WO2017070339A1 - Microfluidic device for enrichment of nucleic acid sequence alterations - Google Patents

Abstract

A method of using a multi-chambered microfluidic device for enrichment of target nucleic acid sequences is provided.

Description

MICROFLUIDIC DEVICE FOR ENRICHMENT OF NUCLEIC ACID SEQUENCE ALTERATIONS

Cross-Reference to Related Applications

This application claims the befit of the filing date of U.S. application

Serial No. 62/243,906, filed on October 20, 2015, the disclosure of which is incorporated by reference herein.

Background

A commonly encountered situation in genetic analysis entails the need to identify a low percent of variant DNA sequences ("target sequences") in the presence of a large excess of non-variant sequences ("reference sequences"). Examples for such situations include: (a) identification and sequencing of a few mutated alleles in the presence of a large excess of normal alleles; (b) identification of a few methylated alleles in the presence of a large excess of unmethylated alleles (or vice versa) in epigenetic analysis; (c) detection of low levels of heteroplasmy in mitochondrial DNA; (d) detection of drug -resistant quasi-species in viral, bacterial or parasitic infections, and (e) identification of tumor-circulating DNA in blood of cancer patients (where people are suspected of having cancer, to track the success of cancer treatment or to detect relapse) in the presence of a large excess of wild-type alleles. Circulating tumor DNA can be isolated from, for example, plasma, serum, circulating tumor cells or exosomes COLD-PCR methods for enriching the concentration of low abundance alleles or target sequences in a sample PCR reaction mixture were initially described in a published patent PCT application entitled "Enrichment of a Target Sequence", International Application No. PCT/US2008/009248, by Gerassimos Makrigiorgos, which is incorporated herein by reference. The described COLD- PCR enrichment methods are based on a modified nucleic acid amplification protocol which incubates the reaction mixture at a critical denaturing temperature "T_c". The PCT application discloses two formats of COLD-PCR, namely full COLD-PCR and fast COLD-PCR. For example, in full COLD-PCR, the reaction mixture is subjected to a first denaturation temperature (e.g. , 94°C) which is chosen to be well above the melting temperature for the reference (e.g., wild- type) and target (e.g., mutant) sequences similar to conventional PCR. Then, the mixture is cooled (e.g., to 70°C) to facilitate the formation of reference-target heteroduplexes by hybridization. In the basic full COLD-PCR method, lowering of the temperature from the first denaturing temperature (e.g., 94 °C) to the hybridization temperature (e.g., 70°C) over a relatively long time period (e.g., 8 minutes) or retaining the reaction mixture at the hybridization temperature for a relatively long time period (e.g., 70°C for 8 minutes) provides for proper hybridization. Once cooled, the reaction mixture contains not only reference- target heteroduplexes but also reference-reference homoduplexes (and to a lesser extent target-target homoduplexes). When the target sequence and reference sequence cross hybridize, minor sequence differences of one or more single nucleotide mismatches or insertions or deletions anywhere along a short (e.g. , <200 bp) double stranded DNA sequence will generate a small but predictable change in the melting temperature (T_m) for that sequence. Depending on the exact sequence context and position of the mismatch, melting temperature changes of 0.1-20°C are contemplated.

Full COLD-PCR, as described in the above referred PCT application, is premised on the difference in melting temperature between the double stranded reference sequence and the hybridized reference-target heteroduplexes. After cooling down to form reference-target heteroduplexes, the reaction mixture is incubated at a critical denaturing temperature (T_c), which is chosen to be less than the melting temperature for the double stranded reference sequence and higher than the lower melting temperature of the reference-target heteroduplexes, thereby preferentially denaturing the cross hybridized target -reference heteroduplexes over the reference-reference homoduplexes. Summary

Devices and methods for enriching a target DNA sequence in a sample are provided herein. The device and methods allow for enriching of a target nucleic acid sequence in a background of related reference nucleic acid sequences by use of a temperature that allows for preferential denaturation of heteroduplexes over duplexes. The target sequences are the sequences that one wants to determine whether or not they are in a mixed or potentially mixed sample including reference sequences. A target sequence is a nucleic acid that may be less prevalent in a nucleic acid sample than a corresponding reference sequence, for example, the target sequence may make-up only about < 0.01% of the total amount of reference sequence plus target sequence in a sample. The lower limit of detection is based on the number of genomic equivalents in the sample, such that the sample must contain at least one target sequence in order to be able to enrich the target sequence. For example, the limit of detection may be lower than 0.01% target in a background of reference sequences, as defined by the total number of target template molecules present, e.g., the relative molarity or genomic equivalents. The target sequence may include, but is not limited to a somatic mutation, a mitochondrial mutation, a strain or species. For example, a sample (e.g., blood sample) may contain numerous normal cells and few cancerous cells and/or circulating free (cf) tumor DNA. The normal cells contain non-mutant or wild-type alleles, while the small number of cancerous cells and low levels of cfDNA contain somatic mutations. In this case the mutant is the target sequence while the wild-type sequence is the reference sequence. The target sequence must differ by at least one nucleotide from the reference sequence, but must be at least 50% homologous to the corresponding reference sequence. The devices and methods described herein may also be combined with PCR or other types of nucleic acid amplification. In one embodiment, the invention provides a method of using a multi- chambered microfluidic device for enrichment of target nucleic acid sequences. The method includes providing a multi-chambered microfluidic device comprising a first chamber configured to be in fluid communication with a second chamber when a gate between the first chamber and the second chamber is adjusted to allow for fluid flow, and a third chamber configured to be in fluid communication with the second chamber when a gate between the third chamber and the second chamber is adjusted to allow for fluid flow. A sample comprising nucleic acid, e.g., isolated nucleic acid in solution, suspected of having one or more target sequences with nucleic acid alterations is introduced to the first chamber when the gate is adjusted to block fluid flow from the first chamber to the second chamber. The first chamber comprises a plurality of different reference sequences, e.g., beads or an array, selected to have reference sequences of interest, such as a portion of a gene that in the human population is

heterogeneous. The sample and beads or array in the first chamber is subjected to denaturation and hybridization conditions, wherein the hybridization conditions allow for one or more target sequences to hybridize with a reference sequence that is at least 50% homologous to the target sequence. The temperature in the first chamber is then increased to a temperature, T_ci, that allows for

heteroduplexes of the reference sequence and a target sequence with an alteration (e.g., a SNP, a deletion or an insertion) to denature but not homoduplexes of the reference sequence. The cycle of denaturation, hybridization and application of T_ci may be repeated in the first chamber. The gate is adjusted to allow for flow of fluid having the denatured heteroduplexes to the second chamber. The second chamber comprises beads or an array with the same (first) or a different (second) set of reference sequences, and the sample is subjected to denaturation and hybridization conditions, wherein the hybridization conditions allow for the one or more target sequences to hybridize with a reference sequence that is at least 50% homologous to the target sequence. The temperature in the second chamber is increased to a temperature T_c2 that is lower than T_cl. The cycle of denaturation, hybridization and application of T_c2 may be repeated in the second chamber. Subsequent chambers contain yet further sets of the first, second or different reference sequences and samples therein are subjected to conditions with lower T_c. The denatured heteroduplexes are collected, e.g., in a collection vessel, which is in fluid communication with the last enrichment chamber in the series, where a gate between the last enrichment chamber and collection vessel allows for control of fluid flow. The collected target sequences from all the chambers may be re-introduced into the device for a second round of enrichment prior to detection of the altered sequence.

Also provided is a method of using a device for enrichment of target nucleic acid sequences. The method includes providing a microfiuidic device comprising a first chamber configured to be in fluid communication with a collection vessel when a gate between the first chamber and the collection vessel is adjusted to allow for fluid flow. An isolated nucleic acid sample suspected of having one or more target sequences with nucleic acid alterations is introduced to the first chamber when the gate is adjusted to block fluid flow from the first chamber to the collection vessel. The first chamber comprises a plurality of different reference sequences, for instance, on beads or in an array. The sample and beads or array in the first chamber are subjected to denaturation and hybridization conditions, wherein the hybridization conditions allow for the one or more target sequences to hybridize with a reference sequence that is at least 50% homologous to the target sequence. The temperature in the first chamber is increased to a T_cl that allows for heteroduplexes of the reference sequence and a target sequence with an alteration to denature but not homoduplexes of the reference sequence. The cycle of denaturation, hybridization and application may be repeated with the same T_c or the cycle may be repeated with a different (lower) T_c. The gate is adjusted to allow for flow of fluid having the denatured heteroduplexes to the collection vessel. In one embodiment, the nucleic acid comprises circulating DNA, exosomal RNA, mitochondrial DNA, bacterial nucleic acid, parasite nucleic acid or viral nucleic acid. In one embodiment, the nucleic acid is obtained from cells in a physiological sample. In one embodiment, the cells are circulating cells in physiological fluid. In one embodiment, plasma is separated from blood and the plasma is used for genotype analysis. In one embodiment, the nucleic acid is introduced to the first chamber using pressure, an electric current or a magnetic field. In one embodiment, a pre-chamber is configured to isolate plasma from blood or to isolate nucleic acid from circulating tumor cells or from plasma. In one embodiment, a pre-chamber is configured to isolate the nucleic acid from cells and optionally to convert RNA to DNA. In one embodiment, the alterations in the target sequences include copy number variations, insertions, deletions or nucleotide substitutions. In one embodiment, applied pressure or an electric current allows for separation of the denatured heteroduplexes from

homoduplexes in an enrichment chamber. In one embodiment, if the beads are magnetic beads, the denatured heteroduplexes are separated from the homoduplexes using a magnetic field. In one embodiment, the gate between the first chamber and the second chamber, is blocked and fluid is added to the first chamber and the beads or array are subjected to a T_c2, which is different than T_ci, thereby allowing for heteroduplexes with a different T_m than T_ci to denature. In one embodiment, the method includes unblocking (e.g., opening) a gate between chambers in the device and subjecting the sample in one chamber to conditions that allow for separation of fluid having the denatured heteroduplexes from the bound homoduplexes to another chamber or to a collection vessel. In one embodiment, to process multiple samples in parallel, the device comprises in parallel a plurality of first chambers. In one embodiment, the nucleic acid is amplified before introduction to the first chamber.

Various gene fusions have been shown to be important in the detection and treatment of various cancers. Reference sequence (RS) oligonucleotides (oligo) may be synthesized that contain the 5 ' sequence of one of the gene regions and the 3 'sequence of the other gene region(s) involved in the fusion (for example, fusions such as ALK fusions with EML4, ROS1 fusions with CD74, EZR; RET fusions with KIF5B, CCD6; TMPRSS2-ERG fusions; and others). Because fusion genes are very far apart on the chromosome, there would be insufficient sequence present in the RS-oligo to compete with the fusion product, that is, the only sequences that could bind tightly to different RS-oligo fusions would be those samples that contained the exact sequence from each gene region at the fusion breakpoints. In one embodiment, each RS-oligo contains the DNA sequences at and surrounding the fusions point for each fusions type. RS-oligos are then attached to beads or in an array. The solution containing any potential fusions is incubated in the device so that the fusion sequence (whether derived from DNA or RNA) is tightly bound to the RS-oligo. The unbound product may be washed out of the device and collected for use in any quantification algorithm, and the bound solution is then eluted or remainsw in the chamber. Fluorescent probes or other detection components may be added to the chamber or to the eluted sample which is enriched for the fusions. Detection of the specific fusions is determined by the differing characteristics of the probes (e.g., if the probe is a fluorescent probe, then the different emission spectra would indicate the specific fusion that has been detected).

In one embodiment, a microfiuidic device is provided that has a plurality of chambers that are serially in fluid communication with each other. The device includes a first chamber configured to be in fluid communication with a second chamber via a conduit and an adjustable gate configured to control fluid flow from the first chamber to the second chamber, where the first chamber comprises beads or an array having a plurality of different reference sequences. Optionally the second chamber is a collection vessel. The device may also include a third chamber configured to be in fluid communication with the second chamber via a conduit and an adjustable gate configured to control fluid flow from the second chamber to the third chamber.

Brief Description of the Figures

Figure 1. An exemplary device for use in the enrichment method. In this example, beads having a first set of RS-oligonucleotides attached thereto are in a first chamber of a microfiuidic device. Subsequent enrichment chambers having beads with the same set as the first chamber or a different set of RS- oligonucleotides are in fluid communication with each other and the first chamber.

Figure 2. An exemplary device for use in the enrichment method. In this example, in a single enrichment chamber of a microfiuidic device, a population of beads is provided where different subsets of the population have different RS- oligonucleotides (having different Tc's) attached thereto.

Figure 3. An exemplary device for use in the enrichment method. In this example, an array has a geometric pattern of different RS-oligonucleotides (having different Tc's) attached thereto, and is placed in a single enrichment chamber of a microfiuidic device.

Figure 4. An exemplary nucleic acid isolation device which may be employed with the enrichment device for use in the methods or for use in the enrichment and detection of mutations in liquids at the time of collection or later. In this example, a flexible design device can be used for a population of beads where different subsets of the population have different RS-oligonucleotides (having different Tc's) attached thereto or an array that has a geometric pattern of different RS-oligonucleotides (having different Tc's) attached thereto, and is placed in a single detection chamber of a microfiuidic device.

Detailed Description

Definitions As used herein, the term "enriching a target sequence" refers to increasing the amount of a target sequence and increasing the ratio of target sequence relative to a corresponding reference sequence in a sample. For example, where the ratio of target sequence to reference sequence is initially 0.01% to 0.1% in a sample, the target sequence may be enriched so as to produce a ratio of 1 % to 5% target sequence to reference sequence.

As used herein the term "target sequence" refers to a nucleic acid that is less prevalent in a nucleic acid sample than a corresponding reference sequence. The target sequence makes-up less than 50% of the total amount of reference sequence + target sequence in a sample. The target sequence may be a mutant allele. For example, a sample (e.g., blood sample) may contain numerous normal cells and few cancerous cells. The normal cells contain non-mutant or wild-type alleles, while the small number of cancerous cells contains somatic mutations. In this case the mutant is the target sequence while the wild-type sequence is the reference sequence. As used herein, a "target strand" refers to a single nucleic acid strand of a double-stranded target sequence. The target sequence is at least 50% homologous to the corresponding reference sequence, but must differ by at least one nucleotide from the reference sequence. Target sequences may be amplifiable via PCR with the same pair of primers as those used for the reference sequence.

As used herein, the term "reference sequence" refers to a nucleic acid that is more prevalent in a nucleic acid sample than a corresponding target sequence. The reference sequence may make-up 0.01% to 99% or more of the total reference sequence plus target sequence in a sample prior to the use of the method described herein. For example, the reference sequence may be over 50% of the total reference sequence + target sequence in a sample. As used herein, a "reference strand" refers to a single nucleic acid strand of a double-stranded reference sequence. The target and reference sequences can be obtained from a variety of sources including, genomic DNA, CTCs from blood, cfDNA from plasma or serum, RNA from exosomes converted to cDNA, RNA from any source converted to cDNA mitochondrial DNA, viral DNA or RNA, mammalian DNA, fetal DNA, parasitic DNA or bacterial DNA. While the reference sequence is generally the wild-type and the target sequence is the mutant, the reverse may also be true. The mutant may include any one or more nucleotide deletions, insertions or alterations including translocations and SNPs (single nucleotide polymorphisms). The target sequence may be a sequence indicative of cancer in a cell, metastases of cancer via detection of cells comprising the mutation in a different tissue or in the blood, prognosis of cancer or another disease, drug or chemotherapeutic sensitivity or resistance of a cancer or a microorganism to a therapeutic, or presence of a disease related to a somatic mutation such as mitochondrial heteroplasmy.

As used herein, the term "wild-type" refers to the most common polynucleotide sequence or allele for a certain gene in a population. Generally, the wild-type allele will be obtained from normal cells. The wild-type is generally the reference sequence.

As used herein, the term "mutant" refers to a nucleotide change (e.g., a single or multiple nucleotide substitution(s), deletion(s), or insertion(s), translocation s), fusion(s), SNPs, CNVs, altered methylation patterns, or combinations thereof) in a nucleic acid sequence.. A nucleic acid which bears a mutation has a nucleic acid sequence (mutant allele) that is different in sequence from that of the corresponding wild-type polynucleotide sequence. The invention is broadly concerned with somatic mutations or polymorphisms. The target sequence may also be referred to as the mutant sequence. The methods described herein are useful in selectively enriching a target strand which contains 1 or more nucleotide sequence changes as compared to the reference strand. As used herein the term "melting temperature" or "T_m" refers to the temperature at which a polynucleotide dissociates from its complementary sequence. Generally, the T_m may be defined as the temperature at which one-half of the Watson-Crick base pairs in a double stranded nucleic acid molecule are broken or dissociated (i.e., are "melted" or "denatured") while the other half of the Watson-Crick base pairs remain intact in a double stranded conformation. In other words, the T_m is defined as the temperature at which 50% of the nucleotides of two complementary sequences are annealed (double strands) and 50% of the nucleotides are denatured (single strands). T_m therefore defines a midpoint in the transition from double-stranded to single- stranded nucleic acid molecules (or, conversely, in the transition from single-stranded to double- stranded nucleic acid molecules).

The T_m can be estimated by a number of methods, for example by a nearest-neighbor calculation as per Wetmur 1991 (Wetmur, J. G. 1991. DNA probes: applications of the principles of nucleic acid hybridization. Crit Rev Biochem Mol Biol 26: 227-259,) and by commercial programs including OligoTM Primer Design and programs available on the internet. Alternatively, the T_m can be determined though actual experimentation. For example, double- stranded DNA binding or intercalating dyes, such as Ethidium bromide or SYBR- green (Molecular Probes) can be used in a melting curve assay to determine the actual T_m of the nucleic acid. Additional methods for determining the T_m of a nucleic acid are well known in the art. Some of these methods are listed in the inventor's prior patent application entitled "Enrichment of a Target Sequence", International Application No. PCT/US2008/009248, incorporated by reference herein.

As used in connection with the present invention, the term "critical temperature" or "T_c" refers to a temperature selected to preferentially denature duplexes of target strands and the reference blocking sequence (heteroduplexes) as compared to the reference blocking sequence -reference strand duplexes. The critical temperature (T_c) is selected so that duplexes of the reference blocking strands and complementary reference strands remain substantially undenatured when the reaction mixture is incubated at T_c yet duplexes of the reference blocking strands and the target strands substantially denature. The term

"substantially" means at least 60%, at least 70%, at least 80%, at least 90% or at least 98% in a given denatured or undenatured form. For instance, the selected critical temperature "T_c" may be about 84.5° C, whereas the first denaturing temperature is 95° C.

As used herein, "homology" refers to the subunit sequence similarity between two polymeric molecules, e.g., two polynucleotides or two polypeptides. An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nucleic Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

Methods and Devices

This application describes methods and the use of a device, e.g., a Point of Care (POC) plasmapheresis device, to isolate, enrich and/or detect nucleic acid, e.g., circulating free tumor DNA (also known as cfDNA or ctDNA or cftDNA), exosomal RNA (RNA to cDNA), DNA isolated from CTCs, mitochondrial DNA, bacterial DNA or RNA, viral DNA, viral RNA, and the like. The methods and device provide for the sensitive detection of nucleic acid alterations including copy number variation, gene expression signatures and mutations (including point mutations, insertions and deletions). The device includes at least one chamber (a first "enrichment" chamber), each chamber having at least two conduits for fluid communication into and out of the chamber, where the contents of the chamber including a nucleic acid sample may be controllably heated and cooled. In one embodiment, the device includes a series of enrichment chambers (in tandem) in fluid communication with each other, e.g., where each chamber subjects the sample to a different T_c. In one embodiment, different enrichment reactions are conducted sequentially in the same enrichment chamber. Individual chambers in the devices can be independently heated and cooled in a controlled fashion. Individual chambers in the device may be isolated from other chambers, e.g. , by gated opening and closing between chamber(s) in a controlled fashion.

The device and methods provides for enrichment of a population of distinct target sequences. Those enriched sequences, after application of a specific T_c to an enrichment chamber having a population of distinct reference sequences and a nucleic acid sample suspected of having one or more target sequences, results in unbound DNA with at least some of the target sequences. The unbound nucleic acid, such as DNA, passes (flows) to the next chamber(s) or a collection vessel using, for example, applied pressure, an electric current or a magnetic field. In one embodiment, the device may also have a pre-chamber for introducing a physiological sample having cells, e.g. , whole blood. This pre- chamber may be configured to capture cells, for instance, circulating tumor cells (CTCs), or to isolate nucleic acid from a sample, such as one having lysed cells or from plasma (see Figure 4). If exosomal RNA is isolated from a physiological sample, a pre-chamber may be employed to convert RNA into cDNA in the device. A nucleic acid containing solution, containing, for example, the exosomes and/or cfDNA, may be transferred via a conduit to a first enrichment chamber. In one embodiment, one of the chambers of the device, e.g., a pre-chamber or a chamber after the last enrichment chamber, may contain beads, e.g. , magnetic beads to determine the concentration of the isolated nucleic acid.

The devices and methods described herein may be used in a variety of situations in which one wants to identify a target nucleic acid from within a mixed population of sequences with some sequence homology. In particular, the devices and methods may be useful to enrich sequences for mutation analysis, in particular somatic mutational analysis, and can be used to identify cells or subjects having mutations related to, for example, development of cancer, prognosis of cancer or small molecule and biologic drug efficacy, mosaicism or mitochondrial myopathies. For other potential applications of this method for somatic mutation analysis, see, for example, Erickson, Mutat Res.. 705 :96 (2010).

For example, assays for detection of mutations in EGFR, KRAS, NRAS, PIK3Ca, and BRAF known to be associated with cancerous transformation of cells may be facilitated using the devices and methods. In addition, the methods and devices are useful for determining strain or species designation in a potentially mixed population, such as during an infection. The methods could also be used to identify antibiotic resistant mutants developing during drug treatment of an infection, such as in a viral e.g. , HIV, or bacterial infection, or to identify chemotherapy resistant mutants during chemotherapy. Those skilled in the art will appreciate other uses of the devices and methods described here.

Samples

Samples useful in the methods and devices described herein include any substance containing or presumed to contain a nucleic acid of interest (target and reference sequences) or which is itself a nucleic acid containing or presumed to contain a target nucleic acid of interest. The term sample thus includes a sample of nucleic acid (genomic DNA, cDNA, RNA, cfDNA), cell, organism, tissue, fluid, or substance including, but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, stool, external secretions of the skin, respiratory, intestinal and genitourinary tracts, saliva, blood cells, biopsy, tumors, organs, tissue, samples of in vitro cell culture constituents, natural isolates (such as drinking water, seawater, solid materials), microbial specimens, and objects or specimens that have been "marked" with nucleic acid tracer molecules. Thus, the nucleic acid sample may be from RNA, mRNA, cfDNA, cDNA and/or genomic DNA, or a combination thereof. These nucleic acids can be isolated from tissues or cells according methods known to those of skill in the art. Complementary DNA or cDNA may also be generated according to methods known to those of skill in the art. Alternatively nucleic acids sequences of the invention can be isolated from blood or other physiological fluids by methods well known in the art

Nucleic acid sequences of the invention can be amplified, e.g., by polymerase chain reaction, prior to or after use in the enrichment methods and devices described herein. The amplification products may be directly sequenced by selectively degrading one strand of the amplified target sequence. One method of selecting a single strand of a double-stranded DNA product is where one strand may be biotinylated and bound to a column or solid support coated with streptavidin. The non-biotinylated strands can then be purified by denaturing the strands and removing the biotinylated strand bound to the avidin coated solid support in order to allow for sequencing of the non-biotinylated strand.

Alternatively, the PCR reaction can be carried out using a 5'-phosphorylated amplification primer in addition to the sequencing primer such that one strand of the product comprises a 5' phosphate. This strand can then be degraded by incubation with a 5'-phosphate dependent exonuclease, such as lambda exonuclease which was used in the Examples..

Exemplary Embodiments

In one embodiment, an enrichment chamber may house different reference sequence oligonucleotide (RS-oligo) groupings, specific for target nucleic acid regions, e.g., attached to beads (magnetic or non-magnetic) or attached to a surface, such as an array. One enrichment chamber may include RS-oligos that have specific critical temperatures (T_c) (e.g., a range of similar T_cs, such as those that vary by, for example, from about 0.2°C to about 10°C, and that chamber is in fluid communication with another (downstream) enrichment chamber that has RS-oligos with a different (lower) T_c or range of lower T_c's. Either a pool of unbound DNAs from all serially connected chambers is collected, or unbound DNAs from different chambers are individually collected. Alternately, all RS- oligos may be in one chamber and multiple elutions of the unbound DNA after each increasing T_c application may be used.

For example, after denaturation and hybridization, an enrichment chamber may be incubated (heated) at the targets' T_c's to detach (denature) any DNA that has alteration(s) in the target sequences as compared to the reference sequences. At the T_c, the downstream gate(s) open and the unbound DNA flows to the next enrichment chamber, or a collection vessel, through use of, for instance, pressure or an electric current or gravity. This may be a multi-chambered microfiuidic device, whereby the flow from one chamber to another is "gated" such that opening and closing of the gates is controlled and the aqueous DNA containing sample is moved from one chamber to the next. If magnetic beads are used, a magnetic field may be generated to separate the beads with the bound DNAs from the unbound DNAs. The unbound DNAs can progress (be transferred to) to the second chamber, e.g., using controlled gating and applied force. In one embodiment, a fluid collection vessel is in fluid communication with the final chamber, e.g., to collect fluid flowing from the enrichment chamber(s).

In one embodiment, a panel of forward and reverse RS-oligos specific to the reference sequence of relevant genes may be covalently bound to a surface, e.g., in a bead format or in an array format. In one embodiment, if the beads containing the bound oligonucleotides are magnetic beads, then the sample may be moved from one chamber to the next using a solution transfer process making sure that the pore size of the gate prevents the transfer of the beads. For example, RS-oligos used in Improved and Complete Enrichment CO-amplification at Lower Denaturation temperature-PCR (ICE COLD-PCR) reactions are bound to the bead surface or to an array. The sample (in solution) that is introduced to the device may be any DNA (or RNA) that can be isolated from a sample. For example, the sample may be DNA (or RNA converted to cDNA) isolated from formalin- fixed, paraffin- embedded (FFPE), plasma, serum, urine or other bodily fluids. If the sample is genomic DNA, it may be sheared or fragmented prior to enrichment. In addition, amplified PCR products from a PCR or multiplex PCR containing one or a plurality of amplicons of interest may be used as the starting material.

In one embodiment, a DNA containing aqueous solution enters the first enrichment chamber, which may contain RS-oligos (both forward and reverse oligos in order to capture both strands of the reference sequence) whose T_c's are the same or within a predetermined range. In some embodiments, the population of forward and reverse RS-oligos may have different T_c's and are located in different T_c-specific enrichment chambers. In one embodiment, a plurality of enrichment chambers in tandem is employed, where the first chamber in the series has RS-oligos with the highest T_c and subsequent chambers have RS-oligos with sequentially lower T_c values. The samples are denatured, hybridized and then incubated at their assigned (predetermined) T_c's to denature those samples that contain mutations in the regions selected by the set of RS-oligos in the chamber. Samples may be cycled in each chamber several times, e.g., using the same denaturation, hybridization and T_c, to get optimal enrichment of the alterations in the regions interrogated.

At the end of the T_c step, the "gate" to the next (downstream) chamber opens and the unbound material present in solution is moved to the next chamber. For example, the fluid may be moved using gates and an electric current in order to transfer to the next chamber any DNA that is not bound to the RS-oligos. Alternatively, the fluid may be moved using a magnetic field that is applied to immobilize the magnetic beads containing the RS-oligos with DNA bound to them and the solution containing the DNAs of interest are moved to the next chamber, for instance, using a microfluidic pump and gates. In one embodiment, a subsequent enrichment chamber may contain RS- oligos bound to a surface, where the RS-oligos are those whose T_c's are slightly lower than the T_c's in the previous enrichment chamber. The subsequent enrichment chamber may be subjected to the same temperature cycling

(denaturation/hybridization) as before (except for T_c) and then the unbound DNA is transferred to the next chamber, or a collection container, by adjusting a gate to the next chamber to open (e.g., while the gate to the prior chamber remains closed) and applying a force so that the unbound DNA flows to the next chamber or collection container. This process can be repeated in the same chamber or may be repeated through multiple chambers depending on the number of T_c's required for enrichment of sequence alterations of multiple genes.

After the last chamber for enrichment cycling, the unbound portion of the sample is collected in a container. The collected sample has DNAs that have been depleted of both the forward and reverse reference sequence DNA strands for the genes whose specific RS-oligos were used in the enrichment chamber(s).

Fragments of DNA that do not contain any of the genes of interest may still be present, but may not interfere with the analysis of the enriched sample, because the genes for those regions may not be analyzed in the downstream mutation detection process.

For quantification, the DNA in the eluted (enriched or unenriched) sample may be incubated with a fluorescent detection system which employs an intercalating dye, antibody, etc. The reagent for detection may be automatically removed prior to final sample collection, e.g., before analysis. The DNA in the eluted, enriched sample may also be tagged with a fluorescent primer or nucleotide. Alternately, the initial amount of DNA can be determined using magnetic bead capture and quantification. As an example of such a methodology, see Leslie et al., J. Am Chem. Soc, 134:5689 (2012). Simple, sequence- independent quantification methods include spectrophotometry and fluorometry. A sequence-independent approach (e.g., chaotrope-driven aggregation, CDA) facilitates a sensitive method to quantify DNA directly in crude samples or seamlessly via interface extraction, purification, and quantification in a single process. With CD A, bead aggregation results from DNA binding to the silica surface of the paramagnetic beads in, e.g., 6 M guanidine (GdnHCl). The aggregation is sensitive to low concentrations of DNA and insensitive to the presence of proteins. CDA may employ large, irregular-shaped 8-μπι beads and highly denaturing conditions.

After the eluted enriched sample is collected, all chambers are incubated at about 95°C to about 98°C to release all (unenriched) DNA bound to the RS- oligos (the eluted unenriched sample). All gates are then opened and the DNA collected, e.g., via gravity, electrophoresis or fluid flow using applied pressure. This DNA may also be quantified, for instance by incubating with a fluorescent compound, as discussed above, and the amount of the bound (unenriched) DNA can be measured. If the DNA has been tagged with a fluorescent primer or nucleotide, it may be directly measured.

In order to have sufficient enriched DNA for analysis of multiple enriched regions, the sample may be subjected to amplification, e.g., PCR, such as whole genome amplification, standard 3 -cycle PCR, touchdown PCR etc. either prior to the chambered enrichment process (CEP), after the CEP, or both before and after the CEP. If the samples were used in a single cell sequencing process, such as that used by NanoString or other methodology for digital profiling of single molecules, amplification is not needed.

If the samples are amplified, e.g., using PCR, prior to the CEP, a universal primer may be added to all the DNA samples whereby the universal primer contains a double stranded portion with a loop. The double stranded portion insures that there would be no interaction between other universal primers and thus prevents primer-dimers, etc. This is in essence a hairpin structure that quickly anneals with itself before annealing with other structures. After the CEP, the hairpin loops are cleaved, e.g., by Uracil-N-glycosylase if the sequence contains uracils, by a restriction enzyme if the hairpin loop contains a restriction enzyme site, or by a similar process. Universal primers may then be added to a standard PCR reaction to produce sufficient quantities for multiple NGS reactions. Other types of DNA may be added to the initial PCR fragment such that only specific primers in any downstream PCR amplify fragments without forming primer-dimers.

If the enriched samples are amplified after CEP, then universal primers may be ligated to the DNA and then the DNA may undergo emulsion PCR. In this example, all the emulsion droplets contain the same primer sets. When these are merged with the droplets containing the CEP-ligated DNAs, all the PCR reactions are the same, so there is no problem with a particular droplet containing an alteration where the primers were not compatible with the alteration. After the emulsion PCR, the droplets are disrupted and the samples may be sequenced using any NGS platform or other methodology for detection of sequence alterations.

As the method and device can be used with DNA isolated from any source, in one embodiment, an automated DNA isolation methodology is used whereby the isolated DNA is directly injected into the first chamber of the CEP device. In one embodiment, there are separate isolation cartridges for different sample sources used.

The method and device(s) is particularly suitable for enrichment of alterations in DNA when these alterations are extremely scarce in the population being analyzed. This is especially useful for monitoring during cancer treatment and detection of drug resistance or detection of new druggable mutations occurring in a bodily fluid of the patient. Thus, because the relative proportion of mutations compared to the reference sequences is extremely low and the available patient sample volume which can be used may be low, the disclosed device allows for an easy, compact method to isolate and enrich for mutations in many different genes. The chambers of the CEP device can be designed to capture complementary nucleic acid to the forward and reverse reference

oligonucleotides, which can correspond to any gene or gene region, resulting in a solution of highly enriched DNA for any and all mutations present in the region(s) interrogated. These enriched samples may be analyzed on various downstream sequence analysis platforms, including but not limited to most Next Generation Sequencing (NGS) platforms.

For instance, the sample is loaded into a CEP device (e.g., one having RS- oligo beads or an RS-oligo array) that contains multiple forward and reverse RS- oligos for multiple regions in one or more genes or gene regions, each in a specific chamber according to the T_c with the highest T_c in the first chamber and the lowest T_c in the last chamber. At least the first chamber in the CEP device is incubated at 95°C then cooled for annealing of reference and target DNA sequences to RS-oligos at 70°C. The temperature is raised to the highest T_c for multiple chambered devices ("A type") and to the lowest T_c for single chambered devices ("B-type") of the RS-oligo groupings to denature the first set of target sequences from the RS-oligos.

For A-type devices, where there are multiple chambers, the solution that moves onto the next chamber contains the mutated sequences for the amplicon(s) with the highest T_c and the mutated and wild-type sequences for all the other amplicons being tested. In this type of device additional solution may not need to be added for each successive T_c.

For B-type devices where there is a single chamber for enrichment, each mutation enriched fraction is collected while the remaining mutant and wild-type sequences stay bound to their respective RS-oligos. In this type of device additional solution may need to be added for each successive T_c.

A first gate between a first chamber and a second chamber, where the first chamber has the mixed bound and unbound DNA sample, is opened thereby providing a conduit for fluid communication between the first and second chambers. Electrophoresis or pressure may be used to transfer the unbound DNA sample to the next (second) chamber which has a second gate for a conduit to a third chamber of a collection vessel. The first gate is closed. The temperature in the next chamber may be raised to 95°C, then annealed at 70°C for heteroduplex formation and then is raised to a predetermined T_c for the RS-oligos in the chamber. The second downstream gate between the mixed bound and unbound sample is opened while the first (upstream) gate remains closed. Electrophoresis or pressure may be used to transfer the unbound sample to the next chamber. The gate is closed. Denaturation, applied T_c, and transfer of unbound sample (e.g., to a collection vessel) using gatings, is repeated.

After collection of the mutant sequences is complete, the sample containing the reference sequence from the particular set of RS-oligos may be denatured using the T_c associated with that chamber or the beads or array removed. This collection is continued through the last chamber. Once all the bound reference sequence is eluted from all chambers, further enrichment could be achieved by introducing the collected target sequences to the above process(es) and have the steps repeated. This may be necessary when the target is present in very low concentrations or when the amount of the input sample is too high and some of the reference sequence(s) escape capture. After repeating the enrichment process, the bound DNA would need to be eluted and this added to the first bound DNA for determination of the amount of target sequence present in the starting material.

After collecting the eluted unbound DNA, the amount may be determined using methodologies such as fluorescence, magnetic beads, Qubit, etc. This fraction should contain all mutated sequences for analysis and little to no wild- type sequence for the amplicons interrogated. This DNA may be used in downstream detection platforms to determine the exact nature of the sequence alterations. After collection of the unbound DNAs, all gates are opened and the amount of the DNA that was bound to the RS-oligos is determined using methodologies such as fluorescence, magnetic beads, Qubit, etc. This fraction should contain all wild-type sequences for analysis and little to no mutant sequence for the amplicons interrogated.

The methods described herein may also be followed by analysis of the eluted enriched DNAs using a mutation detection method. Those skilled in the art will appreciate that many methods may be used to analyze a sample for a particular (target) nucleic acid. Such methods include, but are not limited to, MALDI-TOF, HR-Melting, Di-deoxy- sequencing, next generation sequencing, single-molecule sequencing, pyrosequencing, second generation high-throughput sequencing, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR. These methods may be useful for detecting target sequences that represent a mutant allele of the reference sequence comprising a deletion, insertion or alteration of one or more nucleotides.

The methods described herein may be performed in a quantitative or realtime PCR device. The reaction mixture may contain a nucleic acid detection agent, such as a nucleic acid detection dye (e.g., SYBR Green) or a labeled probe (e.g., a TaqMan probe or other oligonucleotide labeled with a fluorescent marker). The methods described herein may also be used to enrich two or more different target sequences. Such a reaction may include more than one nucleic acid detection agent.

Regardless of the device configuration, the following methods can be used: a Step-Up process can be used to increase incrementally temperatures around the T_c value; colorimetric labels such as intercalating dyes or fluorescent probes may be used for determination of the concentration of the nucleic acid during the PCR cycle(s); and colorimetric labels such as fluorescent probes may be used for detection of nucleic acid alterations/mutations.

In the Example Table below:

1. Each well can have a different MX-ICP mutation label present

2. Each well/mutation can be a simple single color or multiple colors

3. Each well may have a different temperature setting The well strip could be detachable to be used on PCR instruments or can be processed in the device using any means of heat (Infrared (IR) or direct heat)

The strip can have IR or direct heat as a means of controlling temperature during PCR.

6. The colors given Options 1 and 2 in the table below are illustrative of the different colorimetric fluorescent probes that could be used.

The methods described herein may also be followed by analysis of the enriched and optionally amplified sample using a mutation detection method. Those skilled in the art will appreciate that many methods may be used to analyze a sample for a particular nucleic acid. Such methods include, but are not limited to, MALDI-TOF, HR-Melting, Di-deoxy-sequencing, next generation sequencing, single-molecule sequencing, pyrosequencing, second generation high- throughput sequencing, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative- PCR. These methods may be useful for detecting target sequences that represent a mutant allele of the reference sequence comprising a deletion, insertion or alteration of one or more nucleotides.

The methods described herein may be performed in a quantitative or real-time PCR device. The reaction mixture may contain a nucleic acid detection agent, such as a nucleic acid detection dye (e.g., SYBR Green) or a labeled probe (e.g., a TaqMan probe or other oligonucleotide labeled with a fluorescent marker). The methods described herein may be used to enrich two or more different target sequences and the target sequences may be amplifiable with the same primer pair or with different primer pairs. Such a reaction may include more than one nucleic acid detection agent.

The invention will be described by the following non-limiting examples.

Example 1

Figure 1 shows a multi-chambered device useful in an enrichment method. For example, a sample having unamplified isolated DNA or isolated amplified DNA is introduced to a first chamber with beads having multiple forward and reverse reference sequence oligonucleotides (RS-oligos). In the chamber, after denaturation and hybridization, T_cl is applied. If the device has multiple enrichment chambers, each is thermally controlled, e.g., each may be subjected to a different T_c, and has gates which can open and close at specific times to allow unbound DNA to move to the next chamber and the same set or next (different) set of RS-oligos. The size of the beads are selected to prevent the beads from passing into the next chamber. In the final enrichment chamber, T_cn, is applied, a temperature which is lower than Τ __ΐ). Fluorescence detection may be used for quantification of the collected, e.g., enriched, samples. The first elution of DNA for analysis contains enriched target sequences. The last elution is of bound DNA from all chambers. Electrophoresis direction(s) (current) allows for collection of fractions with DNA denatured from chambers. Magnetized cycling using an electromagnet may also be employed.

Example 2

Figures 2 and 3 show a device useful in an enrichment method (single enrichment chamber). A sample with isolated DNA (no amplification) or isolated DNA (with amplification) is added to a first chamber. In Figure 2 beads, or in Figure 3 an array, with all forward and reverse RS-oligos are in the chamber. The single chamber is thermally heated and cooled multiple times. Gates which can open and close at specific times allow unbound DNA to flow out of the chamber. Solution is added after each T_c is applied and the sample is transferred out of the chamber. With a single enrichment chamber, the first applied T_c is the lowest T_c and the T_c is successively raised to successively elute the target sequences. Fluorescence detection may be used for quantification. The sequential elutions of DNA are then used for analysis. The final elution is of bound DNA.

Example 3

Figure 4 shows an exemplary nucleic acid isolation device. The sample may be added to a first chamber, e.g., with a pipette to a column, that is separated from a second chamber with a one way filter. The second chamber includes beads which bind nucleic acid non-specifically (see, e.g., Leslie et al., J. Am Chem. Soc, 134:5689 (2012)), at least one inlet and at least one outlet. The beads may be impregnated in a membrane, or imbedded in but not bound to the membrane. The beads may be washed in the second chamber, for instance, using the inlet and outlet. The beads, or the nucleic acid bound to the beads that is subsequently removed from the beads, may then be transferred to the third chamber (e.g., a detection chamber). The second chamber may be separated from a third chamber by a pressure filter. The third chamber may be separated from a fourth chamber, e.g., nucleic acid amplification chamber, by a pressure filter. The nucleic acid isolation device may be configured (adapted) to be in fluid communication with an enrichment device.

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of using a multi-chambered microfiuidic device for enrichment of target nucleic acid sequences, comprising:

a) providing a multi-chambered microfiuidic device comprising a first chamber flanked by a first gate and a second gate and configured to be in fluid communication with a second chamber when the second gate between the first chamber and the second chamber is adjusted to allow for fluid flow, and a third chamber flanked by a third gate and a fourth gate and configured to be in fluid communication with the second chamber when the third gate between the third chamber and the second chamber and the fourth gate are adjusted to allow for fluid flow to and retention in the third chamber;

b) introducing a fluid or tissue sample comprising nucleic acid suspected of having one or more target sequences with nucleic acid alterations to the first chamber and adjusting the first gate and optionally the second gate to block fluid flow from the first chamber, wherein the first chamber comprises beads or an array having a plurality of different reference sequences that are at least 50% homologous to a plurality of the target sequences;

c) subjecting the sample and beads or array in the first chamber to denaturation and hybridization conditions, wherein the hybridization conditions allow for one or more of the target sequences with a nucleic acid alteration in the sample to hybridize with a reference sequence in the first chamber that is at least 50% but not 100% homologous to the one or more target sequences, thereby forming heteroduplexes, and allow for one or more of target sequences without the alteration to hybridize with a reference sequence in the first chamber that is 100% homologous to the one or more of the target sequences without the alteration, thereby forming homoduplexes;

d) increasing the temperature in the first chamber to a T_ci that allows the heteroduplexes to denature but not the homoduplexes; e) adjusting the second gate and optionally the third gate to allow for flow to and retention of fluid having the denatured heteroduplexes in the second chamber, wherein the second chamber comprises beads or an array having a plurality of reference sequences that are different than the reference sequences in the first chamber;

f) subjecting the sample and beads or array in the second chamber to denaturation and hybridization conditions, wherein the hybridization conditions allow for one or more of the target sequences with a nucleic acid alteration in to hybridize with a reference sequence in the second chamber that is at least 50% but not 100% homologous to the one or more target sequences, thereby forming heteroduplexes, and for allow for one or more of target sequences without the alteration to hybridize with a reference sequence in the second chamber that is 100% homologous to the one or more of the target sequences without the alteration, thereby forming homoduplexes;

g) increasing the temperature in the second chamber to a T_c2 that allows for the heteroduplexes to denature but not homoduplexes, wherein the T_c2 is lower than the T_ci; and

h) adjusting the third gate and optionally the fourth gate to allow for flow to and retention of fluid having the denatured heteroduplexes in the third chamber.

2. A method of using a device for enrichment and detection of target nucleic acid sequences, comprising:

a) providing a microfluidic device comprising a first chamber flanked by a first gate and a second gate, and configured to be in fluid communication with a second chamber when the second gate between the first chamber and the second chamber is adjusted to allow for fluid flow;

b) introducing a fluid sample comprising nucleic acid suspected of having one or more target sequences with nucleic acid alterations to the first chamber and adjusting the first gate and optionally the second gate to block fluid flow from the first chamber, wherein the first chamber comprises beads or an array having a plurality of different reference sequences that are at least 50% homologous to a plurality of the target sequences;

c) subjecting the fluid sample and beads or array in the first chamber to denaturation and hybridization conditions, wherein the hybridization conditions allow for one or more of the target sequences with a nucleic acid alteration in the sample to hybridize with a reference sequence in the first chamber that is at least 50% but not 100% homologous to the one or more target sequences, thereby forming heteroduplexes, and for allow for one or more of target sequences without the alteration to hybridize with a reference sequence in the first chamber that is 100% homologous to the one or more of the target sequences without the alteration, thereby forming homoduplexes;

d) increasing the temperature in the first chamber to a T_ci that allows for the heteroduplexes to denature but not the homoduplexes;

e) adjusting the second gate to allow for flow and retention of fluid having the denatured heteroduplexes in the second chamber;

f) introducing fluid into the first chamber and adjusting the first gate and optionally the second gate to provide for retention of the introduced fluid in the first chamber; and

g) increasing the temperature in the first chamber to a T_c2 that allows for heteroduplexes to denature but not homoduplexes, wherein the T_c2 is higher than

3. The method of claim 1 or 2 wherein the nucleic acid comprises circulating DNA, exosomal RNA,RNA converted to cDNA, mitochondrial DNA, bacterial nucleic acid, parasite nucleic acid or viral nucleic acid.

4. The method of any one of claims 1 to 3 wherein the nucleic acid is obtained from cells in a physiological sample.

5. The method of claim 4 wherein the cells are circulating cells in physiological fluid.

6. The method of any one of claims 1 to 5 further comprising a chamber for nucleic acid isolation or amplification that is in fluid communication with the first chamber when the first gate is adjusted to allow for fluid flow to the first chamber.

7. The method of claim 6 wherein the chamber for nucleic acid isolation or amplification is configured to isolate the nucleic acid from cells and optionally to convert RNA to DNA.

8. The method of any one of claims 1 to 7 wherein the nucleic acid is introduced to the first chamber using pressure, an electric current or a magnetic field.

9. The method of any one of claims 1 to 8 wherein the alterations include copy number variations, insertions, deletions or substitutions.

10. The method of any one of claims 1 to 9 wherein the beads are not magnetic beads.

11. The method of any one of claims 1 to 9 wherein the beads are magnetic beads.

12. The method of any one of claims 1 to 11 further comprising repeating c) and d) before e).

13. The method of any one of claims 1 to 12 further comprising a chamber for quantitation of the heteroduplexes.

14. The method of any one of claims 1 to 13 wherein the nucleic acid is amplified before introduction to the first chamber.

15. The method of any one of claims 1 to 13 wherein the nucleic acid is amplified after g).

16. The method of any one of claims 1 to 15 further comprising collecting the heteroduplexes.

17. A microfluidic device having a plurality of chambers that are serially in fluid communication with each other, comprising:

a first chamber flanked by a first gate and a second gate and configured to be in fluid communication with a second chamber via a conduit when the second gate between the first chamber and the second chamber is adjusted to allow for fluid flow;

a first set of beads or an array having a plurality of different reference sequences in the first chamber and a second set of beads or an array having a plurality of different reference sequences in the second chamber, wherein the reference sequences in the second chamber are different than the reference sequences in the first chamber, wherein the plurality of reference sequences in the first chamber is selected to detect alterations in a first set of target nucleic acids having at least 50% but not 100% homology to the reference sequence, wherein the plurality of reference sequences in the second chamber is selected to detect alterations in a second set of target nucleic acids having at least 50% but not 100% homology to the reference sequences, wherein the selection of the plurality of referen ce sequences in the first chamber provides for detection of a first set of target nucleic acid alterations having substantially the same denaturation temperature, T_ci, for heteroduplexes of target nucleic acid with the alteration and reference sequences, wherein the selection of the plurality of reference sequences in the second chamber provides for detection of a set of target nucleic acid alterations having substantially the same denaturation temperature, T_C2 for heteroduplexes of target nucleic acid with the alteration and reference sequences, and wherein the T_cl is greater than the T_c2;

a third chamber having a third gate and configured to be in fluid communication with the second chamber via a conduit when the third gate is adjusted to allow for fluid flow.

18. The device of claim 17 wherein the third chamber comprises beads or an array having a plurality of reference sequences that are different than the reference sequences in the first and second chambers.

19. The method of any one of claims 1 to 16 wherein a further chamber is in fluid communication with the first chamber.

20. The method of claim 19 wherein the further chamber is employed to

separate

plasma from blood, purify CTCs from blood or isolate exosomes.