CA2922261A1 - Synthesis and enrichment of nucleic acid sequences - Google Patents
Synthesis and enrichment of nucleic acid sequences Download PDFInfo
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- CA2922261A1 CA2922261A1 CA2922261A CA2922261A CA2922261A1 CA 2922261 A1 CA2922261 A1 CA 2922261A1 CA 2922261 A CA2922261 A CA 2922261A CA 2922261 A CA2922261 A CA 2922261A CA 2922261 A1 CA2922261 A1 CA 2922261A1
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Abstract
The present disclosure relates to the enrichment of target nucleic acid sequences present in low-abundance relative to corresponding non-target or reference nucleic acid sequence in a sample. In particular, the methods allow for a substantially greater level of detection sensitivity of target sequence by orders of magnitude enrichment of a low-abundance sequence.
Description
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 ENRICHMENT AND QUANTIFICATION OF NUCLEIC ACID SEQUENCES
FIELD OF THE DISCLOSURE
This disclosure relates to detecting nucleic acid sequence alterations, or mutations, in the presence of highly similar wild-type sequences and to quantification of the sequence alteration or mutation.
BACKGROUND OF THE DISCLOSURE
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.
Cell-free (cf) nucleic acids present in bodily fluids may be an aid in identifying and selecting individuals with cancer or other diseases associated with such genetic alterations.
SUMMARY OF THE DISCLOSURE
The instant disclosure is based in part on the development of an assay using cell-free DNA (cfDNA) that enriches for the extremely low levels of altered, or mutant, DNA to provide high detection sensitivity. The disclosure utilizes a non-limiting example of a KRAS
assay using cfDNA extracted from urine to illustrate aspects and principles of the process.
In a first aspect, 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 temperature that is above the melting temperature (T.) of the reference sequence and above the melting temperature (T.) of the double stranded target sequence so as to form denatured reference strands and denatured target strands;
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 c) reducing the temperature of the reaction mixture so as to permit formation of duplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands;
d) increasing the temperature of the reaction mixture to a critical temperature (Tc) sufficient to permit preferential denaturation of said heteroduplexes of the reference blocking sequence and target strands in preference to denaturation of the duplexes of the reference blocking sequence and reference strands;
e) reducing the temperature of the reaction mixture so as to permit the primer pair to anneal to denatured target strands and any denatured reference strands in the reaction mixture;
t) increasing the temperature of the reaction mixture to a denaturing temperature that is above the melting temperature (T.) of the reference sequence and above the melting temperature (T.) of the double stranded target sequence so as to form denatured reference strands and denatured target strands to extend the primers annealed to the denatured target strands and denatured reference strands in the reaction mixture; and g) repeating c) through 1) for two or more cycles to enrich, in the reaction mixture, the target sequence relative to the reference sequence.
The temperature increase in subparagraph f) is performed without maintenance of any one temperature as a "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). Optionally, the denaturing temperature of subparagraph f) is the same as that in b).
In some embodiments, the reference blocking sequence is complementary to a portion of the denatured target strand that is also complementary to at least a portion of the 3' end of one or both of the primers used. In other embodiments, the reference blocking sequence may include a 3'-end that is blocked to inhibit extension. In further embodiments, the reference blocking sequence strands may include a 5'-end comprising a nucleotide that prevents 5' to 3' exonucleolysis by Taq DNA polymerases. In yet additional embodiments, 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.
Filing Date: October 20, 2013 ENRICHMENT AND QUANTIFICATION OF NUCLEIC ACID SEQUENCES
FIELD OF THE DISCLOSURE
This disclosure relates to detecting nucleic acid sequence alterations, or mutations, in the presence of highly similar wild-type sequences and to quantification of the sequence alteration or mutation.
BACKGROUND OF THE DISCLOSURE
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.
Cell-free (cf) nucleic acids present in bodily fluids may be an aid in identifying and selecting individuals with cancer or other diseases associated with such genetic alterations.
SUMMARY OF THE DISCLOSURE
The instant disclosure is based in part on the development of an assay using cell-free DNA (cfDNA) that enriches for the extremely low levels of altered, or mutant, DNA to provide high detection sensitivity. The disclosure utilizes a non-limiting example of a KRAS
assay using cfDNA extracted from urine to illustrate aspects and principles of the process.
In a first aspect, 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 temperature that is above the melting temperature (T.) of the reference sequence and above the melting temperature (T.) of the double stranded target sequence so as to form denatured reference strands and denatured target strands;
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 c) reducing the temperature of the reaction mixture so as to permit formation of duplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands;
d) increasing the temperature of the reaction mixture to a critical temperature (Tc) sufficient to permit preferential denaturation of said heteroduplexes of the reference blocking sequence and target strands in preference to denaturation of the duplexes of the reference blocking sequence and reference strands;
e) reducing the temperature of the reaction mixture so as to permit the primer pair to anneal to denatured target strands and any denatured reference strands in the reaction mixture;
t) increasing the temperature of the reaction mixture to a denaturing temperature that is above the melting temperature (T.) of the reference sequence and above the melting temperature (T.) of the double stranded target sequence so as to form denatured reference strands and denatured target strands to extend the primers annealed to the denatured target strands and denatured reference strands in the reaction mixture; and g) repeating c) through 1) for two or more cycles to enrich, in the reaction mixture, the target sequence relative to the reference sequence.
The temperature increase in subparagraph f) is performed without maintenance of any one temperature as a "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). Optionally, the denaturing temperature of subparagraph f) is the same as that in b).
In some embodiments, the reference blocking sequence is complementary to a portion of the denatured target strand that is also complementary to at least a portion of the 3' end of one or both of the primers used. In other embodiments, the reference blocking sequence may include a 3'-end that is blocked to inhibit extension. In further embodiments, the reference blocking sequence strands may include a 5'-end comprising a nucleotide that prevents 5' to 3' exonucleolysis by Taq DNA polymerases. In yet additional embodiments, 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.
2 Attorney Docket No. 032035-052600US
Filing Date: October 20,2013 In cases of a peptide nucleic acid or locked nucleic acid, the position of the peptide nucleic acid or locked nucleic acid on the chimera sequence are selected to match positions where mutations are suspected to be present, thereby maximizing 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.
In other embodiments, 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 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 a concentration of about 25 nM.
In some versions of the method, the temperature reducing in c) is less than one minute. 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 T, is maintained for a period from 1 second to 60 seconds.
Including in the disclosure are methods wherein 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.
In further embodiments, the target sequence may be that of a homozygous mutation in a subject, such as a human patient. In some cases, the reference and target sequences are KRAS sequences, optionally human KRAS sequences. In additional cases, the reference and target sequences comprise at least 25 base pairs. In further cases, the reference and target sequences are cell-free DNA (c1DNA), optionally obtained from a bodily fluid such as urine, blood, serum, or plasma.
In other embodiments, the disclosed method is performed in a real-time PCR
device, optionally utilizing a labeled probe. In additional embodiments, the reaction mixture in the method contains a nucleic acid detection dye.
In a second aspect, the above enrichment method may be combined with a detection method for assessing one or more mutations post-enrichment. 'The disclosed methods may thus 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,
Filing Date: October 20,2013 In cases of a peptide nucleic acid or locked nucleic acid, the position of the peptide nucleic acid or locked nucleic acid on the chimera sequence are selected to match positions where mutations are suspected to be present, thereby maximizing 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.
In other embodiments, 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 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 a concentration of about 25 nM.
In some versions of the method, the temperature reducing in c) is less than one minute. 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 T, is maintained for a period from 1 second to 60 seconds.
Including in the disclosure are methods wherein 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.
In further embodiments, the target sequence may be that of a homozygous mutation in a subject, such as a human patient. In some cases, the reference and target sequences are KRAS sequences, optionally human KRAS sequences. In additional cases, the reference and target sequences comprise at least 25 base pairs. In further cases, the reference and target sequences are cell-free DNA (c1DNA), optionally obtained from a bodily fluid such as urine, blood, serum, or plasma.
In other embodiments, the disclosed method is performed in a real-time PCR
device, optionally utilizing a labeled probe. In additional embodiments, the reaction mixture in the method contains a nucleic acid detection dye.
In a second aspect, the above enrichment method may be combined with a detection method for assessing one or more mutations post-enrichment. 'The disclosed methods may thus 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,
3 Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR. These analytical techniques may be used to detect specific target (mutant) sequences as described herein.
In a third aspect, the assessment described above may be performed with 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 Figures 5 and 6. 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.
In some embodiments, 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 is provided. The method for determining may comprise performance of a disclosed enrichment method followed by a 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 and the one or more control samples by comparison to the measurement(s) of one or more known samples of target sequence in the sample. In some cases, the sample is urine, and the target sequence is cfDNA.
The number of control samples may be two or more, optionally selected to include a first control with an amount higher than that expected in the test sample and a second control with an amount that is lower than that expected in the test sample. Without limiting the disclosure, the control samples may be considered "markers" that are measured at the same time as the test sample.
Of course the disclosure further provides computer readable media comprising program instructions for performing any disclosed method.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the two-step assay design for a 28-30 bp footprint in the target gene sequence.
Figure 2 illustrates the establishment of detection cutoffs using MAD scores.
Filing Date: October 20, 2013 SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR. These analytical techniques may be used to detect specific target (mutant) sequences as described herein.
In a third aspect, the assessment described above may be performed with 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 Figures 5 and 6. 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.
In some embodiments, 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 is provided. The method for determining may comprise performance of a disclosed enrichment method followed by a 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 and the one or more control samples by comparison to the measurement(s) of one or more known samples of target sequence in the sample. In some cases, the sample is urine, and the target sequence is cfDNA.
The number of control samples may be two or more, optionally selected to include a first control with an amount higher than that expected in the test sample and a second control with an amount that is lower than that expected in the test sample. Without limiting the disclosure, the control samples may be considered "markers" that are measured at the same time as the test sample.
Of course the disclosure further provides computer readable media comprising program instructions for performing any disclosed method.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the two-step assay design for a 28-30 bp footprint in the target gene sequence.
Figure 2 illustrates the establishment of detection cutoffs using MAD scores.
4 Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 Figure 3 illustrates that a cell line spike at 0.2% mutant indicates a highly specific assay.
Figure 4 illustrates KRAS detection in the urinary cfDNA of cancer patients.
Figure 5 shows a curve fit of KRAS enrichment data from known amounts of mutant sequence relative to wildtype sequence.
Figure 6 shows a log curve fit with 95% confidence bands and calculated input mutation level of a cancer patient DETAILED DESCRIPTION OF MODES OF PRACTICING THE DISCLOSUFtE
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. In some embodiments, the test is based on KRAS mutant detection. In other embodiments, the test may be for the BRAF V600E mutation.
The disclosed method for enriching a target nucleic acid sequence in an amplification reaction mixture was developed in part to achieve a high sensitivity, such as the ability to detect a target (mutant) sequence at a concentration between 0.01 to 0.05% or higher in the presence of excess reference (wildtype) sequence. In some embodiments, this was achieved in combination with a short DNA footprint of about 28 basepairs to about 50 basepairs. In the non-limiting description of the KRAS assay herein, the footprint was about 30 basepairs.
The KRAS assay detects at least 7 different KRAS mutations in codons 12 and 13 of human KRAS. A list of nucleotide substitutions for each of the KRAS mutations tested in the assay are shown in the following Table. Seven mutations were validated using mutation containing cell line DNA.
Table 1
Filing Date: October 20, 2013 Figure 3 illustrates that a cell line spike at 0.2% mutant indicates a highly specific assay.
Figure 4 illustrates KRAS detection in the urinary cfDNA of cancer patients.
Figure 5 shows a curve fit of KRAS enrichment data from known amounts of mutant sequence relative to wildtype sequence.
Figure 6 shows a log curve fit with 95% confidence bands and calculated input mutation level of a cancer patient DETAILED DESCRIPTION OF MODES OF PRACTICING THE DISCLOSUFtE
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. In some embodiments, the test is based on KRAS mutant detection. In other embodiments, the test may be for the BRAF V600E mutation.
The disclosed method for enriching a target nucleic acid sequence in an amplification reaction mixture was developed in part to achieve a high sensitivity, such as the ability to detect a target (mutant) sequence at a concentration between 0.01 to 0.05% or higher in the presence of excess reference (wildtype) sequence. In some embodiments, this was achieved in combination with a short DNA footprint of about 28 basepairs to about 50 basepairs. In the non-limiting description of the KRAS assay herein, the footprint was about 30 basepairs.
The KRAS assay detects at least 7 different KRAS mutations in codons 12 and 13 of human KRAS. A list of nucleotide substitutions for each of the KRAS mutations tested in the assay are shown in the following Table. Seven mutations were validated using mutation containing cell line DNA.
Table 1
5 Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 todbn 12 Cexion 13 . . . . . . . . . . .
WI GGTGGC
GI2A. .
.G C I ...... G. .
.
= === .===== ==
.................... .. . ...
............. .......... . ........ . . ..........
..............
. . ..
.
= = . = =
. ..
.. . .
These mutations may be assessed by use of the disclosed enrichment method followed by next generation sequencing as a non-limiting example. This assessment, or assay, provides the ability to detect multiple mutations in a single run. Nine (9) KRAS mutations that were assessed in a single assay were Gl2A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, and G13D.
In addition to KRAS mutations, 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. In many embodiments, 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, rectral 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
Filing Date: October 20, 2013 todbn 12 Cexion 13 . . . . . . . . . . .
WI GGTGGC
GI2A. .
.G C I ...... G. .
.
= === .===== ==
.................... .. . ...
............. .......... . ........ . . ..........
..............
. . ..
.
= = . = =
. ..
.. . .
These mutations may be assessed by use of the disclosed enrichment method followed by next generation sequencing as a non-limiting example. This assessment, or assay, provides the ability to detect multiple mutations in a single run. Nine (9) KRAS mutations that were assessed in a single assay were Gl2A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, and G13D.
In addition to KRAS mutations, 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. In many embodiments, 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, rectral 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
6 =
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 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 (SMZL), splenic diffuse red pulp small B-cell lymphoma (SDRPSBCL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, low grade lymphoma, systemic mastocytosis, or splenic lymphoma/leukemia unclassifiable (SLLU)), 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, stomach cancer, small intestine cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.
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).
In additional embodiments, the disclosed methods are used in human subjects, such as those undergoing therapy or treatment for a disease or disorder associated with a gene alteration as described herein. Subjects may be of any age, including, but not limited to infants, toddlers, children, minors, adults, seniors, and elderly individuals.
In many eases, the sample containing a target sequence is a bodily fluid. Non-limiting examples of a bodily fluid include, but are not limited to, peripheral blood, serum, plasma, urine, lymph fluid, amniotic fluid, and spinal fluid. .
The disclosure demonstrates that 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.
Preliminary 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
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 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 (SMZL), splenic diffuse red pulp small B-cell lymphoma (SDRPSBCL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, low grade lymphoma, systemic mastocytosis, or splenic lymphoma/leukemia unclassifiable (SLLU)), 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, stomach cancer, small intestine cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.
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).
In additional embodiments, the disclosed methods are used in human subjects, such as those undergoing therapy or treatment for a disease or disorder associated with a gene alteration as described herein. Subjects may be of any age, including, but not limited to infants, toddlers, children, minors, adults, seniors, and elderly individuals.
In many eases, the sample containing a target sequence is a bodily fluid. Non-limiting examples of a bodily fluid include, but are not limited to, peripheral blood, serum, plasma, urine, lymph fluid, amniotic fluid, and spinal fluid. .
The disclosure demonstrates that 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.
Preliminary 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
7 Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 tumor heterogeneity of these samples. Using massively parallel DNA sequencing to detect mutations from cell-free urinary DNA non-invasively monitors metastatic patients for response, non-response and the emergence of resistance mechanisms of molecularly targeted therapies.
The present disclosure also provides, in part, a kit for performing the disclosed methods. The kit may include a specific binding agent that selectively binds to a BRAF
mutation, and instructions for carrying out the method as described herein.
As used herein the term "sample" refers to anything which may contain an analyte for which an analyte assay is desired. In many cases, the analyte is a cf nucleic acid molecule, such as a DNA or cDNA molecule encoding all or part of BRAF. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. Biological tissues are aggregate 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).
As used herein, a "subject" includes a mammal. The mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, home, goat, camel, sheep or a pig. In many cases, the mammal is a human being.
Cancer is a group of diseases that may cause almost any sign or symptom. The signs and symptoms will depend on where the cancer is, the size of the cancer, and how much it affects the nearby organs or structures. If a cancer spreads (metastasizes), then symptoms may appear in different parts of the body.
One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005);
Sambrook et al., Molecular Cloning, A Laboratory Manual (ri edition). Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et at., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990).
These texts can, of course, also be referred to in making or using an aspect of the disclosure.
Filing Date: October 20, 2013 tumor heterogeneity of these samples. Using massively parallel DNA sequencing to detect mutations from cell-free urinary DNA non-invasively monitors metastatic patients for response, non-response and the emergence of resistance mechanisms of molecularly targeted therapies.
The present disclosure also provides, in part, a kit for performing the disclosed methods. The kit may include a specific binding agent that selectively binds to a BRAF
mutation, and instructions for carrying out the method as described herein.
As used herein the term "sample" refers to anything which may contain an analyte for which an analyte assay is desired. In many cases, the analyte is a cf nucleic acid molecule, such as a DNA or cDNA molecule encoding all or part of BRAF. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. Biological tissues are aggregate 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).
As used herein, a "subject" includes a mammal. The mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, home, goat, camel, sheep or a pig. In many cases, the mammal is a human being.
Cancer is a group of diseases that may cause almost any sign or symptom. The signs and symptoms will depend on where the cancer is, the size of the cancer, and how much it affects the nearby organs or structures. If a cancer spreads (metastasizes), then symptoms may appear in different parts of the body.
One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005);
Sambrook et al., Molecular Cloning, A Laboratory Manual (ri edition). Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et at., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990).
These texts can, of course, also be referred to in making or using an aspect of the disclosure.
8 Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the disclosure.
EXAMPLES
Example 1: Materials and Methods:
The following methods described herein were utilized in the examples that follow.
Patient urine samples A total of 9 patients with advanced cancers, who were previously tested for mutations in KRAS by a CLIA certified laboratory, were tested for codon 12 and 13 KRAS
mutations along with 27 healthy controls. Cell free urinary DNA was collected from each patient.
Two-step assay design A two-step assay design was developed for a 28-30 basepair footprint in the target mutant gene sequence. Figure 1 summarizes the assay design, which includes a first pre-amplification step to increase the number of copies of a target mutant gene sequence relative to wild-type gene sequences that are present in the sample. The pre-amplification is conducted in the presence of a wild-type (non-mutant) suppressing "WT blocker"
oligonucleotide that is complementary to the wild-type sequence (but not the mutant sequence) to decrease amplification of wild-type DNA. The pre-amplification is performed with primers that include adapters (or "tags") at the 5' end to faciliatate amplification in the second step.
"lhe second step is additional amplification with primers complementary to the tags on the ends of the primers used in the first step and substitution of multiplex sequencing for the illustrated use of digital droplet PCR with a Taqman (reporter) probe oligonucleotide complementary to the mutant sequence,.
Assay development Figure 2 illustrates the establishment of 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).
Filing Date: October 20, 2013 Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the disclosure.
EXAMPLES
Example 1: Materials and Methods:
The following methods described herein were utilized in the examples that follow.
Patient urine samples A total of 9 patients with advanced cancers, who were previously tested for mutations in KRAS by a CLIA certified laboratory, were tested for codon 12 and 13 KRAS
mutations along with 27 healthy controls. Cell free urinary DNA was collected from each patient.
Two-step assay design A two-step assay design was developed for a 28-30 basepair footprint in the target mutant gene sequence. Figure 1 summarizes the assay design, which includes a first pre-amplification step to increase the number of copies of a target mutant gene sequence relative to wild-type gene sequences that are present in the sample. The pre-amplification is conducted in the presence of a wild-type (non-mutant) suppressing "WT blocker"
oligonucleotide that is complementary to the wild-type sequence (but not the mutant sequence) to decrease amplification of wild-type DNA. The pre-amplification is performed with primers that include adapters (or "tags") at the 5' end to faciliatate amplification in the second step.
"lhe second step is additional amplification with primers complementary to the tags on the ends of the primers used in the first step and substitution of multiplex sequencing for the illustrated use of digital droplet PCR with a Taqman (reporter) probe oligonucleotide complementary to the mutant sequence,.
Assay development Figure 2 illustrates the establishment of 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).
9 =
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 Figure 3 illustrates that a cell line spike at 0.2% mutant indicates a highly specific assay. Figure 4 illustrates KRAS detection in the urinary cfDNA of cancer patients.
Together, these figures show results from sample libraries that were combined and sequenced on an Illumina MiSeq instrument (mean sample sequence counts of 129k).
Following demultiplexing, KRAS fragments were identified by matching left and right flanking regions (mean counts of 117k). The target mutant variants were quantified by computing the frequency of occurrence of each 5 bp sequence (Table 1) in the KRAS identified samples. For each targeted mutation, the frequency of non-target nucleotides (not including wildtype) was also computed. These values were used to normalize against variation in the nucleotide substitution rates inherent in the enrichment, library prep, and sequencing steps of sample processing. The ratio of target to non-target frequencies was used as a test statistic for KRAS
mutation detection. Target to non-target ratios were standardized by converting them to robust z-scores. The robust z-score of a raw score x is:
robust z = (x ¨ m) /1.4826 MAD
where m is the median of the healthy control sample population and MAD is the median absolute deviation of the healthy control sample population. Using the median and MAD of the population produce a z-score that is more robust to outliers than z-scores computed using the mean and standard deviation.
Quantifation The following table and Figure 5 illustrate curve fit and calculated input mutation level of a cancer patient containing the KRAS G12D mutation.
Table 2 Sample Actual% Calculated Up-per Lower Output %Input etiltiletitiFINISMOSEUINICASSIMPIPR.RIVA4SME041351(GIZD
MiiinigeningiginaMeMEM:EMONWSMIMMONN:
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 The raw data plot (Figure 5) 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 nonlinear enrichment of low level mutant species. Mutant DNA input at known amounts of 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. Using urinary DNA from a stage IV colorectal carcinoma patient with a known mutation at the 612D site it was found that the mutation accounted for 13.06%
of the total sequence after enrichment, corresponding to an input amount of approximately 0.14%
mutational load in the patient's urine.
The following table and Figure 6 illustrate curve fit and calculated input mutation using a log transformed axis and showing the 95% confidence bands. Figure 6 is a log transformed plot of the data in Figure 5 with inclusion of the 95% confidence bands based on repeated assessment of known amounts of mutant sequence.
Table 3 Sample Actual %
Calculated Output % Inpuõ t =
liC01005012A9 EiRIV803.1108"EN02261548EM
Patient signals whose z score is above the cutoff (Figure 2, >2.0, 95%
confidence) can be quantified using the ratio of mutant to wildtype sequence counts for that position. These are converted to a percentile and plotted to a reference curve to interpolate the input mutation level of the original sample.
Example 2: KRAS mutations in cfDNA
The agreement between tumor tissue and cfKRAS is shown in the table below.
KRAS
mutations were detected in the urine of 8 out of 9 metastatic cancer patients previously detected in tumor tissue by a CLIA certified laboratory. A z score of 2.0 or more indicates a 95% or greater confidence level of a true call compared to background.
=
Attorney Docket No. 032035-052600US
Filing Date: October 20,2013 Table 4:
Detection or mutant , Urtriary tfDNA
1 Cance=rType Tumorl KRAS in Urinary z :score, (WA) KRAS Mutation cfONA
12004001111111110001111111101111113101.110ind06,11,311015.411511 = 7,$$:;9;5.6. =
= = ...... :er&ta. . . . .
. . . ...........:
LungAdenocarcinoma 612V
:V G12V: =
i...004V:N.e.fi:i.:.M61.20ipffigngiONVEREM:MUMBEHO13OMagganglit %VOA
G2V 243.:
id3.iglligg.Nt'it;,:.O.,.lgling4Uki,,oaIV:A.
C910.r.0;ta.V .;. .:.:=:=:
.=::GiI5 32 gOlgIffailOMPF!.MigianT"''''''Vigg2.EIN giiiiiiMMUIREGItigHORG.Eii.E10014.M
'Me citation of documents herein is not to be construed as reflecting an admission that any is relevant prior art. Moreover, their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.
All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not.
Having now fully described the inventive subject matter, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the disclosure and without undue experimentation.
While this disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications.
This application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth.
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 Figure 3 illustrates that a cell line spike at 0.2% mutant indicates a highly specific assay. Figure 4 illustrates KRAS detection in the urinary cfDNA of cancer patients.
Together, these figures show results from sample libraries that were combined and sequenced on an Illumina MiSeq instrument (mean sample sequence counts of 129k).
Following demultiplexing, KRAS fragments were identified by matching left and right flanking regions (mean counts of 117k). The target mutant variants were quantified by computing the frequency of occurrence of each 5 bp sequence (Table 1) in the KRAS identified samples. For each targeted mutation, the frequency of non-target nucleotides (not including wildtype) was also computed. These values were used to normalize against variation in the nucleotide substitution rates inherent in the enrichment, library prep, and sequencing steps of sample processing. The ratio of target to non-target frequencies was used as a test statistic for KRAS
mutation detection. Target to non-target ratios were standardized by converting them to robust z-scores. The robust z-score of a raw score x is:
robust z = (x ¨ m) /1.4826 MAD
where m is the median of the healthy control sample population and MAD is the median absolute deviation of the healthy control sample population. Using the median and MAD of the population produce a z-score that is more robust to outliers than z-scores computed using the mean and standard deviation.
Quantifation The following table and Figure 5 illustrate curve fit and calculated input mutation level of a cancer patient containing the KRAS G12D mutation.
Table 2 Sample Actual% Calculated Up-per Lower Output %Input etiltiletitiFINISMOSEUINICASSIMPIPR.RIVA4SME041351(GIZD
MiiinigeningiginaMeMEM:EMONWSMIMMONN:
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013 The raw data plot (Figure 5) 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 nonlinear enrichment of low level mutant species. Mutant DNA input at known amounts of 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. Using urinary DNA from a stage IV colorectal carcinoma patient with a known mutation at the 612D site it was found that the mutation accounted for 13.06%
of the total sequence after enrichment, corresponding to an input amount of approximately 0.14%
mutational load in the patient's urine.
The following table and Figure 6 illustrate curve fit and calculated input mutation using a log transformed axis and showing the 95% confidence bands. Figure 6 is a log transformed plot of the data in Figure 5 with inclusion of the 95% confidence bands based on repeated assessment of known amounts of mutant sequence.
Table 3 Sample Actual %
Calculated Output % Inpuõ t =
liC01005012A9 EiRIV803.1108"EN02261548EM
Patient signals whose z score is above the cutoff (Figure 2, >2.0, 95%
confidence) can be quantified using the ratio of mutant to wildtype sequence counts for that position. These are converted to a percentile and plotted to a reference curve to interpolate the input mutation level of the original sample.
Example 2: KRAS mutations in cfDNA
The agreement between tumor tissue and cfKRAS is shown in the table below.
KRAS
mutations were detected in the urine of 8 out of 9 metastatic cancer patients previously detected in tumor tissue by a CLIA certified laboratory. A z score of 2.0 or more indicates a 95% or greater confidence level of a true call compared to background.
=
Attorney Docket No. 032035-052600US
Filing Date: October 20,2013 Table 4:
Detection or mutant , Urtriary tfDNA
1 Cance=rType Tumorl KRAS in Urinary z :score, (WA) KRAS Mutation cfONA
12004001111111110001111111101111113101.110ind06,11,311015.411511 = 7,$$:;9;5.6. =
= = ...... :er&ta. . . . .
. . . ...........:
LungAdenocarcinoma 612V
:V G12V: =
i...004V:N.e.fi:i.:.M61.20ipffigngiONVEREM:MUMBEHO13OMagganglit %VOA
G2V 243.:
id3.iglligg.Nt'it;,:.O.,.lgling4Uki,,oaIV:A.
C910.r.0;ta.V .;. .:.:=:=:
.=::GiI5 32 gOlgIffailOMPF!.MigianT"''''''Vigg2.EIN giiiiiiMMUIREGItigHORG.Eii.E10014.M
'Me citation of documents herein is not to be construed as reflecting an admission that any is relevant prior art. Moreover, their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.
All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not.
Having now fully described the inventive subject matter, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the disclosure and without undue experimentation.
While this disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications.
This application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth.
Claims (29)
1. A method for enriching a target nucleic acid sequence in an amplification reaction mixture, the method comprising a) preparing an amplification reaction mixture comprising a nucleic acid sample comprising a reference sequence and at least suspected of having one or more target 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 temperature that is above the melting temperature (Tm) of the reference sequence and above the melting temperature (Tm) of the double stranded target sequence so as to form denatured reference strands and denatured target strands;
c) reducing the temperature of the reaction mixture so as to permit formation of duplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands;
d) increasing the temperature of the reaction mixture to a critical temperature (Tc) sufficient to permit preferential denaturation of said heteroduplexes of the reference blocking sequence and target strands in preference to denaturation of the duplexes of the reference blocking sequence and reference strands;
e) reducing the temperature of the reaction mixture so as to permit the primer pair to anneal to denatured target strands and any denatured reference strands in the reaction mixture;
f) increasing the temperature of the reaction mixture to a denaturing temperature that is above the melting temperature (Tm) of the reference sequence and above the melting temperature (Tm) of the double stranded target sequence so as to form denatured reference strands and denatured target strands to extend the primers annealed to the denatured target strands and denatured reference strands in the reaction mixture; and g) repeating c) through f) for two or more cycles to enrich, in the reaction mixture, the target sequence relative to the reference sequence.
b) increasing the temperature of the reaction mixture to a first denaturing temperature that is above the melting temperature (Tm) of the reference sequence and above the melting temperature (Tm) of the double stranded target sequence so as to form denatured reference strands and denatured target strands;
c) reducing the temperature of the reaction mixture so as to permit formation of duplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands;
d) increasing the temperature of the reaction mixture to a critical temperature (Tc) sufficient to permit preferential denaturation of said heteroduplexes of the reference blocking sequence and target strands in preference to denaturation of the duplexes of the reference blocking sequence and reference strands;
e) reducing the temperature of the reaction mixture so as to permit the primer pair to anneal to denatured target strands and any denatured reference strands in the reaction mixture;
f) increasing the temperature of the reaction mixture to a denaturing temperature that is above the melting temperature (Tm) of the reference sequence and above the melting temperature (Tm) of the double stranded target sequence so as to form denatured reference strands and denatured target strands to extend the primers annealed to the denatured target strands and denatured reference strands in the reaction mixture; and g) repeating c) through f) for two or more cycles to enrich, in the reaction mixture, the target sequence relative to the reference sequence.
2. The method of claim 1, wherein a 3'-end on the reference blocking sequence is blocked to inhibit extension.
3. The method of claim 1 or 2, wherein a 5'-end on the reference blocking sequence strands comprises a nucleotide that prevents 5' to 3' exonucleolysis by Taq DNA
polymerases.
polymerases.
4. The method of claim 1 or 2 or 3, wherein the reference blocking sequence provided in a) is a single-stranded nucleic acid reference blocking sequence.
5. The method of claim 1 or 2 or 3 or 4, wherein the reference blocking sequence in a) is 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.
6. The method of claim 1 or 2 or 3 or 4, wherein the reference blocking sequence is one of single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid.
7. The method of claim 1 or 2 or 3 or 4, wherein the reference blocking sequence is a chimera between single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid or another modified nucleotide.
8. The method of claim 7, wherein the position of the peptide nucleic acid or locked nucleic acid on the chimera sequence are selected to match positions where mutations are suspected to be present, thereby maximizing 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.
9. The method of any preceding claim, wherein 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 the primer binding sites.
10. The method of any preceding claim, wherein the reference blocking sequence is equal to or shorter than the reference sequence.
11. The method of any preceding claim, wherein the temperature reducing in c) is less than one minute.
12. The method of any preceding claim, wherein the reference blocking sequence is present in the reaction mixture at a concentration of about 25 nM.
13. The method of any preceding claim, wherein the melting temperature of the double-stranded target sequence is greater than or equal to the melting temperature of the double-stranded reference sequence.
14. The method of any preceding claim, wherein 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 of claim 1.
15. The method of any preceding claim, wherein the target sequence comprises a homozygous mutation.
16. The method of any preceding claim, wherein the reference and target sequences comprise at least 25 base pairs.
17. The method of any preceding claim, wherein the reference and target sequences are KRAS sequences.
18. The method of claim 17, wherein the KRAS sequences are human KRAS
sequences.
sequences.
19. The method of any preceding claim, further comprising the step of 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.
20. The method of any preceding claim, wherein Tc is maintained for a period from 1 second to 60 seconds.
21. The method of any preceding claim, wherein the reaction mixture contains a nucleic acid detection dye.
22. The method of claim 14, wherein the method is performed in a real-time PCR device.
23. The method of any preceding claim, wherein the method is performed under a real-time reaction conditions utilizing a labeled probe.
24. The method of any preceding claim, wherein the reference and target sequences are cell-free DNA (cfDNA).
25. The method of claim 24, wherein the cfDNA is obtained from a urine sample.
26. The method of claim 24, wherein the cfDNA is obtained from a sample selected from blood, serum, or plasma.
27. A method for determining the amount of a target sequence in a sample containing a reference sequence, the method comprising performing the method of claim 19 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;
calculating the amount of the target sequence and the one or more control samples by comparison to the measurement(s) of one or more known samples of target sequence in the sample.
calculating the amount of the target sequence and the one or more control samples by comparison to the measurement(s) of one or more known samples of target sequence in the sample.
28. The method of claim 27, wherein the sample is urine.
29. A computer readable medium comprising program instructions for performing the method of any preceding claim.
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| US201461893283P | 2014-10-20 | 2014-10-20 | |
| PCT/US2014/061435 WO2015073163A2 (en) | 2013-11-14 | 2014-10-20 | Synthesis and enrichment of nucleic acid sequences |
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| US20220348606A1 (en) * | 2019-06-26 | 2022-11-03 | Nanjing GenScript Biotech Co., Ltd. | 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 |
| US8455190B2 (en) * | 2007-08-01 | 2013-06-04 | Dana-Farber Cancer Institute, Inc. | Enrichment of a target sequence |
| DK2545189T3 (en) * | 2010-03-08 | 2018-04-23 | Dana Farber Cancer Inst Inc | COMPLETE COLD-PCR ENRICHMENT WITH REFERENCE BLOCKING SEQUENCE |
-
2014
- 2014-10-20 CA CA2922261A patent/CA2922261A1/en not_active Abandoned
- 2014-10-20 WO PCT/US2014/061435 patent/WO2015073163A2/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2015073163A8 (en) | 2015-12-17 |
| WO2015073163A2 (en) | 2015-05-21 |
| WO2015073163A3 (en) | 2015-11-05 |
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