced DNA amplification of twelve different
CODIS loci.
[0024] FIG. 12 illustrates the varying efficiencies of ligase-assisted wholegenome
amplification in different reaction and buffer conditions.
[0025] FIG. 13 illustrates the inhibition of amplification of high molecular
weight genomic DNA in ligase-assisted whole-genome amplification.
[0026] FIG. 14 illustrates a single-tube ligase-assisted amplification reaction
using male-female plasma/blood, wherein DYS14 male-specific marker is detected
using a library created from the input CNA.
DETAILED DESCRIPTION
[0027] The following detailed description is exemplary and is not intended to
limit the claimed invention or uses of the claimed invention. Furthermore, there is no
intention to be limited by any theory presented in the preceding background of the
claimed invention or the following detailed description.
[0028] In the following specification and the claims which follow, the singular
forms "a", "an" and "the" include plural referents unless the context clearly dictates
otherwise. Approximating language, as used herein throughout the specification and
claims, may be applied to modify any quantitative representation that could
permissibly vary without resulting in a change in the basic function to which it is
related. Accordingly, a value modified by a term such as "about" is not to be limited
to the precise value specified. Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight, reaction conditions, so
forth used in the specification and claims are to be understood as being modified in all
instances by the term "about." In some instances, the approximating language may
correspond to the precision of an instrument for measuring the value.
[0029] As used herein, the term "biological sample" refers to any type of
biological fluid obtained from a biological subject of eukaryotic origin. Non-limiting
examples of biological samples include whole blood, urine, saliva, sweat, tears,
amniotic fluid, breast milk, nasal wash or bronchoalveolar lavage fluids. In some
embodiments, the biological sample is of mammalian (e.g., rat, mouse, cow, dog,
donkey, guinea pig, or rabbit) origin. In certain embodiments, the biological sample
is of human origin.
[0030] As used herein, the term "intact cell" refers to non-disrupted cells that
may be present in a biological sample (i.e., a biological fluid). Since intact cells are
not disrupted, no nucleic acids and/or nucleic acid fragments are released from inside
of an intact cell into non-cellular fractions of the biological sample. The intact cells
may include resident eukaryotic cells (e.g., blood cells in a whole blood) and/or
circulating cells (e.g., circulating tumor cells in a whole blood). In some
embodiments, the intact cells may include other pathogenic cells (e.g., bacterial or
viral cells) that may be present in the biological sample.
[0031] As used herein, the term "non-cellular fraction" refers to the
component of a biological sample that is devoid of intact cells. For example, the noncellular
fraction of whole blood comprises plasma and serum, which are devoid of
intact blood cells (e.g. white blood cells, red blood cells and platelets). Based on the
pore size of the filtration membrane used for the generation of non-cellular fraction,
the non-cellular fraction may be devoid of eukaryotic cells, prokaryotic cells and/or
viral cell particles.
[0032] As used herein, the term "circulating nucleic acid" or "CNA" refers to
cell-free nucleic acids that are found in the non-cellular fraction of a biological
sample. The cell-free nucleic acids are those nucleic acids that are not restricted to an
inside cellular compartment (e.g., nucleus, mitochondria etc.) of a biological cell. The
circulating nucleic acid may be a deoxyribonucleic acid (DNA) or a ribonucleic acid
(RNA).
[0033] As used herein, the term "direct contact" refers to a contiguous contact
between two components. The direct contact between two components is achieved by
placing the two components such that they directly touch each other.
[0034] As used herein, the terms "ssLigase" or "single-strand specific ligase"
refers to a ligase that is capable of intra-molecular ligation of single-stranded nucleic
acids.
[0035] In some embodiments, the invention is directed to a method for
collecting and amplifying CNAs from biological samples. Elevated concentrations of
CNAs are often found in the non-cellular fraction of a biological sample that is
collected from patients with several pathologies when compared with healthy
individuals, indicating their potential as disease biomarkers. For example, tumorderived
circulating nucleic acids that are found in the plasma or serum fraction of
whole blood may be used to detect, monitor, or evaluate cancer and pre-malignant
states. Methods for amplification of CNAs in the non-cellular fraction of biological
sample may therefore aid in the detection, diagnosis, monitoring, treatment, and/or
evaluation of diseases such as neoplastic diseases, inflammation, myocardial
infarction, autoimmune disorders, transplanted organ/tissue rejection, pregnancyassociated
complications, and so forth. The neoplastic diseases may include, but not
limited to, early cancer, premalignant states or advanced cancer.
[0036] Some embodiments of the invention relate to methods and devices for
separation and collection of non-cellular fraction of a biological sample that contains
circulating nucleic acids. After separating and collecting the non-cellular fraction
from intact cells, the method further includes the steps of extracting circulating
nucleic acids from the non-cellular fraction and amplifying these nucleic acids to
create an amplified CNA library. The method and device described herein provide a
simplified and integrated solution for CNA collection and amplification. The method
and device may be suitable for use at a point-of-collection, and may be employed with
low sample volumes (e.g., less than about 150 ) . Thus devices and the associated
methods described herein reduce sample processing time and minimize sample
artifacts related to genomic DNA or RNA contamination, and help increase the
sensitivity of CNA amplification and/or detection.
[0037] In some embodiments, CNAs may be a tumor-derived circulating
nucleic acid. In some other embodiments, CNAs may be derived from a fetus, a
donated organ after implantation, a transplanted cell, a transplanted tissue, or a
diseased state. In some embodiments, the circulating nucleic acids comprise
circulating DNA or a circulating RNA. The circulating DNAs may include, but not
limited to, a tumor-derived DNA, a fetus-derived DNA, a donated organ-derived
DNA, a transplant cell-derived DNA, a transplanted tissue-derived DNA or a
combination thereof.
[0038] One aspect of the invention relates to a method for amplification of
circulating nucleic acids that are present in the non-cellular fraction of a biological
sample. The method comprises the steps of filtering the biological sample to separate
the non-cellular fraction from intact cells, collecting the separated, non-cellular
fraction onto a dry solid matrix and extracting the CNAs from the collected, noncellular
fraction. The method further includes the steps of circularizing the extracted
circulating nucleic acids by using a single-strand-specific ligase to form singlestranded
nucleic acid circles, and amplifying the single-stranded nucleic acid circles
by random-primed rolling circular amplification to form an amplified, CNA product.
In some embodiments, a method for amplification and detection of a tumor-derived
circulating DNA in the non-cellular fraction of a biological sample is provided.
[0039] FIG. 1 represents a flow diagram illustrating an embodiment of the
invention. The biological sample is applied on to a device that comprises a filtration
membrane membrane configured to separate the non-cellular fraction from intact
cells, and a dry solid matrix configured to collect the separated non-cellular fraction.
As shown in FIG. 1, the biological sample is applied on to the filtration membrane
(102). Upon filtration, intact cells of the biological sample are retained on the
upstream side/surface of filtration membrane and the non-cellular fraction is collected
onto a dry solid matrix (104), which may be located either on the downstream side (in
a lateral flow device) or on the downside surface (in a vertical flow device). The dry
solid matrix containing the non-cellular fraction may then be stored (106) or may be
directly used for extraction of circulating nucleic acids (108). The extracted
circulating nucleic acids are then subsequently circularized by a ligase that is capable
of intra-molecular ligation of single-stranded nucleic acids to form single-stranded
nucleic acid circles (110). In some embodiments, the method further comprises drying
the collected non-cellular fraction to a substantially dry state prior to extraction. If
the CNAs are in the double-stranded form, prior denaturation of the circulating
nucleic acids may be necessary before the ligation reaction. The single-stranded
nucleic acid circles are then subsequently amplified by a random-primed, rolling
circular amplification to form an amplified, CNA product.
[0040] FIG. 2 depicts a schematic representation of one embodiment of a
device that may be used for separating the non-cellular fraction of a biological
sample. The filtration membrane (202) has a sample application zone (210) and a
transfer zone (212). Filtration membrane is in direct contact with a solid matrix (204)
via the transfer zone (212). The filtration step includes providing a biological sample
at the sample application zone of the filtration membrane and passing the biological
sample through the filtration membrane. The filtration membrane has a plurality of
pores. Once the biological sample passes through the filtration membrane, resident
intact cells within the biological sample are retained by the filtration membrane,
mostly at the sample application zone (210) itself, and the non-cellular fraction are
passed through the pores to reach the transfer zone (212) and gets transferred and
collected onto the dry solid matrix. In some embodiments, a filtration membrane
having pore size in the range of about 0.01 micron to about 5 micron may be
employed. In some other embodiments, pore size of the filtration membrane may
vary between about 0.22 micron to about 2 micron. In one example embodiment, the
filtration membrane has a pore size between about 1 micron to about 2 micron. When
a filtration membrane of 1 micron pore size is used, any other circulating eukaryotic
cells and/or pathogenic cells having diameters greater than 1 micron will be retained
in the filtration membrane and so will not reach the dry solid matrix upon filtration.
[0041] In some embodiments, the non-cellular fraction may be filtered out
from the biological sample at the point-of-collection itself. Filtration may be
performed without any prior pre-treatment of the biological sample. Further filtration
may be performed in absence of any stabilizing reagent. After filtration, the
separated, non-cellular fraction may be collected onto a dry solid matrix by means of
physical interaction. The non-cellular fraction may be collected on to dry solid matrix
by means of adsorption or absorption.
[0042] Filtration membrane may be made from a variety of materials. The
materials used to form the filtration membrane may be a natural material, a synthetic
material, or a naturally occurring material that is synthetically modified. Suitable
materials that may be used to make the filtration membrane include, but are not
limited to, glass fiber, polyvinlyl alcohol-bound glass fiber, polyethersulfone,
polypropylene, polyvinylidene fluoride, polycarbonate, cellulose acetate,
nitrocellulose, hydrophilic expanded poly(tetrafluoroethylene), anodic aluminum
oxide, track-etched polycarbonate, electrospun nanofibers or polyvinylpyrrolidone. In
one example, the filtration membrane is formed from polyvinlyl alcohol-bound glass
fiber filter (MF1™ membrane, GE Healthcare). In another example, filtration
membrane is formed from asymmetric polyethersulfone (Vivid™, Pall Corporation).
In some embodiments, filtration membrane may be formed by a combination of two
or more different polymers. For example, filtration membrane may be formed by a
combination of polyethersulfone and polyvinylpyrrolidone (Primecare™, iPOC).
[0043] The non-cellular fraction that is collected on to the dry solid matrix
upon filtration may then be dried to a substantially dry state and stored for later
analysis. The term "substantially dry state" as used herein refers to conditions
wherein the dried sample contain less than about 10% (wt/wt) water content. In some
embodiments, the sample may be dried such that it contains less than about 5% water.
In some other embodiments, the sample may be dried such that it contains less than
about 2% water. In this way, CNAs that may be present in the non-cellular fraction
of a biological sample may be stored in a dried form which is suitable for later
subsequent analysis. The dried non-cellular fraction may be stored for long periods,
for example, for at least 24 hours, for at least 7 days, for at least 30 days, for at least
90 days, for at least 180 days, for at least one year, or for at least 10 years. In one
embodiment, non-cellular fraction is stored on the dry solid matrix for at least 30
minutes. Typically, samples are stored at temperatures ranging from -80 C to 40 C.
In addition, samples may be optionally stored under dry or desiccated conditions or
under inert atmospheres. Drying may be done by air-drying under ambient condition
or by vacuum-assisted evaporation. In some embodiments, the non-cellular fraction is
dried under ambient conditions by normal evaporation and maintained in a lowhumidity
environment. The removal of water from the collected non-cellular fraction
aids in stabilizing the circulating nucleic acids that are present in the non-cellular
fraction.
[0044] A dry solid matrix suitable for this purpose includes, but is not limited
to, a natural material, a synthetic material, or a naturally occurring material that is
synthetically modified. Suitable materials that can act as dry solid matrix include, but
are not limited to, cellulose, cellulose acetate, nitrocellulose, carboxymethylcellulose,
quartz fiber, hydrophilic polymers, polytetrafluroethylene, fiberglass and porous
ceramics. Hydrophilic polymers may be polyester, polyamide or carbohydrate
polymers. In some embodiments, the dry solid matrix is comprised of cellulose. The
cellulose-based dry solid matrix is devoid of any detergent. In some embodiments,
cellulose-based dry solid matrix may not be impregnated with any reagent. In other
embodiments, cellulose-based dry solid matrix may be impregnated with a chaotropic
salt. Examples of chaotropic salt include, but are not limited to, guanidine
thiocyanate, guanidine chloride, guanidine hydrochloride, guanidine isothiocyanate,
sodium thiocyanate, and sodium iodide. In some embodiments, the cellulose-based
dry solid matrix is FTATM Elute (GE Healthcare).
[0045] After collection of non-cellular fraction onto the dry solid matrix,
CNAs are extracted from this collected non-cellular fraction. The extraction may be
performed using any of the conventional nucleic acid extraction method. Nonlimiting
examples of extraction methods that may be used include, but are not limited
to, electroelution, gelatin extraction, silica or glass bead extraction, guanidinethiocyanate-
phenol solution extraction, guanidinium thiocyanate acid-based
extraction, centrifugation through sodium iodide or similar gradient, or phenolchloroform-
based extraction. The extraction step helps to remove impurities such as
proteins and concentrates the circulating nucleic acids. Extracted circulating nucleic
acids may be inspected using methods such as agarose gel electrophoresis,
spectrophotometry, fluorometry, or liquid chromatography.
[0046] The extracted CNAs are then converted to single-stranded nucleic acid
circles via an intra-molecular ligation reaction after extraction. The CNAs may either
be in a double-stranded or in a single-stranded form. Furthermore, CNAs may often
be highly fragmented. The double-stranded CNAs are denatured to a single-stranded
form prior to the intra-molecular ligation reaction. This denaturation of doublestranded
nucleic acids to single-stranded form may be achieved by using any of the
art-recognized methods. For example, the double-stranded nucleic acid may be
thermally denatured, chemically denatured, or both thermally and chemically
denatured. The double- stranded nucleic acid may be chemically denatured using a
denaturant (e.g., glycerol, ethylene glycol, formamide, or a combination thereof) that
reduces the melting temperature of double- stranded nucleic acid. The denaturant may
reduce the melting temperature by 5°C to 6°C for every 10% (vol./vol.) of the
denaturant added to the reaction mixture. The denaturant or combination of
denaturants (e.g., 10% glycerol and 6-7% ethylene glycol) may comprise 1%, 5%,
10%, 15%, 20%, or 25% of reaction mixture (vol./vol.). For example, salts that
reduce hybridization stringency may be included in the reaction buffers at low
concentrations to chemically denature the double-stranded circulating DNAs at low
temperatures. The double- stranded circulating DNA may also be thermally denatured
by heating at 95°C to form single- stranded DNA (ssDNA). After the denaturing step,
the generated single- stranded nucleic acids may be treated with a single-strand
specific ligase that is capable of intra-molecular ligation of single-stranded nucleic
acid substrates to form single-stranded nucleic acid circles.
[0047] Intra-molecular ligation of single-stranded circulating nucleic acids
may be performed in the presence or absence of a template by employing any of the
conventional methods used for intra-molecular ligation of single- stranded nucleic
acids. For example, conversion of linear, single-stranded DNA molecules to singlestranded
DNA circles is conventionally performed via a template-dependent intra
molecular ligation reaction using a ligation enzyme such as T4 RNA ligase.
However, template-dependent intra-molecular ligation of single- stranded DNA or
single-stranded RNA has met only with limited success, particularly when the
circularization of single-stranded DNA molecules is to be performed in a population
of single-stranded DNA molecules of unknown sequence and/or size. Even though
bacteriophage T4RNA ligase I exhibits a template-independent intra-molecular
ligation activity, this activity is far too low and inefficient for practical use in
generating circular single- stranded DNA molecules from linear, single-stranded DNA
molecules. In some embodiments, intra-molecular ligation of the extracted singlestranded
circulating nucleic acids is performed in the absence of any template. For
example, single-stranded DNA sequences that even are shorter than 500 nucleotides
may be circularized using template-independent intra-molecular ligation. Further, no
prior knowledge of the target sequence is needed to create DNA circles when the
ligation of the single stranded DNA (ssDNA) is performed in a template-independent
manner.
[0048] In some embodiments, conversion of the linear single-stranded
circulating nucleic acids to single-stranded nucleic acid circles is performed with a
thermostable RNA ligase that has good template-independent, intra-molecular ligation
activity for linear single-stranded DNA and/or single-stranded RNA substrates that
have 5' phosphoryl and 3' hydroxyl groups. Suitable ligases that may be used for
template-independent intra-molecular ligation of extracted single-stranded circulating
nucleic acids include, but are not limited to, TS2126 RNA ligase, T4 DNA ligase, T3
DNA ligase or E. coli DNA ligase. For example, TS2126 RNA ligase derived from
the Thermus bacteriophage TS2126 that infects the thermophilic bacterium, Thermus
scotoductus, may be employed for template-independent circularization of the linear
circulating ssDNA to generate circular, single-stranded DNA. TS2126 RNA ligase is
more thermostable (stable up to about 75 °C) than many of the mesophilic RNA
ligases such as the T4 RNA ligase. As a result, TS2126 RNA ligase may be used at
higher temperatures, which further reduce undesirable secondary structures of
ssDNA. HEPES buffer having a pH of 8.0 may be used for increasing the efficiency
of TS2126 RNA ligase-mediated intra-molecular ligation. The circularization of
extracted single-stranded circulating DNA may also be achieved using a ligase other
than TS2126 RNA ligase or by employing any other enzyme having DNA joining
activity such as topoisomerase. In some embodiments, circularization of ssDNA
molecule may be achieved by an RNA ligase 1 derived from thermophilic
archeabacteria, Methanobacterium thermoautotrophicum (Mthl) that has high
template-independent ligase activity in circularizing linear, fragmented single
stranded DNA molecules.
[0049] The single-stranded nucleic acid circles may then be amplified under
isothermal conditions by employing rolling circle amplification (RCA) methods. The
amplification of single-stranded nucleic acid circles may be performed in the same
reaction vessel in which the intra-molecular ligation is performed. Isolation or
purification of single-stranded nucleic acid circles and/or removal of the ligase may
not be necessary prior to the amplification reaction. In some embodiments, the entire
process of single-stranded nucleic acid ligation and amplification may be performed
in a single tube without any intermediate purification or isolation steps.
[0050] In some embodiments, the method further comprises detecting nucleic
acids from the amplified, circulating nucleic acid product. Detection of nucleic acids
from the amplified, circulating nucleic acid product is done by methods known in the
art. Various methods of detection of amplified product includes, but are not limited
to, PCR, RT-PCR, qPCR, RT-qPCR, restriction enzyme-based methods, agarose gel
electrophoresis, ELISA detection methods, electrochemiluminescence, high
performance liquid chromatography, Southern blot hybridization, Northern blot
hybridization, or reverse dot blot methods. In one embodiment, the detection is
performed by quantitative PCR using specific primers that amplify a specified target
within the circulating nucleic acid amplification product. The detection may be
performed to identify the presence, absence and/or quantity of a specific circulating
nucleic acid sequence in the amplified, circulating nucleic acid product.
[0051] The whole genome amplification methods disclosed herein improve
amplification sensitivity, reduce sequence dropout and allow more balanced
amplification. The described methods are advantageous especially when limited
quantities of biological sample are available. In some embodiments, a non-cellular
fraction is isolated from a total biological sample volume of about IO to about
500m . Further, both the circularization and amplification reactions may be
performed in a single reaction vessel without any intermediated purification or
isolation steps thereby reducing the chances of contamination and simplifying the
amplification workflow.
[0052] In some embodiments, whole genome amplification of fragmented,
circulating DNAs via multiple displacement amplification (MDA) are provided. The
circulating DNAs, by its nature of origin, are often highly fragmented. Furthermore,
the amount of circulating DNAs in the non-cellular fraction of a biological sample is
generally very low. Conventional methods of MDA, when attempted on linear
fragmented DNA, result in decreased amplification speed, significant sequence
dropout and lead to highly sequence-biased amplification. To overcome these
limitations, after extraction of the circulating DNAs from the dry solid matrix, the
fragmented double-stranded circulating DNAs are first converted to their singlestranded
form. The single-stranded circulating DNAs are then converted to singlestranded,
DNA circles via a template-independent intra-molecular ligation reaction,
thereby eliminating the problematic DNA ends. After circularization of the
fragmented single-stranded circulating DNA, MDA is performed on the circularized
DNA.
[0053] The MDA reaction of the extracted circulating DNAs may be
performed under isothermal conditions via employing rolling circle amplification
(RCA) methods. For amplification of single-stranded DNA circles, amplification
reagents including a DNA polymerase, primers and dNTPs may be added to the same
reaction vessel where ligation is performed to produce an amplification reaction
mixture to initiate an RCA reaction. The amplification reaction mixture may further
include reagents such as single-stranded DNA binding proteins and/or suitable
amplification reaction buffers. RCA may be performed by using any of the strand
displacing DNA polymerases that are known in the art such as a Phi29 DNA
polymerase. RCA may be performed using commercially available RCA
amplification kits such as TempliPhi™ RCA kit (GE Healthcare). The TempliPhi™
rolling-circle amplification employs locked nucleic acid-containing random primers,
which provide higher sensitivity and amplification balance. In some embodiments,
random primers are used for the RCA reaction. The primer sequences comprising one
or more nucleotide analogues (e.g., LNA nucleotides) may be used. In some
embodiments, nuclease-resistant primers (e.g., primer sequences comprising
phosphorothioate groups at appropriate positions) are employed for amplification
reaction (e.g., NNNN*N*N, where *N represents a random nucleotide with a
phosphorothioate linkage). In some embodiments, rolling circle amplification may
be performed by contacting the single-stranded DNA circles with a primer solution
comprising a random primer mixture to form a DNA template-primer complex;
contacting the DNA template-primer complex with a DNA polymerase and
deoxyribonucleoside triphosphates; and amplifying the DNA template. Since
template-independent circularization of single-stranded DNA may be achieved on
short sequences even at low concentrations, a more balanced DNA amplification with
faster kinetics and improved sequence coverage may be achieved when ligase-assisted
whole-genome amplification is employed for amplification of highly fragmented
circulating DNAs (e.g., circulating DNAs that are present in whole blood).
[0054] FIG. 7 depicts a schematic representation of an embodiment of ligaseassisted
whole-genome amplification of a fragmented double-stranded circulating
DNA. The persistence length of double-stranded DNA is much higher (-150 bp) than
single-stranded DNA, and its innate stiffness makes circularization of fragments less
than 500 bp highly inefficient. Further, with small double-stranded fragmented DNA
molecules of about 250 bp range, circularization is inefficient unless the ends are in
proper alignment (-10.5 bp/turn). In contrast, the persistence length of the
circularization of single-stranded fragmented DNA is very small, approximately 15
nucleotides, as compared to the double-stranded fragmented DNA. As depicted in
FIG. 7, in ligase-assisted whole-genome amplification, fragmented double-stranded
circulating DNA is first converted into single-stranded DNA circles. This may be
achieved by incubating the fragmented double-stranded circulating DNA at 95 °C for
sufficient period in order to denature the double stranded DNA into single strands.
The fragmented single stranded circulating DNA is then treated with a DNA or RNA
ligase that is capable of template-independent, intra-molecular ligation of singlestranded
circulating DNA substrates to generate single-stranded DNA circles.
Amplification reagents, including DNA polymerase, random primers, and dNTPs are
then added to initiate a RCA reaction on the single-stranded DNA circles. This
ligase-assisted whole-genome RCA amplification produces large quantities of DNA
with reduced sequence dropout and amplification bias in contrast to the conventional
whole-genome amplification methods. Therefore, it may be used to amplify and
detect even highly fragmented circulating DNA. In some embodiments, the entire
process of generation of the single-stranded DNA circles and its subsequent
amplification by RCA is done in a single tube without any intervening purification
steps.
[0055] Ligation-assisted whole-genome amplification methods provided
herein, which comprise prior ligation of single-stranded circulating DNA fragments to
DNA circles followed by rolling circle amplification, provides preferential
amplification of fragmented CNA over high molecular weight genomic DNA. For
example, plasma preparations comprising CNA may often be contaminated with
genomic DNA that are released from blood cells during the purification process.
Conventional methods of whole-genome amplification via MDA amplify both the
circulating DNA and the genomic DNA. In contrast, when fragmented, CNA
molecules are first circularized using TS2126 RNA ligase followed by amplification
via RCA employing a Phi29 DNA polymerase, circulating DNAs were preferentially
amplified over the high molecular weight genomic DNA. Such preferential
amplification of fragmented circulating DNA over the genomic DNA is particularly
suitable for diagnostic applications since diagnostically relevant DNA may be
preferentially amplified for downstream analysis (FIG. 13). Further, ligase-assisted
whole-genome amplification allows more robust amplification of fragmented DNA as
compared to conventional MDA-based whole-genome amplification.
[0056] In some embodiments, sensitivity of circulating DNA amplification
and detection in the non-cellular fraction of a biological sample may further be
increased by phosphorylating the extracted circulating DNAs with a polynucleotide
kinase (PNK) prior to the ssDNA ligation step and RCA. Intra-molecular ligation of
DNA is not feasible unless the ssDNA template has a 5' phosphate group and a 3'
hydroxyl group. A variety of conditions (e.g., DNase II enzymatic cleavage, and
phosphatase activity in blood) may lead to the generation of circulating DNAs with
non-ligatable DNA sequences having either 5' hydroxyl groups or 3' phosphate groups
or both. The PNK treatment converts these non-ligatable DNA sequences to ligatable
DNA sequences by phosphorylating the 5' end or dephosphorylating the 3' end. This
improves the diversity of rolling-circle amplified CNA library. Upon incorporating
the PNK step in the work flow, ligase-assisted whole-genome amplification methods
presented herein could detect male circulating DNA in female whole blood when
spiked at 1% levels (triplicate repeats, FIG. 14).
[0057] In one embodiment, a method for amplification and detection of
circulating nucleic acids that are present in whole blood is provided. Whole blood
comprises a cellular fraction (i.e. white blood cells, red blood cells and platelets) and
a non-cellular fraction (e.g., plasma or serum). Circulating DNA is amplified from
the non-cellular fraction of the whole blood (e.g., plasma or serum). In a preferred
embodiment, plasma or serum is separated from a fingerstick volume of blood. The
method comprises the steps of collecting the non-cellular fraction of the whole blood,
extracting the circulating DNAs (mostly presented in its native double-stranded form)
from the non-cellular fraction, denaturing the double-stranded circulating DNAs to
generate single-stranded DNAs, circularizing the circulating single-stranded DNAs to
generated single-stranded DNA circles, and amplifying the single-stranded DNA
circles via rolling circle amplification to form an amplified circulating nucleic acid
product. While filtering the whole blood using the device described herein, plasma or
serum passes through the pores of filtration membrane and gets collected onto a dry
solid matrix. The intact blood cells are retained by the filtration membrane. Plasma
or serum may be separated from the whole blood sample by filtration in the absence
of an anticoagulant. Therefore, no extra steps are required to maintain the integrity of
the whole blood sample prior to filtration. In some embodiments, biological sample
may be pre-treated with reagents like anticoagulant before filtration. The genomic
contamination from intact blood cells may be minimized by filtering the whole blood
at the point-of-collection. In one embodiment, the separated plasma or serum,
containing CNAs, is adsorbed on to a dry solid matrix by passive wicking. In one
embodiment, circulating DNAs are extracted from the plasma or serum previously
collected onto a solid matrix using sodium iodide and alcohol (DNA Extractor SP™,
Wako Chemical). In one example, the plasma is separated from less than 150m of
whole blood.
[0058] It is often not possible to circularize double-stranded DNA that has a
sequence length smaller than 150 bp, and it is very difficult to circularize double
stranded DNA until the DNA is longer than 200 bp. In contrast, linear single stranded
DNA molecules having a sequence length of 15 nucleotides or more are very
efficiently circularized by a suitable ligase as long as the 5' end is phosphorylated and
the 3' end is hydroxylated. The circularization of the single-stranded DNA to
generate single-stranded DNA circle may be achieved by employing a ligase that is
capable of template-independent intra-molecular ligation of single-stranded DNA. In
some embodiments, the circularization of the single-stranded DNA molecules is
performed by treating the single-stranded linear DNA with an RNA ligase such as
CircLigase II™.
[0059] Another aspect of the invention relates to a method for processing
whole blood at a point-of-collection itself to collect plasma or serum. The method
comprises the steps of filtering the whole blood to separate the plasma or serum at the
point of sample collection, collecting the separated plasma or serum on to a dry solid
matrix, wherein the solid matrix is devoid of any detergent and drying the collected
plasma or serum in the solid matrix. In some embodiments, filtration is done by using
MF1™ membrane and collection is done using a cellulose-based solid matrix
arranged laterally to MF1™ membrane. In other embodiments, Vivid™ or
Primacare™ membrane and a Cellulose-based solid matrix are arranged vertically. In
one example, either the whole blood or the filtration membrane is not pre-treated with
any anticoagulant. In another example, blood and/or filtration membrane is pre-treated
with an anticoagulant. In some embodiments, a cellulose matrix that is impregnated
with a chaotropic salt may be used to collect the plasma or serum at the point-ofcollection.
Suitable chaotropic salts that may be employed includes, but not limited
to, guanidine thiocyanate, sodium thiocyanate, potassium thiocyanate, or guanidine
hydrochloride. The solid matrix that contains dried plasma or serum may be stored
for longer periods, and the circulating nucleic acids may be extracted, amplified and
detection from this dried plasma or serum at a later point in time.
[0060] In some aspects, a method for detecting circulating nucleic acids from
a dried sample of plasma or serum is provided. The method comprises the steps of
extracting the circulating nucleic acids from a dried plasma or serum sample,
performing a whole genome amplification of the extracted circulating nucleic acids to
generate an amplified circulating nucleic acid product and then detecting the presence,
absence, or quantity of a specific circulating nucleic acid sequence within the
amplified circulating nucleic acid product. Whole genome amplification of the
extracted circulating nucleic acids may be achieved by first circularizing the extracted
circulating nucleic acids by a single-stranded specific ligase to form single-stranded
nucleic acid circles and amplifying the single stranded nucleic acid circles by randomprimed
rolling circular amplification to form the amplified circulating nucleic acids
product. The detection of specific circulating nucleic acid sequences in the amplified
library may be achieved by any of the conventional nucleic acid detection
technologies. The method may further include the step of denaturing double-stranded
CNAs to their single-stranded form prior to the intra-molecular ligation reaction by a
single-strand specific ligase.
[0061] Another aspect of the invention relates to a device for collecting the
non-cellular fraction of a biological sample, which contains the circulating nucleic
acids. The device comprises a filtration membrane configured to separate the noncellular
fraction of the biological sample from intact cells, and a dry solid matrix
configured to collect the separated non-cellular fraction. The solid matrix is devoid of
any detergent and is in direct contact with the filtration membrane. The device may
be a lateral flow device or a vertical flow device.
[0062] FIG. 2 depicts a schematic representation of a lateral flow device (200)
as described in one embodiment of the invention. The lateral flow device contains a
filtration membrane (202) and a dry solid matrix (204). The filtration membrane and
the dry solid matrix are arranged laterally such that the non-cellular fraction of the
biological sample passes through the filtration membrane to the solid matrix in a
lateral direction. The filtration membrane has a sample application zone (210) and a
transfer zone (212). Filtration membrane is in direct contact with the solid matrix via
the transfer zone. Essentially, the transfer zone of the filtration membrane is the part
of the filtration membrane that touches the dry solid matrix when the filtration
membrane is in direct contact with the solid matrix. The sample application zone is
used for receiving biological sample and the transfer zone is used for delivering noncellular
fraction of biological sample to the dry solid matrix. In some embodiments,
the filtration membrane and dry solid matrix are arranged such that they may partially
overlap each other. In other embodiments, the filtration membrane and dry solid
matrix are arranged such that they may not overlap each other but still biological
sample passes through the filtration membrane to the solid matrix in the lateral
direction. In such case, the dry solid matrix is positioned downstream of the filtration
membrane, touching the filtration membrane but not overlapping. In some
embodiments, the filtration membrane is disposed on a first solid support (206) and
the dry solid matrix is disposed on a second solid support (208). In some
embodiments, the first solid support and the second solid support are arranged facing
opposite to each other. In other embodiments, the first solid support and the second
solid support may be arranged next to each other. In some embodiments, the filtration
membrane and dry solid matrix are laterally disposed on a solid support (206). In
some embodiments, a second solid support (208) is included over the dry solid matrix
to sandwich the dry solid matrix against the filtration membrane and establish an
effective transfer zone, for example, as in FIG. 2.
[0063] The first solid support may be connected to the second solid support
via a means for establishing a direct contract of the filtration membrane with the dry
solid matrix. The means for establishing direct contact may be a hinge, foldable
indentation, or an otherwise pliable connection. In some embodiments, the lateral
flow device may be configured by a process (300) as shown in FIG. 3. The first solid
support (306) and the second solid support (308) are connected to each other via a
foldable hinge (310). The first solid support has a filtration membrane (302) disposed
on it and the second solid support has a dry solid matrix (304) disposed on it. The
filtration membrane may be brought in direct contact with the dry solid matrix by
folding the hinge such that the filtration membrane and the dry solid matrix partially
overlap each other. In one embodiment, the lateral flow device comprises an MF1™
filtration membrane and a cellulose-based dry solid matrix. In embodiments where
plasma/serum is collected from whole blood, whole blood is applied and passed
across the filtration membrane, and non-cellular plasma or serum is collected or
wicked on to the dry solid matrix.
[0064] In some embodiments, the device may be a vertical flow device. A
schematic representation of the vertical flow device (400) is illustrated in FIG. 4. The
device (400) contains the filtration membrane (402) and the dry solid matrix (404),
wherein the filtration membrane is disposed on to the dry solid matrix. The filtration
membrane is in direct contact with the solid matrix. The filtration membrane has a
sample application zone (406) and a transfer zone (408). The sample application zone
is used for receiving biological sample and the transfer zone is used for delivering a
filtered non-cellular fraction of the biological sample to the dry solid matrix. The
transfer zone of the filtration membrane is defined by a zone which is in touch with
the dry solid matrix. As shown, the filtration membrane and the dry solid matrix are
arranged such that non-cellular fraction of the biological sample can pass through the
filtration membrane to the solid matrix in a vertical direction. In some embodiments,
the dry solid matrix is disposed on to a third solid support (410). In one embodiment,
the vertical flow device comprises a Vivid™ or Primecare™ filtration membrane and
a cellulose-based dry solid matrix. In the embodiments where plasma/serum is
collected from whole blood, whole blood is applied and passed through the filtration
membrane, and non-cellular plasma or serum is collected or wicked on to the dry
solid matrix.
[0065] As described above, a first solid support may carry the filtration
membrane and a second and third solid support may carry the dry solid matrix. The
solid support may be positioned directly adjacent to the filtration membrane or dry
solid matrix membrane as shown in FIG. 2, FIG. 3 or FIG. 4. In some embodiments,
one or more intervening layers may be positioned between the solid support and the
filtration membrane and/or the dry solid matrix. The solid support may be formed
from any material that is able to carry the filtration membrane and/or dry solid matrix.
The support may be formed from a material that is transmissive to light, such as
transparent or optically diffuse (e.g., transluscent) materials. It may be desirable that
the solid support is liquid-impermeable so that the fluid flowing through the
membrane or solid matrix does not leak through the solid support. Examples of
suitable materials for the solid support include, but are not limited to, glass, polymeric
materials such as polystyrene, polypropylene, polyester, polybutadiene,
polyvinylchloride, polyamide, polycarbonate, epoxides, methacrylates or
polymelamine. To provide a sufficient structural backing for the membrane or solid
matrix, the solid support is generally selected to have a certain minimum thickness.
For example, the solid support may have a thickness that ranges from about 1/16 inch
to about 1/4 inch. In one embodiment, the solid support is polycarbonate-based
(Clear Lexan™) having a thickness of about 0.10 inch.
[0066] Detergents may precipitate out of solution while employing the nucleic
acid precipitation methods described above and therefore would interfere with the
method of nucleic acid preparation and analysis. Therefore, the dry solid matrix of
the device described herein is devoid of any detergent such as sodium dodecyl sulfate
(SDS), SLS (lauryl), alkyl aryl sulfonates, long chain alcohol sulfates, olefin sulfates,
sulfosuccinates, phosphate esters, sodium 2-ethylhexysulfate, polyvinyl sulfate,
polyacrylate, polyphosphate, sodium polyacrylate or sodium polyvinyl sulfate. In
some embodiments, the dry solid matrix may be impregnated with a chaotropic salt.
[0067] In some embodiments, the device is designed such that the dry solid
matrix is well suited for direct downstream analysis (e.g. nucleic acid extraction)
without any further processing such as coring or punching. In particular, the dry solid
matrix of the device has a dimension which makes it suitable to fit entirely into
standard laboratory extraction vessels (e.g., microcentrifuge tube, centrifuge tubes).
In one embodiment, the dimensional width of the dry solid matrix is up to about 8
millimeters so that it fits entirely inside an extraction vessel. Such device design aids
in eliminating the requirement for coring or punching the material prior to sample
extraction, and therefore minimizes DNA contamination from the surrounding sample
environment that might feed into whole genome amplification.
[0068] In some embodiments, the filtration membrane is also scaled
proportionally to a maximum dimensional width of 8 mm in order to establish even
sample wicking with the solid matrix. The dimensional lengths of the filtration
membrane and solid matrix are dictated by the desired input volume of the biological
sample. In one embodiment, for lateral flow separation of 100 m whole blood, the
optimal dimension of the MF1™ filtration membrane is 8mm wide x 20mm long. At
this dimension, the red blood cell front arrests near the interface of the solid matrix,
thereby minimizing the volume of plasma retained on the filtration membrane and
maximizing transfer of the plasma onto the dry solid matrix. For vertical flow
separation of 100 m whole blood, the optimal dimension of Vivid™ or Primecare™
filtration membrane is 8mm wide x 32mm long.
[0069] In some embodiments, the solid matrix may be de-coupled from the
upstream filtration membrane and stored at ambient temperature for long-term
archiving after sample filtration and transfer of non-cellular fraction onto the solid
matrix. Furthermore, the non-cellular fraction that is transferred to the dry solid
matrix may be dried so that it can be stored for longer periods without damaging the
circulating nucleic acids present therein. At the time of analysis, circulating nucleic
acid can be extracted from the solid matrix by transferring the solid matrix into a
conventional extraction vessel (e.g. a microcentrifuge tube) and rehydrating the
matrix in a suitable extraction buffer.
[0070] The device as described above may be used for collecting a noncellular
fraction of a biological sample at the point-of-collection of said biological
sample. Biological sample may be provided directly on the filtration membrane
without any pre-treatment at the point of sample collection. Once the filtration step is
complete, a non-cellular fraction may be collected on to the dry solid matrix and
stored. In some embodiments, a method for collecting plasma or serum from whole
blood using the device is described. The method comprises the steps of providing the
whole blood at the sample application zone of the filtration membrane, allowing the
whole blood to pass through the filtration membrane to separate the plasma or serum
from blood cells, and collecting the separated plasma or serum on the dry solid
matrix. Once collected, the plasma or serum may be dried on the solid matrix for long
term storage. The entire process may be done at the point-of-collect of the whole
blood sample. Later on, the dried plasma or serum fraction having circulating nucleic
acids may be further processed by the methods described herein for downstream
analysis. In some embodiments, less than 100 m of whole blood sample may be used
to collect plasma or serum.
[0071] The device presented herein may further include additional functional
components that do not affect the basic functionality of the device, namely the
collection of a non-cellular fraction having circulating nucleic acids from the
biological sample. For example, additional filtration membranes with different pore
size may be included in the device. In some embodiments, the device includes a
single filtration membrane configured to separate a non-cellular fraction from intact
cells and a single dry solid matrix configured to collect the separated non-cellular
fraction. In some other embodiments, the device may include a filtration membrane
configured to separate a non-cellular fraction from intact cells, a dry solid matrix
configured to collect the separated non-cellular fraction, and other functional
components that do not alter the basic functionality of the device. Examples of such
other functional components include, but not limited to, a solid support, casing for the
device, holding rings and/or covering membranes.
[0072] Practice of the invention will be still more fully understood from the
following examples, which are presented herein for illustration only and should not be
construed as limiting the scope of the present invention as defined by the appended
claims. Some abbreviations used in the examples section are expanded as follows:
"mg": milligrams; "ng": nanograms; "pg": picograms; "fg": femtograms; "mL":
milliliters; "mg/mL": milligrams per milliliter; "mM": millimolar; "mmol":
millimoles; "pM": picomolar; "pmol": picomoles; "mI7': microliters; "min.": minutes
and "h.": hours.
EXAMPLES
[0073] Example 1: Circulating DNA collection on plasma collection
membranes following lateral or vertical separation of human whole blood:
[0074] For lateral flow devices, MF1™ membrane was used as a filtration
membrane and 903 cellulose paper was used as a dry solid matrix. For vertical flow
devices, Primecare™ and Vivid™ membranes were used as filtration membranes and
903 cellulose paper was used as a dry solid matrix. 100 E of human whole blood
was applied on to the filtration membranes of the lateral or vertical flow devices and
plasma was collected on to the dry solid matrix. The collected plasma was placed into
a desiccator cabinet and dried at room temperature to form a dried plasma sample.
Following 24 hours of storage, plasma DNA was extracted from each solid matrix by
adaptation of DNA Extractor SP (Wako Chemical), and precipitated plasma DNA was
analyzed using gel electrophoresis. For comparative purposes, whole blood was
centrifuged with three-step gentle protocol (1600 x g, 10 minutes; collect and re-spin
plasma at 1600 x g, lOminutes; collect and spin at 16,000 x g, 10 minutes for cell-free
plasma), and 50 m of centrifuged plasma was spotted onto identical 903 cellulose
paper, extracted, and analyzed in parallel. FIG. 5 demonstrates that circulating
plasma DNA is efficiently collected and stabilized from a dry solid matrix overlapped
downstream of commercially-available filtration membranes. The yield of plasmacirculating
DNA was measured with a PicoGreen assay and demonstrated similar
DNA recovery between MF1-filtered whole blood (175 pg/ E) and centrifuged
plasma (179 pg/mE) . In contrast, a small amount of genomic contamination (DNA >
lOkB) was visible following filtration using Primecare™ and Vivid™ membranes
(vertical flow filtration). However, no genomic contamination was seen after MF1
lateral-flow filtration or gentle centrifugation.
[0075] Example 2 : Ligase-assisted whole genome amplification for detection
of four different chromosomal loci from plasma DNA separated from whole blood by
lateral or vertical flow:
[0076] Plasma DNA extracted from the dry solid matrix (903 cellulose paper)
from Example 1 was amplified using rolling circle amplification techniques in the
absence or presence of a commercial single strand-specific ligase (CircLigase,
EpiCentre), and four random STR chromosomal loci (vWA, TPOX, D8S1129, and
D13S317) were interrogated to assess genomic coverage. FIG. 6 demonstrates that
rolling circle amplification techniques combined with single-strand-specific ligase
activity enables sensitive detection of all four chromosomal STR loci from picogram
quantities of plasma-circulating DNA. The experiment was performed using mini-
STR primer sets, since traditional STR primer pairs typical amplify regions of DNA
that are larger than circulating DNA itself. Single-strand-specific ligase activity in
combination with rolling circle amplification technique permitted detection of plasma
STR loci with qPCR CT values close to that of unamplified genomic DNA from buffy
coat fractions, which were isolated by centrifugation and extracted using QIAamp
DNA blood mini kit (Qiagen) (FIG. 6). Without single-strand-specific ligase activity,
only two out of four plasma STR markers could be detected using the rolling circle
amplification technique alone. Using ligase-assisted whole genome amplification,
STR detection levels from plasma appeared similar between membrane-filtered blood
and centrifuged blood.
[0077] Example 3 : Whole-genome amplification of circulating nucleic acid
from blood plasma:
[0078] Circulating DNA was isolated from citrate-phosphate-dextrose (CPD) -
stabilized blood plasma of apparently healthy individuals using the Wako DNA
extractor SP kit (Wako Pure Chemical Industries). Approximately 1.3 ng was
analyzed by electrophoresis through a 2% agarose gel using TBE buffer, stained with
SYBR Gold and visualized using a Typhoon imager. As depicted in FIG. 8, the
majority of the circulating DNA was approximately 180 bp in length, with an
additional smaller amount of sequences that were approximately 370 bp long, and a
substantially smaller amount of higher molecular weight sequences.
[0079] 350 pg circulating DNA from plasma was heated at 95 °C to denature
the template. The denatured, single-stranded DNA template was then treated with an
RNA or DNA ligase to generated single-stranded DNA circles. ATP-dependent T4
DNA ligase, cell-encoded NAD-dependent E. coli DNA ligase or a thermostable
RNA ligase (CircLigase II) were used for these ligation reactions. 100 pg of ligated
single-stranded DNA circles were then subjected to whole-genome amplification
using GenomiPhi kit (GE Healthcare) employing a Phi29 DNA polymerase. The
amplification was performed using the primer mixture +N+N(at N)(at N)(at N)*N
where +N represents an LNA nucleotide and "at N" represents a random mixture
containing 2-amino dA, 2-thio dT, normal G and normal C. Real time amplification,
wherein the amplification and quantification of the target nucleic acid is done
simultaneously, was performed by adding a small amount of SYBR green I to the
amplification mixture and monitoring the fluorescence signal increase over time in a
Tecan plate reader (Tecan Sniper, Amersham Pharmecia Biotech). For comparison,
an equivalent concentration of untreated genomic DNA, untreated plasma DNA, and a
sample without DNA template (No template amplification) were included.
[0080] As depicted in FIG. 9, the amplification kinetics of the untreated,
fragmented plasma DNA was much lower when compared with an equivalent amount
of high molecular weight genomic DNA, indicating a defect in amplification.
However, when the fragmented plasma DNA was pre-treated and converted to
single-stranded DNA circles using the CircLigase ™ II, rapid amplification kinetics
was achieved (FIG. 9A). Other ligases, including the ATP-dependent T4 DNA ligase
(FIG. 9B) and the cell-encoded NAD-dependent E. coli DNA ligase (FIG. 9C) were
also effective in restoring amplification kinetics of the fragmented plasma DNA. In
these examples, the relative increase in amplification kinetics indicates the
effectiveness of each of the ligases in promoting the intra-molecular ligation of the
single-stranded DNA template.
[0081] Example 4 : Analysis of amplified circulating nucleic acids from blood
plasma by ligase-assisted whole-genome amplification.
[0082] The amplified DNA generated in Example 3 was further analyzed by
quantitative PCR using primers targeting four different CODIS loci (vWA, TPOX,
D8S1129, and D13S317) in order to sample the effectiveness of the ligase-assisted
whole-genome amplification method for promoting sensitive and balanced DNA
amplification. These DNA levels were compared with the values from unamplified
DNA to determine the relative representation levels after amplification. As illustrated
in FIG. 10, in both examples, qPCR analysis of untreated plasma DNA led to
sequence dropout or produced DNA that was highly under-represented at the tested
loci. In contrast, including either CircLigase™ II or T4 DNA ligase in the method
prevented the sequence dropout of the four loci and produced DNA that was more
similar in representation to the amplified high molecular weight genomic DNA. In a
further example using CircLigase™ II as the single-stranded DNA ligase, out of 12
different CODIS loci tested by quantitative PCR (qPCR), 11 were detected after
ligase-assisted whole genome amplification, whereas only 4 were present in the
untreated plasma DNA (FIG. 11). In FIG. 11, the Ct values reported are an average of
two replicates. PCR reactions where the Ct value was undetermined are marked by an
[0083] Example 5 : Optimization of reaction conditions for ligase-assisted
whole-genome amplification.
[0084] The ligase-assisted DNA amplification reaction was further optimized
by optimizing the efficiency of ligation reaction of single-stranded DNA molecules by
TS2126 RNA ligase. The presence of metal ion was essential for the ligation reaction
since eliminating manganese from the standard manufacturer-recommended buffer
reduced amplification rates to background levels. Untreated genomic DNA and
untreated plasma DNA were compared with CircLigase II™ -treated plasma DNA
samples using modified buffer conditions. All buffer conditions contained 33 mM
KoAc, 0.5 mM DTT, and 1M betaine. Where indicated, the buffers contained 33 mM
Tris-acetate (pH 7.5) or 33 mM HEPES-KOH (pH 8.0) and additionally contained 2.5
mM MgCl2 or 2.5 mM MnCl2. Real time amplification was performed by adding a
small amount of SYBR green I to the amplification mixture and monitoring
fluorescence increase over time in a Tecan plate reader. Amplification threshold is
the time at which fluorescence rises above background levels (2000 RFU).
[0085] A comparison of amplification kinetics of ligase-assisted wholegenome
amplification reactions (100 pg input of circulating DNA) is depicted in FIG.
12. Both magnesium and manganese produced similar effects in the presence of the
standard TRIS buffer. However, a combination of manganese and magnesium in the
presence of HEPES buffer, pH 8.0 promoted higher amplification rates. HEPES
buffer increased circularization efficiency of the plasma DNA in these reactions by
decreasing the oxidation rate of the manganese cations compared to TRIS buffer.
[0086] Example 6 : Inhibition of amplification of high molecule weight
genomic DNA in ligase-assisted whole-genome amplification.
[0087] The amplification kinetics of whole-genome amplification of untreated
genomic DNA was compared to CircLigase™ I and CircLigase™ Il-treated genomic
DNA samples (100 pg DNA input). The results are illustrated in FIG. 13. As
depicted in FIG. 13, CircLigase™ treatment of single-stranded genomic DNA
produced an inhibitory effect on the amplification rate of high molecular weight
genomic DNA (unlike the positive effects on plasma DNA such as illustrated in FIG.
9A). The inhibition was apparent for both CircLigase™ I and CircLigase™ II.
[0088] To investigate if Phi29-based amplification was inhibited by the ligase,
untreated double-stranded genomic DNA was amplified in the presence of active
ligase. Real time amplification was performed by adding a small amount of SYBR
green I to the amplification mixture and monitoring fluorescence increase over time in
a Tecan plate reader. Amplification threshold is the time at which fluorescence rises
above background levels (2000 RFU). It was observed that the genomic DNA
amplification inhibition was not a consequence of active ligase being present during
the amplification.
[0089] A preference for the amplification of circulating over high molecular
weight genomic DNA might be an advantage for certain applications, as genomic
DNA from blood cells often contaminates preparations of circulating nucleic acids,
and is of less diagnostic value.
[0090] Example 7 : Single-tube amplification of fragmented DNA employing
ligase-assisted whole-genome amplification - Effect of phosphorylation of circulating
DNA fragments with kinase prior to intra-molecular ligation.
[0091] Phosphorylation of circulating DNA fragments with kinase was
discovered to elicit more sensitive detection of circulating DNA in blood plasma. A
male-female plasma/blood mixing experiment demonstrated that the DNA library
created from input CNA treated with kinase was more representative, allowing for
more sensitive detection of the DYS14 male-specific marker (3/3 replicates, whereas
only 1/3 was detected if phosphorylation was not done). 100 uL of blood/plasma
mixtures were prepared as follows: 100A: 100% male plasma; 5A-C: male plasma
spiked into female whole blood at 5% v/v; 1A-C: male plasma spiked into female
whole blood at 1% v/v; and OA: 100% female blood. The plasma was separated from
the blood cells by lateral flow through an MF1 membrane followed by collection onto
a 903 cellulose pad, which was subsequently dried and stored overnight. Circulating
DNA was then extracted from the cellulose pad by a modification of the Wako
extractor SP kit (Wako Pure Chemical Industries), a standard sodium iodide/detergent
based method. Approximately 1.8 ng of DNA was then treated with or without T4
polynucleotide kinase in the presence of GTP, manganese, and betaine and then
treated with CircLigase IITM to circularize the single-stranded DNA fragments.
DNA was then subjected to GenomiPhi whole-genome amplification (GE Healthcare)
and products were analyzed by quantitative PCR to assess the detection of two
markers: Dysl4, which is a multi-copy gene located on the Y-chromosome and
should be detectible from the male fraction only, and D16S539 which is an STR locus
located on chromosome 16 and should be detectible from both male and female
fractions. The reaction was performed in a single reaction vessel, without any
intermediate purification or isolation steps in the workflow. This was achieved by
performing the phosphorylation reaction at a relatively low concentration of GTP.
[0092] FIG. 14 illustrates that inclusion of a kinase in the reaction allows for
circularization and amplification of CNA fragments that do not necessarily contain a
5' phosphate, thereby creating a more representative library. This would include DNA
fragments containing a 5' hydroxyl, which are specifically generated by DNase II
digestion during cell death. Using a male-female plasma/blood mixing experiment, it
is demonstrated that the library created from the input DNA treated with kinase is
more representative, allowing for more sensitive detection of the DYS14 malespecific
marker (3/3 replicates, whereas only 1/3 was detected if phosphorylation was
not done).
[0093] The claimed invention may be embodied in other specific forms
without departing from the spirit or essential characteristics thereof. The foregoing
embodiments are selected embodiments or examples from a manifold of all possible
embodiments or examples. The foregoing embodiments are therefore to be
considered in all respects as illustrative rather than limiting on the invention described
herein. While only certain features of the claimed invention have been illustrated and
described herein, it is to be understood that one skilled in the art, given the benefit of
this disclosure, will be able to identify, select, optimize or modify suitable
conditions/parameters for using the methods in accordance with the principles of the
present invention, suitable for these and other types of applications. The precise use,
choice of reagents, choice of variables such as concentration, volume, incubation
time, incubation temperature, and the like may depend in large part on the particular
application for which it is intended. It is, therefore, to be understood that the
appended claims are intended to cover all modifications and changes that fall within
the true spirit of the invention. Further, all changes that come within the meaning and
range of equivalency of the claims are intended to be embraced therein.

CLAIMS
1. A method for amplification of circulating nucleic acids that are present
in the non-cellular fraction of a biological sample, the method comprising:
filtering the biological sample to separate the non-cellular fraction from
intact cells;
collecting the separated, non-cellular fraction onto a dry solid matrix;
extracting the circulating nucleic acids from the collected, non-cellular
fraction;
circularizing the extracted, circulating nucleic acids to form singlestranded
nucleic acid circles; and
amplifying the single-stranded nucleic acid circles via random-primed
rolling circle amplification to form an amplified, circulating nucleic acid product.
2. The method of claim 1, further comprising drying the collected, noncellular
fraction to a substantially dry state prior to extraction.
3. The method of claim 1, further comprising denaturing the extracted,
circulating nucleic acids prior to circularization.
4 The method of claim 2, wherein the circularization is performed using
a TS2126 RNA ligase.
5. The method of claim 1, further comprising detecting the presence,
absence or quantity of a specific circulating nucleic acid sequence in the amplified,
circulating nucleic acid product.
6. The method of claim 1, wherein the biological sample is whole blood
and the non-cellular fraction is plasma or serum.
7. The method of claim 6, wherein the plasma or serum is collected from
less than 150m of the whole blood.
8. The method of claim 6, wherein the plasma or serum is separated from
the whole blood in the absence of an anticoagulant.
9. The method of claim 1, wherein the circulating nucleic acids are
circulating DNAs or circulating RNAs.
10. The method of claim 9, wherein the circulating DNAs comprise
a tumor-derived DNA, a fetus-derived DNA, a donated organ-derived DNA, a
transplanted cell-derived DNA, a transplanted tissue-derived DNA, or combinations
thereof.
11. The method of claim 9, wherein the circulating DNAs comprise
a tumor-derived DNA.
12. The method of claim 1, wherein the filtration of the biological sample
is performed by using a membrane having a pore size between 0.01 micron and 5
micron.
13. The method of claim 12, wherein the filtration of the biological sample
is performed by using a membrane having a pore size between 1 micron and 2 micron.
14. The method of claim 1, wherein the dry solid matrix is a cellulose
matrix that is devoid of any detergent.
15. The method of claim 1, wherein the dry solid matrix is impregnated
with a chaotropic salt.
16. The method of claim 4, wherein the biological sample is whole blood,
the circulating nucleic acids are cirulating DNAs present in the whole blood and the
dry solid matrix is a cellulose matrix that is devoid of any detergent, and wherein the
non-cellular fraction of the whole blood is separated via filtration through a filtration
membrane having a pore size between 1 micron and 2 micron.
17. A method for processing whole blood at a point-of-collection to collect
plasma or serum, the method comprising:
filtering the whole blood to separate the plasma or serum at the point-ofcollection;
collecting the separated plasma or serum on to a dry solid matrix, wherein the
dry solid matrix is devoid of any detergent; and
drying the collected plasma or serum in the dry solid matrix.
18. The method of claim 17, wherein the plasma or serum is separated
from the whole blood via filtration through a filtration membrane in the absence of an
anticoagulant.
19. The method of claim 18, wherein the dry solid matrix is a cellulose
matrix that is impregnated with a chaotropic salt.
20. The method of claim 19, further comprising storing the dried plasma or
serum.
21. A method for detection of circulating nucleic acids from a dried
sample of plasma or serum, the method comprising:
extracting the circulating nucleic acids from the dried sample of plasma or
serum;
performing a whole genome amplification of the extracted circulating nucleic
acids to generate an amplified, circulating nucleic acid product; and
detecting the presence, absence, or quantity of a specific circulating nucleic
acid sequence in the amplified, circulating nucleic acid product.
22. The method of claim 21, wherein the whole genome amplification of
the extracted circulating nucleic acids comprises:
circularizing the extracted circulating nucleic acids using a single strandspecific
ligase to form single-stranded nucleic acid circles; and
amplifying the single-stranded nucleic acid circles via random-primed rolling
circle amplification.
23. A device for collecting a non-cellular fraction containing circulating
nucleic acids from a biological sample, the device comprising:
a filtration membrane configured to separate the non-cellular fraction from
intact cells; and
a dry solid matrix configured to collect the separated, non-cellular fraction,
wherein the dry solid matrix is devoid of any detergent, and wherein the filtration
membrane and the dry solid matrix are configured to establish a direct contact
between them.
24. The device of claim 23, wherein the filtration membrane comprises a
sample application zone and a transfer zone, and wherein the solid matrix directly
touches the transfer zone of the filtration membrane.
25. The device of claim 24, wherein the filtration membrane and the dry
solid matrix are arranged laterally such that the non-cellular fraction of the biological
sample passes from the sample application zone to the dry solid matrix via the transfer
zone in a lateral direction.
26. The device of claim 23, wherein the filtration membrane is disposed on
a first solid support and the dry solid matrix is disposed on a second solid support, and
wherein the first solid support is connected to the second solid support via a means for
establishing the direct contact of the filtration membrane with the dry solid matrix.
27. The device of claim 23, wherein the filtration membrane and the dry
solid matrix are arranged vertically such that the non-cellular fraction of the biological
sample passes through the filtration membrane to reach the dry solid matrix in a
vertical direction.
28. The device of claim 23, wherein the dry solid matrix is impregnated
with a chaotropic salt.
29. The device of claim 23, wherein the dry solid matrix has a width of up
to 8 millimeters.
30. A method for collecting plasma or serum from whole blood using the
device of claim 23, the method comprising:
providing the whole blood at a sample application zone of the filtration
membrane;
passing the whole blood through the filtration membrane to separate the
plasma or serum from blood cells; and
collecting the separated plasma or serum on the dry solid matrix.
31. The method of claim 30, wherein the plasma or serum is collected
from less than 150m of whole blood.
32. The method of claim 31, further comprising drying the collected
plasma or serum on the dry solid matrix for storage.