WO2015073163A2 - Synthesis and enrichment of nucleic acid sequences - Google Patents
Synthesis and enrichment of nucleic acid sequences Download PDFInfo
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- WO2015073163A2 WO2015073163A2 PCT/US2014/061435 US2014061435W WO2015073163A2 WO 2015073163 A2 WO2015073163 A2 WO 2015073163A2 US 2014061435 W US2014061435 W US 2014061435W WO 2015073163 A2 WO2015073163 A2 WO 2015073163A2
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- Mutations in BRAF and KRAS are examples of genetic alterations that confer a survival and growth advantage to cancer cells. Such genetic alterations can be used for selection of targeted therapies. But in a subject, the alterations are present with a large excess of non-altered, wild-type sequences.
- This disclosure relates to synthesizing and enriching target nucleic acid sequences containing one or more alterations, or mutation; the target sequence present in low abundance relative to highly similar wild-type sequences present in a biological sample. Preferential and specific enrichment of the target nucleic acid sequence provides a substantially sensitive level of detection not previously achieved. The disclosed methods also allow quantitative detection of the target sequence.
- PCR polymerase chain reaction
- COLD-PCR technique is relatively simple to perform, but has a low amplification factor (3-100x) and a low sensitivity towards minute temperature changes (Li, J., et al., 2008.
- Nucleic acids in cancerous tissues, circulating cells, and cell-free (cf) nucleic acids present in bodily fluids can aid in identifying and selecting individuals with cancer or other diseases associated with such genetic alterations.
- Mutations in BRAF and KRAS are examples of genetic alterations that confer a survival and growth advantage to cancer cells. Such genetic alterations can be used for selection of targeted therapies. But in a subject, the alterations are present with a large excess of non-altered, wildtype sequences.
- the instant disclosure is based in part on the development of a method for substantially enriching for and detecting low-abundance nucleic acid sequences (target sequence) such as altered, mutant, non-wildtype nucleic acid sequence or other nucleic acid not normally present in a biological sample having a background of native nucleic acid, DNA, or RNA sequence, that utilizes fewer steps and greatly reduced reaction assay times while still allowing for orders of magnitude greater sensitivity in detecting a low-abundance nucleic acid sequence.
- the method is developed to enable enrichment of mutant sequences present in a short, fragmented form ( ⁇ 50bp) and is also applicable for amplification of less fragmented sequences (>50bp).
- Application of the present method allows for enrichment of low-abundance nucleic acid sequence within a high background of non-target or wildtype nucleic acid such that as few as a single copy of target sequence can be detected within a biological sample.
- the enrichment is based on a relative increase in the amount of the target sequence, via its preferential amplification and thus substantially greater (700X - 10000X fold and greater) enrichment relative to other nucleic acids present within the same biological sample.
- the disclosure provides a method for enriching a target nucleic acid sequence in an amplification reaction mixture.
- the method may comprise a) preparing an amplification reaction mixture comprising a nucleic acid sample comprising a reference (optionally wild-type) sequence and at least suspected of having one or more target (optionally mutant) sequences that are at least 50% homologous to the reference sequence and are also amplifiable by the same primer pair as the reference sequence, and an excess amount of reference blocking nucleic acid sequence which is fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between its primer sites; b) increasing the temperature of the reaction mixture to a first denaturing
- the temperature increase in subparagraph f) is performed without maintenance of any one temperature as a discrete "step" for extension of the annealed primers. Stated differently, the temperature of the reaction mixture is continually increased, after reaching the temperature of subparagraph e), until reaching the denaturing temperature in f).
- the denaturing temperature of subparagraph f) is the same as that in b).
- the actions in subparagraphs f) and g) are replaced by the act of repeating b) through e) for two or more cycles to enrich, in the reaction mixture, the target sequence relative to the reference sequence.
- the instant disclosure also provides an allele specific competitive cycling assay (ASCC) design based on kinetics of an amplicon and/or a target sequence allowing for a two-step, a three- step or a four- step amplification cycle which reduces method reaction times while substantially enriching for and detecting low-abundance nucleic acid sequence (target sequence) contained in a high background of non-target nucleic acid sequence.
- ASCC allele specific competitive cycling assay
- the instant disclosure also provides an allele specific competitive cycling assay (ASCC) design for short amplicons ( ⁇ 50bp), also suitable for large amplicons (>50 bp) based on a reference blocker oligonucleotide and primer binding kinetics.
- a reference blocker is a short blocker sequence, allowing for a two-step, a three-step or a four-step amplification cycle, which reduces method reaction times while substantially enriching for and detecting low-abundance nucleic acid sequence (target sequence) contained in a high background of non-target nucleic acid sequence.
- the instant disclosure is also based in part on the discovery that for short amplicons, a significant differential in melting temperature can be obtained between reference blocker- reference sequence Tm and reference blocker-target sequence Tm due to a mismatch at the position with variable sequence.
- the instant disclosure also provides a method for enriching and detecting low-abundance nucleic acid sequences (target sequence) utilizing short reference blockers of about 80bp or less or about 60bp or less, or 40bp or less or about between 12 to 35bp in length.
- the disclosure provides a quantitative method for substantially enriching for and detecting low-abundance nucleic acid sequence (target) present in a sample having a greater abundance of non-target sequence such as native, wild-type, or reference sequence.
- the substantially enriched target nucleic acid sequence may be used to provide high detection sensitivity for monitoring or detecting a cancer in a patient by detecting or quantifying the presence of a mutant nucleic acid sequence (e.g. target sequence) in the patient.
- Target sequence includes, for example, cancer-associated mutant forms of BRAF, EGFR, c-MET, HER- 2, HER-3, NRAS, KRAS, PIK3CA, AKT-1, MAP2PK, ER, AR, FGFRl, FGFR2, FGFR3, KIT, PDGFR1, PDFGR2, PDGFR3, TP53, SMAD1 and others.
- cfDNA cell-free DNA
- a method for enriching a target nucleic acid sequence in a nucleic acid sample suspected of containing one or more low abundance target sequence comprising: a. preparing a reaction mixture comprising: a reference sequence, an excess of blocking sequence relative to the amount of reference sequence in the mixture, and suspected of containing one or more target sequence, wherein:
- the target sequence is at least 50% homologous to the reference sequence
- the blocking sequence is fully complementary with region of the reference sequence, the region of the reference sequence being between or overlapping the target sequence;
- a method for enriching a target nucleic acid sequence in a sample suspected of having one or more low abundance target sequence comprising: a) preparing a reaction mixture including a reference sequence, an excess of blocking sequence that is fully complementary to the reference sequence, and a primer pair that is fully complementary with a region of the target sequence wherein the target sequence has at least 50% complementarity to the reference sequence and subjecting the reaction mixture to two or more cycles of: i) a selective temperature (T sd ) that is above the melting temperature of the target sequence and below the melting temperature of reference sequence homoduplex or blocker-reference sequence duplex so as to allow denaturation of target sequence
- T sd selective temperature
- a method for enriching a target nucleic acid sequence in a sample suspected of having one or more low abundance target sequence comprising: a) preparing a reaction mixture including a reference sequence, an excess of blocking sequence that is fully complementary to the reference sequence, and a primer pair that is fully complementary with a region of the target sequence between or overlapping the reference sequence, wherein the target sequence has at least 50% complementarity to the reference sequence and subjecting the reaction mixture to two or more cycles of: i) heating the reaction mixture to a first temperature sufficient to allow denaturation of homoduplexed reference and target sequences; ii) cooling the reaction mixture to a temperature that allows preferential formation of reference-blocker duplexed sequences relative to target-blocker duplexed sequences; iii) heating the reaction mixture to a selective denaturation temperature (T s d) so as to allow denaturation of the blocker-target duplexed sequences; and iv) cooling the reaction mixture to a temperature that is below the
- a method for optimizing the design of primer sequences allowing for preferential amplification and substantially greater enrichment of a low-abundance target sequence is provided. Also provided are single-stranded oligonucleotide DNA
- primers for amplification of a low-abundance target sequence present in sample having a majority background of reference, native, or wild-type nucleic acid are primers for amplification of a low-abundance target sequence present in sample having a majority background of reference, native, or wild-type nucleic acid.
- the temperature selected for selective denaturation (T S( j) may be at or above the melting temperature of a target sequence.
- the temperature selected for selective denaturation may be substantially above the melting temperature of the target sequence.
- the temperature selected for selective denaturation may be below the melting temperature of target sequence.
- the reference blocking sequence, or short blocking sequence may be complementary to a portion of the denatured target strand that is itself also complementary to at least a portion of the 3' end of one or both of the primers used.
- the reference blocking oligonucleotide may include a 3'-end that is blocked to inhibit extension.
- the 5'end of the same oligonucleotide may also be blocked.
- the reference blocking oligonucleotide strand(s) may include a 5'-end comprising a nucleotide that prevents 5' to 3' exonucleolysis by Taq DNA polymerases.
- the reference blocking sequence may be a single- stranded nucleic acid reference blocking sequence; a double- stranded nucleic acid reference blocking sequence which denatures to form single strand reference blocking sequences in b) when the reaction mixture is heated to the first denaturing temperature; single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid; or a chimera between single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid or another modified nucleotide.
- the reference blocking oligonucleotide may contain DNA residues with one or more locked nucleic acid (LNA) nucleotides having a ribose sugar moiety that is 'locked" in the 3'-endo conformation.
- LNA locked nucleic acid
- the use of such an LNA reference blocking oligonucleotide may be used to increase the melting temperature of the oligonucleotide for both a reference sequence and target sequence of the disclosure.
- the reference blocking oligonucleotide may comprise a shorter sequence of base pairs wherein the blocking oligonucleotide sequence is complementary or specific to the wildtype (non-target) DNA sequence or allele.
- Such short sequence oligonucleotides (“short blockers") may be complementary to either the forward or reverse strand.
- the short blockers have a melting temperature that is about at or above the melting temperature of its wildtype oligonucleotide sequence.
- the short sequence blocking oligonucleotide may contain LNA(s) as desired.
- the short sequence is preferably about 80 bp in length or less, or about 60 bp in length or less, or about 40bp in length or less, or between about 12 to about 60 bp in length.
- the position(s) of the peptide nucleic acid or locked nucleotide on the chimeric blocking oligonucleotide are selected to match position(s) where mutations are suspected or known to be present, thereby increasing the difference between the temperature needed to denature heteroduplexes of the reference blocking sequence and target strands and the temperature needed to denature heteroduplexes of the reference blocking sequence and the complementary reference strand.
- the reference blocking sequence is fully complementary with one of the strands of the reference sequence between its primer binding sites, or overlapping at either end with the primer binding sites. In further embodiments, the reference blocking sequence is equal to or shorter than the reference sequence. In yet additional embodiments, the reference blocking sequence is present in the reaction mixture at molar excess in comparison to the primers of the primer pair. In other versions, the melting temperature of the double-stranded target sequence is greater than or equal to the melting temperature of the double-stranded reference sequence. In additional versions, the Tsd is maintained for a period from 1 second to 60 seconds.
- the method includes use of a reaction mixture comprising a nucleic acid sample having a reference sequence and suspected of having one or more target sequence that are at least 50% homologous to the reference sequence and are also amplifiable by the same primer pair as the reference sequence, an excess amount of blocking nucleic acid sequence .
- primer sequence is complementary to reference sequence and also contains tag sequences for integration with subsequent mutation detection methods including but not limited to next generation sequencing.
- the reference and target sequences are first amplified by subjecting the reaction mixture to PCR and then subjecting at least a portion of the reaction mixture to the enrichment method described above.
- the first amplification by PCR may be for 10 cycles or less, 8 cycles or less, or 6 cycles or less. In other cases, the first amplification by PCR may be for 10 cycles or more.
- the target sequence may be that of a homozygous mutation in a subject, such as a human patient.
- the reference and target sequences are KRAS sequences, optionally human KRAS sequences.
- the target sequence may contain a mutation in the BRAF sequence.
- the mutation is in a human BRAF sequence.
- the mutation may be the V600E, V600K, V600D, or V600R mutation known to the skilled person as a valine mutation at position 600 of the BRAF amino acid sequence.
- the target sequence may be a cancer-associated mutant sequence of BRAF, EGFR, c-MET, HER-2, HER- 3, NRAS, PIK3CA, KRAS, AKT-1, MAP2PK, ER, AR, FGFR1, FGFR2, FGFR3, KIT, PDGFR1, PDFGR2, PDGFR3, TP53, SMAD1 or other genes.
- the reference and target sequences are cell-free DNA (cfDNA), optionally obtained from a bodily fluid such as urine, blood, serum, or plasma.
- cfDNA cell-free DNA
- a disclosed primer pair are two oligonucleotide primers wherein each contains a sequence at its 3'-end that is complementary to one strand of a duplex target sequence. Additionally, one or both of the oligonucleotide primers contains a heterologous sequence at its 5'-end that is not found in the target sequence.
- the heterologous sequence may be artificial, synthetic, manmade, or from a source that is exogenous to the target sequence. The use of such a primer converts the target sequence into a chimeric molecule that is artificial and the result of performing the disclosed synthesis of nucleic acid molecules.
- a disclosed enrichment method is used as part of a method for determining the amount of a target sequence in a sample containing a reference sequence.
- the method for determining may comprise performance of a disclosed enrichment method followed by an assessment or detection method, such as sequencing or massively parallel sequences ass non-limiting example, with a sample from a subject and one or more control samples with a known amount of the target sequence to measure the amount of the target sequence; and then calculating the amount of the target sequence relative to the one or more control samples by comparison to the measurement(s) of one or more known samples of target sequence in the sample.
- the sample is urine
- the target sequence is cfDNA.
- Figure 1 illustrates one embodiment of the present method.
- Figure 2 illustrates one embodiment of the present method.
- Figure 3 illustrates one embodiment of the present method.
- Figure 4 illustrates melting temperature profiles.
- Figure 5 illustrates an exemplary kinetics profile of different blocker.
- Figure 6 illustrates Poisson distribution utilized to verify single copy assay sensitivity.
- Figure 7 illustrates demonstration of single copy sensitivity of KRAS assay.
- Figure 8 illustrates substantial enrichment of a low-abundance target sequence.
- Figure 9 provides a schematic of one embodiment of the present method.
- Figure 10 provides a schematic of one embodiment of the present method.
- Figure 11 illustrates one embodiment of the present method.
- Figure 12 illustrates clinical application of one embodiment of the present method.
- Figure 13 illustrates clinical application of one embodiment of the present method. DETAILED DESCRIPTION OF THE INVENTION
- the present invention is directed to methods, compositions, software and kits for high enrichment of low abundance target nucleic acid sequences from a cell-free sample.
- the method is based in part on a nucleic acid sequence amplification protocol that is more efficient, has fewer steps and requires shorter reaction times than previous methods.
- the disclosed method allows orders of magnitude (700X - 1000X+) greater detection sensitivity while maintaining target specificity.
- disclosed herein are methods which employ optimized blocker sequence and primer design as well as requiring far fewer cycle steps.
- the method provided also allows detection of a greater range of target sequence and is not restricted to identifying only those low-abundance target sequences having a melting temperature that is lower than the melting temperature of the reference sequence.
- the present invention can be performed on multiple sample type such as cfDNA (urine, serum, plasma), CTCs, body fluids (saliva, sputum, pancreatic juice, semen, cerebrospinal fluid, tears, mucus) or tissue biopsy (FNA, FFPE, TMA); requires small amounts of tissue or DNA; is quantifiable; has ability to multiplex preferential enrichment of all mutations and detects all mutations in amplified region.
- the present invention is not restricted to detection of allele- specific mutations).
- the term “enriching a target sequence” or “enrichment” of a low- abundance target nucleic acid refers to increasing the amount of a target sequence and increasing the ratio of target sequence relative to the corresponding reference sequence in a sample.
- the ratio of target sequence to reference sequence is initially 5% to 95% in a sample
- the target sequence may be preferentially amplified in an amplification reaction so as to produce a final ratio of 99.99999% and 0.00001% reference sequence, or the target sequence may be preferentially amplified in an amplification reaction so as to increase its presence within the sample by orders of magnitude.
- target sequence refers to a nucleic acid that is in low- abundance or is less prevalent in a nucleic acid sample than a corresponding reference sequence.
- the target sequence will make up less than 50% of the total amount of reference sequence + target sequence in a sample.
- the target sequence may be an abnormal or mutant allele.
- a sample e.g. tissue, blood, plasma, urine or other bodily fluid
- the normal cells contain non-mutant or wild- type alleles, while the cancerous cells contain somatic mutations and/or differences in sequence as compared to their counterpart non-mutant or wild-type allele.
- the mutant is the target sequence while the wild-type sequence is the reference sequence.
- a "target strand” refers to a single nucleic acid strand of a double-stranded target sequence.
- Target sequence must be at least 50% homologous to the corresponding reference sequence, but must differ by at least one nucleotide from the reference sequence.
- Target sequences are amplifiable via PCR with the same pair of primers as those used for the reference sequence but need not be so restricted.
- Target sequences may also be amplifiable via PCR with primer pairs not used for the reference sequence so long as the primers are selected to amplify at region of sequence containing the target sequence.
- amplicon refers to a nucleic acid that is the product of amplification.
- an amplicon may be homologous to a reference sequence, a target sequence, or any sequence of nucleic acid that has been subjected to amplification.
- concentration of amplicon sequence will be significantly greater than the concentration of original (template) nucleic acid sequence.
- 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 makes-up over 50% of the total reference sequence+target sequence in a sample.
- the reference sequence is expressed at the DNA and/or RNA level lOx, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x lOOx, 150x, 200x or more than the target sequence.
- a "reference strand” refers to a single nucleic acid strand of a double- stranded reference sequence.
- 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. Depending on the particular purpose or desire of the practitioner, the reference sequence is generally a wild-type sequence.
- mutant refers to a nucleotide change (i.e., a single or multiple nucleotide substitution, deletion, or insertion) 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 and polymorphisms. The methods of the invention are especially useful in selectively enriching a mutant allele which contains between about 1 and 10 nucleotide sequence changes, although it is useful even with a higher number of sequence changes.
- a mutant allele will typically be obtained from diseased tissues or cells and is associated with a disease state.
- T m refers to the temperature at which a polynucleotide dissociates from its complementary sequence.
- 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") while the other half of the Watson- Crick base pairs remain intact in a double stranded conformation.
- 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).
- a “selected denaturation temperature” or "T s d" is a temperature determined utilizing a preselected design including parameters and calculated T m according to one aspect of an embodiment as disclosed herein.
- a selected temperature will be a preselected temperature that is below the melting temperature of a blockenreference sequence, or above the melting temperature of a blockenreference sequence or about at the melting temperature of a blockenreference sequence.
- the Tm can be determined though actual experimentation.
- 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, now U.S. Ser. No. 12/671,295, incorporated by reference herein.
- reference blocking sequence is an engineered single stranded or double stranded nucleic acid sequence, such as an oligonucleotide and preferably has a length smaller than the amplified section of the target sequence.
- the reference blocking sequence is several bases smaller than the amplified section of the reference sequence, on each side of the sequence so that the primers do not bind appreciably to the reference sequence.
- the reference blocking sequence may overlap with a primer binding site.
- the 3' OH end of the reference blocking sequence is blocked to DNA-polymerase extension.
- the 5 '-end is modified to prevent 5' to 3' exonucleolysis by Taq DNA polymerases.
- the reference blocking sequence can also take other forms which remain annealed to the reference sequence when the reaction mixture is subject to the critical temperature "T c ", such as a chimera between single stranded DNA, RNA, peptide nucleic acid (PNA) or locked nucleic acid (LNA), or another modified nucleotide.
- T c critical temperature
- a PNA or LNA is used in the reference blocking sequence at a positions which flank and/or include the nucleotide in the reference sequence differs from that in the target sequence.
- Such a construction will increase the difference in the melting temperature of the reference blocking sequence-reference sequence and the reference blocking sequence- target sequence heteroduplexes to further favor denaturation of reference blocking sequence- target sequence heteroduplexes at the T S( j and enrichment of the target sequence.
- PNA or LNA modifications may be added to other positions with the reference blocking sequence as to elevate and adjust the melting temperatures of the reference blocking sequence with the reference sequence and with the target-sequence.
- the position of the modified nucleotide, LNA or PNA may be selected to match at least one position where a mutation (i.e. a difference in sequence between the target and reference sequences) is suspected to be present.
- a mutation i.e. a difference in sequence between the target and reference sequences
- the difference between the temperature needed to denature duplexes of the reference blocking sequence and the complementary reference strand and that required to denature heteroduplexes of the reference blocking sequence and the partially complementary target sequence is maximized.
- a “reference blocking” sequence or “short blocking” sequence or “blocker” sequence may be fully complementary with one strand of the reference sequence (between primer binding sites or partially overlapping the primer binding sites).
- the reference blocking sequence and short blocking sequence are shorter than the reference sequence.
- a blocker sequence may exceed the length of a primer. For example, (depending upon a target sequence length) in the case where a blocker sequence fully extends along the length of a forward or a reverse primer including into a sequenced region and includes overlap with a reverse blocker.
- a KRAS blocker having a 13 base pair forward primer may be 13 nucleotide bases from a forward primer + 5 bases in the sequencing region + up to 3 bp complementary to the reverse primer.
- a blocker can include a length of about 21 base pair with longer primers and longer sequenced regions extending the total length.
- short blocking sequence or “short blocker” is a reference blocking sequence having 80 bp length or less, or 60 bp length or less, or between 10 bp and 63 bp in length.
- a short blocker sequence include "hot blocker sequences” or sequences having a melting temperature that is above the melting temperature of the reference sequence or a WT-WT duplex nucleotide strand.
- a short blocking sequence when duplexed with a reference sequence will have a blockenreference sequence melting temperature that is greater than the melting temperature of at least one primer of the pair included in a reaction mixture.
- a reference blocking sequence or short blocking sequence may also be designed so as to allow amplification of fragments (amplicons) of any size length. Such blocking sequences are preferably designed so as to have a length sufficient to amplify short fragmented nucleic acids such as those fragmented DNA sequences present in a cell-free DNA sample.
- a reference blocking sequence is preferably designed so as to allow a differential between the melting temperature of blocking sequence-reference sequence and melting temperature of blocking sequence-target sequence and melting temperature of a primer in the reaction mixture.
- the length of a reference blocking sequence has no maximum or upper limit as the kinetics of the method are based in part on a relationship between a primer-blocker binding temperature and a native-denatured conformation of a target nucleic acid.
- a blocker having a high melting temperature and present in excess quantity in a reaction mixture allows achievement of its preferential binding or annealing to a reference nucleic acid or wildtype nucleic acid sequence.
- a target:blocker melting temperature is lower or substantially lower due to nucleic acid sequence mismatch it will result in a less favored kinetics or binding rate - allowing the forward or reverse primer to
- target nucleic acid sequence preferentially anneal to the target nucleic acid sequence relative to or as compared to the kinetics or rate of blocker binding/annealing to a target sequence.
- a “critical temperature” or “T c " refers to a temperature below the melting temperature "Tm" of the reference sequence.
- the T c is below the T m of both the reference and the target sequence (T c ⁇ T re f or T tg t).
- the critical temperature takes advantage that at a temperature lower than T m , a double stranded target sequence and target-reference sequence (bl:ref) cross hybridized to form double stranded DNA duplex so as to preferentially denature these heteroduplexes over the reference/reference homoduplexes.
- a “primer sequence” includes nucleic acid sequences of 9 - 30bp, or 10-25 bp, or 11-22 bp or 13-16 bp in length.
- a primer sequence is a synthetically engineered nucleic acid sequence that anneal to opposite strands of a target and reference sequence so as to form an amplification product during a PCR reaction.
- the target and the reference sequence should be at least 25 bases in order to facilitate primer attachment.
- a primer sequence may include tag or adapter sequence.
- Adapters are engineered nucleic acid which may be 15 - 30 bp, or 20-25 bp, or 18-23bp in length.
- the primer pair may be designed so as to have a T m lower than the T S( j of the reaction.
- “primer pair” refers to two primer sequences designed so as to anneal to and extend from complementary nucleic acid strands and may be up to 45 bp.
- 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., Nuc. Acids Res. 25:3389- 3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively.
- the disclosure provides a sensitive, easy and inexpensive test for the routine clinical detection of a gene alteration in cell-free nucleic acids from a sample of a subject.
- the test is based on KRAS mutant detection.
- the test may be for a BRAF mutation, such as the BRAF V600E mutation or an EGFR mutation, c-met, MET, HER-2, HER-3, NRAS, PIK3CA 1047, KRAS 161H and others.
- the gene alteration may be a substitution, insertion, deletion, or translocation resulting in a difference between a target sequence and the corresponding reference sequence.
- the disclosure provides for the use of the disclosed methods for any cellular or mitochondrial mutation associated with a disease or disorder in the presence of wildtype sequences.
- the disclosed methods may be performed in cases of cancer, including primary cancer or cancer that has metastasized. In other cases, the methods may be used in cases of a malignant, or non- malignant, tumor.
- Non-limiting examples of cancer include, but are not limited to, adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain or a nervous system cancer, breast cancer, cervical cancer, colon cancer, recta; cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing family of tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal cancer, Hodgkin Disease, intestinal cancer, Kaposi Sarcoma, kidney cancer, large intestine cancer, laryngeal cancer, hypopharyngeal cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), non-HCL lymphoid malignancy (hairy cell variant, splenic marginal zone
- lymphoma/leukemia unclassifiable liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, nasal cavity cancer, paranasal sinus cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity cancer, oropharyngeal cancer, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, adult soft tissue sarcoma, skin cancer, basal cell skin cancer, squamous cell skin cancer, basal and squamous cell skin cancer, melanoma,
- Non-limiting examples of non-HCL lymphoid malignancy include, but are not limited to, hairy cell variant (HCL-v), splenic marginal zone lymphoma (SMZL), splenic diffuse red pulp small B-cell lymphoma (SDRPSBCL), splenic leukemia/lymphoma unclassifiable (SLLU), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, low grade lymphoma, systemic mastocytosis, and splenic lymphoma/leukemia unclassifiable (SLLU).
- HCL hairy cell variant
- SDRPSBCL splenic diffuse red pulp small B-cell lymphoma
- SLLU splenic leukemia/lymphoma unclassifiable
- CLL chronic lymphocytic leukemia
- prolymphocytic leukemia low grade lymphoma
- systemic mastocytosis systemic mastocytosis
- a "patient” includes a mammal.
- the mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. In many cases, the mammal is a human being.
- the disclosed methods are used with human subjects, such as those undergoing therapy or treatment for a disease or disorder associated with a gene alteration as described herein, or subjects surveyed for residual disease or recurrence.
- Subjects may be any individual of any age, sex or race.
- the sample from a subject, and containing a target sequence is a bodily fluid.
- a bodily fluid include, but are not limited to, peripheral blood, serum, plasma, urine, sputum, saliva, pancreatic juice, cerebrospinal fluid, tears, mucus, semen, lymph fluid, amniotic fluid, and spinal fluid.
- the disclosure demonstrates that substantial (100X-11000X fold) enrichment of target sequence can be achieved thereby allowing for down to single-copy mutant sequence (e.g. EGFR deletion, EGFR T790M, KRAS single base substitution), within a biological sample containing a background of non-target nucleic acid can be detected.
- single-copy mutant sequence e.g. EGFR deletion, EGFR T790M, KRAS single base substitution
- massively parallel sequencing can be an effective tool to monitor mutation status of the KRAS gene in urinary cfDNA.
- the assay is selective and highly specific for all seven KRAS mutations within KRAS codons 12 and 13. Results show that mutated KRAS could be detected in the urine of 8 out of 9 patients whose tumor tissue contained a KRAS mutation.
- the discrepancy of the called nucleotide in 4 of the 8 detectable tumor samples may highlight discrepancies in patient tumor heterogeneity of these samples.
- sample refers to anything which may contain an analyte for which an analyte assay is desired.
- the analyte is a cell-free (cf) nucleic acid molecule, such as a DNA or cDNA molecule encoding all or part of BRAF.
- sample includes a sample of nucleic acid (genomic DNA, cDNA, RNA, mRNA).
- the sample may be a biological sample, such as a biological fluid or a biological tissue.
- biological fluids include urine, blood, plasma, serum, saliva, pancreatic juice, semen, stool, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid or the like.
- Biological tissues are aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
- a "sample” also includes a sample of in vitro cell culture constituents, natural isolates (such as drinking water, seawater, solid materials), microbial specimens or specimens that have been "spiked” with nucleic acid tracer molecules.
- Nucleic acid sequences of the invention can be amplified from genomic DNA.
- Genomic DNA can be isolated from tissues or cells according to the following method or an alternative method. Such methods are well known in the art.
- nucleic acids sequences of the invention can be isolated from blood or another fluid by methods well known in the art.
- a disclosed enrichment method is used as part of a method for determining the amount of a target sequence in a sample containing a reference sequence.
- the enrichment method may also be combined with a detection method for assessing one or more mutations post-enrichment.
- the method may comprise performance of a disclosed
- the disclosed methods may further include analyzing the reaction mixture with enriched target sequence using one or more methods selected from MALDI-TOF, HR-melting, di-deoxy-sequencing, single-molecule sequencing, pyrosequencing, second generation high-throughput sequencing, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR. These analytical techniques may be used to detect specific target (mutant) sequences within synthesized nucleic acids as described herein.
- the sample is urine
- the target sequence is cfDNA.
- the PCR amplifies a sequence of less than about 50 nucleotides, e.g., as described in US Patent Application
- the PCR is performed using a blocking oligonucleotide that suppresses amplification of a wildtype version of the gene, e.g., as described in US Patent 8,623,603 or US Provisional Patent Application No. 62/039,905.
- one or more primers contains an exogenous or heterologous sequence (such as an adapter or "tag" sequence), as is known in the art, such that the resulting amplified molecule has a sequence that is not naturally occurring.
- a disclosed primer pair are two oligonucleotide primers wherein each contains a sequence at its 3'-end that is complementary to one strand of a duplex target sequence. Additionally, one or both of the oligonucleotide primers contains a heterologous sequence at its 5'-end that is not found in the target sequence.
- the heterologous sequence may be artificial, synthetic, manmade, or from a source that is exogenous to the target sequence. The use of such a primer results converts the target sequence into a chimeric molecule that is artificial and the result of performing the disclosed synthesis of nucleic acid molecules.
- a primer may be up to 45 bp or about 9 - 30, 10-25, 11-22 or 13-16 bp in length.
- a primer may include an adapter sequence. An adapter sequence may be about 15 - 30 bp, 20-25 bp or 18- 23bp in length.
- the described method may also be performed as a quantitative assay allowing for quantification of the detected target (mutant) sequences.
- the quantification provides a means for determining a calculated input percentage of the target sequence prior to enrichment based upon the output signal (optionally as a percentage) from the assessment. This may be performed by reference to a fitted curve like those illustrated in Figure 12.
- the actual output from an assessment of a test sample is determined in combination with one or more control reactions containing a known quantity of target sequence DNA.
- the outputs from the test sample and the control(s) are compared to a fitted curve to interpolate or extrapolate a calculated input for the test sample. This permits a quantitative determination of the amount of a target sequence in a sample pre- enrichment based upon a post-enrichment detection.
- the detection limits for the presence of a gene alteration (mutation) in cf nucleic acids may be determined by assessing data from one or more negative controls (e.g. from healthy control subjects or verified cell lines) and a plurality of patient samples.
- the limits may be determined based in part on minimizing the percentage of false negatives as being more important than minimizing false positives.
- One set of non-limiting thresholds for BRAF V600E is defined as less than about 0.05% of the mutation in a sample of cf nucleic acids for a determination of no mutant present or wild-type only; the range of about 0.05% to about 0.107% as "borderline", and greater than about 0.107% as detected mutation.
- a no-detection designation threshold for the mutation is set at less than about 0.1%, less than about 0.15%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% detection of the mutation relative to a corresponding wildtype sequence.
- One set of non-limiting thresholds for KRAS Exon 2 mutation assay is defined as less than 1 copy of the mutation for quantitative determination of mutation.
- Prior PCR-based methods of enrichment for low-abundance target sequence employ standard amplification protocols and are based solely on the theoretical difference in melting temperature between the double stranded reference sequence and the double stranded target sequence.
- amplification temperature typically the mutant sequence
- the method preferentially amplifies a sequence having a lower denaturation temperature (typically the mutant sequence) than that of the reference sequence by cycling the denaturation temperature at or above the melting temperature of the target temperature but below the reference temperature.
- T c 83.5° C
- T m the melting temperature for the double stranded reference sequence and higher than the lower melting temperature of the double stranded target sequence, or; (b) to be lower than the T m of both reference and target sequences, whilst still creating a differential between the degree of denaturation of reference and target sequences.
- Makrigiorgos et al. to be "inefficient and “time consuming” and Fast COLD-PCR is unable to detect "mutant sequences having the same or higher than" Tm of the wild-type sequence (See, US20140106362, Background of the Invention).
- U.S. Patent No. 8623603 and US20140106362 by Makrigiorgos et al. (“Full cold-PCR enrichment with reference blocking sequence") attempts to address the problems of Full and Fast COLD-PCR include use of "excess amount of reference blocking sequence" in the reaction mixture to improve efficiency and reduce cycle time.
- the methods allow detection only of those mutant sequences having a Tm that is lower than the wild-type Tm.
- Tm target sequence melting temperature
- the methods are restricted to conditions where the Tm of the double stranded target sequence is lower than the Tm for the double stranded reference sequence.
- the blockenreference sequence can be any of the three categories (above, below or at the Tm) our method is effective independent of the melting temperature of the PCR product. Enrichment becomes independent of the melting temperature, utilizing advantageous aspects of short amplicon binding kinetics between a blocker and a reference or blocker and target strand; an aspect independent of prior methods.
- the present disclosure provides a method that allows detection of a greater range of target sequence and is not limited to identification of only those target sequences having a melting temperature that is lower than the reference sequence melting temperature.
- the present method allows for a substantially greater (700X - 1000X) enrichment of low-abundance nucleic acid sequence within a sample and particularly a sample containing fragmented DNA and/or containing a majority of wild-type or non-target sequence. For example, a single copy mutation in high background (e.g., 10,000 or greater) wild type molecules.
- high background e.g. 10,000 or greater
- the methods provided herein employ sets of amplification cycles utilizing specifically designed reference blocker sequence and primer sequence.
- the blocker sequences are a short nucleotide sequence having complementarity to a selected original or wild-type nucleotide sequence.
- the short blockers include LNAs as predetermined or desired by the practitioner.
- Short blocker sequence may be complementary to either the forward or reverse strand are preferably are preselected based upon a design method provided herein and below. If desired, there may be overlap of the blocker sequence with the same-stranded primer.
- a set of amplification cycles may include 2 or more amplification cycles, 3 or more amplification cycles, suitably 5 or more amplification cycles, suitably 7 or more amplification cycles, suitably 10 or more amplification cycles.
- a set of amplification cycles may include 2-30 cycles, 4-25 cycles, 5- 20 cycles, or 40 cycles or greater.
- the target and reference sequences in the nucleic acid sample may but need not be amplified by a pre- amplification method such as PCR prior to inclusion in the methods.
- the PCR may be completed by using the first denaturing temperature that is higher than the melting temperature of a reference sequence such that both the reference sequence and the target sequence are amplified.
- Reaction mixture including excess blocker may also be used. Excess blocker aids in preventing strand binding and further drives the selective amplification process.
- PCR may also be used subsequent to employing a method described herein to further amplify the nucleic acids in the sample after the enrichment procedure.
- the methods described herein may also be followed by analysis of the amplification reaction mixture using a mutation detection method.
- a mutation detection method Those skilled in the art will appreciate that many methods may be used to analyze a sample for a particular (i.e. target) nucleic acid. Such methods include, but are not limited to, MALDI-TOF, HR-Melting, Di-deoxy-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).
- 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 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 present disclosure also provides, in part, a kit for performing the disclosed methods.
- various chemical reagents to be included in a kit are: packaged in suitable containers - one or more target sequence primer oligonucleotide for amplifying the target nucleic acid, one or more blocker oligonucleotide, a DNA polymerase, a buffer solution for nucleic acid amplification reaction, and control reagents (e.g., positive and/or negative control target nucleic acid and positive and/or negative control wild-type or reference nucleic acid at a standard concentration) and/or instructions for using the kit to detect and optionally quantitate one or more low-abundance target nucleic acid.
- control reagents e.g., positive and/or negative control target nucleic acid and positive and/or negative control wild-type or reference nucleic acid at a standard concentration
- the kit may also include various chemical reagents or appliances, as well as a unit for detection comprising a solution and/or a substance reactable with a dye, tag, fluorescent label or other such marker; the solution containing a dye which binds to a nucleic acid.
- kits can be conveniently packaged in kit form. Such kits can be used in various research and diagnostic applications as described herein.
- FIG. 1 An example of a two-step PCR enrichment assay (EGFR Exon 19 deletions) is provided in Figure 1.
- a selective denaturation step precedes the annealing step.
- the reaction ramps to the annealing temperature of 62.4°C, any complementary wildtype strands generated in the previous PCR cycle bind blocker before the primers anneal.
- the primers then anneal to the complementary mutant strand and bar the possibility of any blocker binding to that mutant strand due to sequence overlap. (For most deletions the likelihood of any blocker binding to the mutant is extremely low as there is actually very little common sequence).
- blockentarget and reference (wt):target heteroduplex formation is likely to be extremely low (below detectable or calculable levels) because only a minor percentage will be complementary.
- FIG. 2 A schematic example of a four-step PCR enrichment assay (EGFR Exon 20 T790M) is provided in Figure 2.
- a 98°C denaturation step ensures that all duplexes denature.
- the second (optional) step 70°C blocker-wildtype duplexes form, but few blocker- mutant duplexes form (as 70°C is above the blocker-mutant Tm).
- step 3 selective denaturation, many of the blocker-wildtype will denature (along with any blocker-mutant duplexes that may exist).
- Figure 3 provides an example of a four-step PCR enrichment assay for KRAS Exon 2 single-base substitution.
- This assay proceeds in a similar fashion as Example 2 except that the selective denaturation step is chosen below the wildtype-blocker Tm.
- the temperature differential between the blocker- wildtype Tm, the primer-template Tm and the blocker-mutant Tm is much greater than in example 2, leading to a more efficient enrichment.
- Tm 80°C
- Tm 64°C
- Tm 64°C
- Tm 64°C
- Tm 69°C
- the given curves are illustrated using the KRAS assay - but the principle is general.
- Each oligonucleotide has an approximately sigmoid (logistic) melting temperature profile with an oligonucleotide's Tm (under assay conditions) being the temperature at which 50% of the molecules are on average single- stranded, and 50% are bound to their template as a double- stranded formation.
- Tm under assay conditions
- preferred melting temperature of preselected primer is designed so as to include:
- c) blocker can overlap with one primer or two primers
- d) blocker need not have overlap with a primers
- an empirically derived selective denaturation can be higher (e.g., EGFRdel and EGFR T790) or lower (e.g.,KRAS) than the melting temperature of a blocker-wt product but generally will be higher than the temperature allowing annealing of the primer to a fully complementary sequence.
- Figure 5 provides a diagram illustrating how the percentage of a blocker that is bound to a target sequence (e.g., a wildtype blocker with a Tm of 76°C) changes over time as the temperature of the reaction approaches a selective denaturation temperature of 78.9°C.
- a target sequence e.g., a wildtype blocker with a Tm of 76°C
- Figure 6 provides a Normal distribution histogram and Poisson probabilities table.
- the table on the right is a Poisson distribution table of probabilities.
- the columns of the table are the number of observed events in a discrete interval.
- the rows are the average number of events per interval (0.13 events per interval, 0.25 events per interval, etc.)
- Each cell in the table is the probability of observing a given number of events (columns) given an average expected number of events (rows) in a single interval. For example, with regards to a cancer mutation detection test, if we expect to detect 2 mutants DNA strands per milliliter (interval), a single milliliter would have a 14% chance of containing no mutant DNA strands at all.
- Figure 7 Curve fit and calculated input mutation level of a cancer patient was detected in a biological fluid sample from the patient containing the KRAS G12D mutation.
- the raw data plot of the enriched reference data shows a best fit to a hyperbolic curve (also known as a saturation binding or dose response curve) demonstrating a strong non-linear enrichment of low level mutant species.
- Mutant DNA input at 0.2%, 0.05%, 0.01% and 0.0% of the total DNA returned observed detection levels of 18.25%, 4.45%, 1.84% and 0.54% respectively as a percentage of the total sequence reads.
- Figure 7 demonstrates an example of theoretical and experimental distribution results of an assay verifying single copy sensitivity of a target KRAS G12A.
- On the left is divided into 14 sub plot depicting distributions.
- the top row of distributions depict a theoretical expectation for 20 measurements given the average copy spiked-in input labeled above each distribution.
- the bottom row depicts actual experimental results.
- the x-axis of each subplot is the number of mutant sequence reads (log scale) detected by the assay.
- the y-axis of each subplot is a density measure of the data points along the x-axis, the closer the points are together, the higher the density.
- the area under the density curve is one.
- the bimodal distribution of points represented by the density curve, provides an intuitive cutpoint (blue line) between detected (MT) and not detected (WT) samples.
- the numerals in each subplot indicate the number of samples (of 20) which were detected (right side of cutpoint) and not detected (left side of cutpoint).
- Figure 8 provides results of an assay wherein mutant was detected at background levels of starting 60ng and 360ng total nucleic acid in the sample used for reaction. Detection of 7 mutant copies in a background of either 60ng (17,400 copies) wildtype genomic DNA or 360ng (104,400 copies) wildtype genomic DNA. The figure s is divided into 7 subplots, each depicting the results for one of the seven KRAS mutations detected by the assay
- each subplot lists the three sample types tested: 7 mutant copies in 17,400 wildtype background, 7 mutant copies in 104,400 wildtype DNA background, and wildtype only (17,400 copies).
- the y-axis of each subplot is the number of mutant sequence reads (log scale) detected by the assay.
- Figure 9 shows BRAF V600E mutation detection assay design for integration with droplet digital PCR (RainDance, Billerica, MA).
- the first step involved pre-amplification with two primers flanking the BRAF V600E locus, where both primers contain non- complementary 5' tags which hybridize to second round primers.
- a complementary blocking oligonucleotide suppressed wt BRAF amplification, achieving enrichment of the mutant BRAF V600E sequence within the pre- amplification step.
- the second step entailed a duplex ddPCR reaction using FAM (V600E BRAF) and VIC (wt BRAF) TaqMan probes to enable differentiation of mutant versus wild-type quantification, respectively.
- the RainDrop ddPCR instrument (RainDance; Billerica, MA) was used for PCR droplet separation, fluorescent reading, and counting droplets containing mutant sequence, wt sequence, or unreacted probe.
- Figure 10 Designed of an ultra-short assay to detect the KRAS gene mutations in codons 12 and 13. Assay utilizes a 31bp footprint, contains a pre-amplification step that specifically enriches mutated DNA fragments and detects at least 7 different KRAS mutations in Exon 2 region. Wiletype sequence blocker and mutation specific amplification provide increased specificity and enrichment of mutated loci.
- Figure 11 Establishing detection cutoffs using MAD scores. Dotted vertical lines represent z-score cutoffs of 2 sigma. z score density distribution of KRAS G12V target/non- target ratios observed in a healthy control (grey) with mutation detection results from colon cancer patient (h.), forward reads (gold point) and reverse reads in (blue point).
- Figure 12 provides results of an assay of the method allowing quantitating input copy number: Standard curves were generated for each mutation at levels varying from 5 to 500 input mutant copies. Both the unknown samples and standard samples were enriched with the same method. Following sequencing mutant read counts above a cutoff value were plotted onto their respective standard curve to calculate the input copy number. Unknown samples copy number input was calculated based on plotting to the standard curve for the mutation of interest. From left to right figures show the same data scaled up to 3000 mutations on the left and 30 mutations on the right demonstrating excellent linearity at lower level inputs. As the mutant copy drops the curve linearizes and tightens to provide better quantitation at low mutational load.
- Example 9 Example 9
- Figure 13 depicts detection of KRAS G12S mutant DNA in fluid samples from an Erdheim Chester Patient cancer patient.
- Urine and blood were collected from a single Erdheim Chester patient at several time points: November 13, 2013 (urine and blood), April 22, 2014 (urine) and April 30, 2014 (blood).
- the KRAS assay was run on DNA extracted from all samples in the same run with 20 wildtype standard control (STD_WT) and 3 no-template control
- the y-axis is the number of mutant sequence reads (log scale) detected by the assay.
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CA2922261A CA2922261A1 (en) | 2013-10-20 | 2014-10-20 | Synthesis and enrichment of nucleic acid sequences |
EP14861883.8A EP2951321A4 (en) | 2013-10-20 | 2014-10-20 | Synthesis and enrichment of nucleic acid sequences |
CN201480013111.4A CN105283555A (en) | 2013-10-20 | 2014-10-20 | Synthesis and enrichment of nucleic acid sequences |
US15/027,688 US20160273022A1 (en) | 2013-10-20 | 2014-10-20 | Synthesis and enrichment of nucleic acid sequences |
JP2016524530A JP2016535987A (en) | 2013-10-20 | 2014-10-20 | Synthesis and enrichment of nucleic acid sequences |
HK15112569.9A HK1211628A1 (en) | 2013-11-14 | 2015-12-21 | Synthesis and enrichment of nucleic acid sequences |
US15/256,523 US9725755B2 (en) | 2013-10-20 | 2016-09-03 | Synthesis and enrichment of nucleic acid sequences |
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CN107604053A (en) * | 2017-08-16 | 2018-01-19 | 金绍莲 | Application and its kit and detection method of a kind of wild-type amplification blocker in detection gene trace mutation reagent is prepared |
WO2020259678A1 (en) * | 2019-06-26 | 2020-12-30 | 南京金斯瑞生物科技有限公司 | Oligonucleotide containing blocker |
CN112375808A (en) * | 2020-11-18 | 2021-02-19 | 合肥欧创基因生物科技有限公司 | Blocker design and screening method of ARMS-TaqMan Blocker system |
CN117025765A (en) * | 2023-02-01 | 2023-11-10 | 珠海圣美生物诊断技术有限公司 | Multiplex digital PCR detection kit and detection method thereof |
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US6391592B1 (en) * | 2000-12-14 | 2002-05-21 | Affymetrix, Inc. | Blocker-aided target amplification of nucleic acids |
KR101376359B1 (en) * | 2007-08-01 | 2014-03-27 | 다나-파버 캔서 인스티튜트 인크. | Enrichment of a target sequence |
CA2792433C (en) * | 2010-03-08 | 2016-10-11 | Gerassimos Makrigiorgos | Full cold-pcr enrichment with reference blocking sequence |
-
2014
- 2014-10-20 WO PCT/US2014/061435 patent/WO2015073163A2/en active Application Filing
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Cited By (4)
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CN107604053A (en) * | 2017-08-16 | 2018-01-19 | 金绍莲 | Application and its kit and detection method of a kind of wild-type amplification blocker in detection gene trace mutation reagent is prepared |
WO2020259678A1 (en) * | 2019-06-26 | 2020-12-30 | 南京金斯瑞生物科技有限公司 | Oligonucleotide containing blocker |
CN112375808A (en) * | 2020-11-18 | 2021-02-19 | 合肥欧创基因生物科技有限公司 | Blocker design and screening method of ARMS-TaqMan Blocker system |
CN117025765A (en) * | 2023-02-01 | 2023-11-10 | 珠海圣美生物诊断技术有限公司 | Multiplex digital PCR detection kit and detection method thereof |
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