Abstract:
The disclosed edge-blocker oligonucleotide based AS-NEPB-PCR method amplifies allele specific DNA (or RNA) while dramatically blocking amplification of wild type (WT) DNA (or RNA). The AS-NEPB-PCR design allows ready modification of an existing PCR reaction setup with an edge-blocker oligonucleotide together with an allele specific primer complementary to the mutant sequence to achieve allele specific amplification. The method simplifies assay optimization procedures and achieved sensitivity sufficient to detect a signal present at 0.1% level with close to 100% specificity, which is useful in detecting SNP or genetic mutations. The method was used to detect three different genetic mutations in cancer, in KRAS, BRAF, and EGFR, with three different types of modified edge-blocker oligonucleotides (phosphate, inverted dT and amino-C7). It was possible to detect one copy of mutant DNA in 1000-copy of normal DNA background of a heterogeneous sample, and was far more sensitive than the other blocking method.

Description:
BACKGROUND 
       [0001]    Use of molecular assays is increasing rapidly in the diagnostic field. Molecular testing has been utilized by clinical scientists and physicians for better understanding of patient disease profiles as well as resistance to diseases at the molecular level. In particular, genetic differences between individuals are considered to be significant factors in evaluating response to treatment or disease and determine response to therapy. 
         [0002]    The holy-grail in treating many diseases is to target particular aberrant and harmful cell types. Often such aberrant cells display mutations not present in the surrounding normal tissue. This is particularly true in the case of cancer where actual clones of cancerous cells may be far fewer in number than the normal cells in the surrounding normal tissue. Sometimes, and with increasing success, such aberrant cells can be targeted by therapies before they expand—provided their presence is detectable. Thus, in combination with clinical parameters molecular profiles can be used to assist in selecting a treatment for a patient, predicting the response of a patient to a particular therapy or predict the disease-free and overall survival for a patient. 
         [0003]    In evaluating disease conditions like cancer, a patient may harbor cells that have one or more single point substitution, insertion, or deletion mutations that play a deleterious role. Different patients may have different mutations even when having the same ‘type’ of cancer. Each mutation or group of mutations may be best treated by different therapeutic regimes. But, such point mutations are often far more difficult to detect than large insertions or deletions or germ line mutations since they are camouflaged by normal tissue which yields a large signal due to the identity of virtually all of nucleic acid sequence flanking the mutation site with genetic material obtained from surrounding normal tissue in a sample. In other words, a sample of tissue including some cancerous tissue but also much normal tissue will, upon using Polymerase Chain Reaction (“PCR”) techniques, generally yield a signal corresponding to normal tissue rather than the mutant sequence. 
         [0004]    Although with a sensitive technique like PCR, the usual admonition is to adopt special laboratory practices to avoid false positive amplifications because the high throughput and repetition of PCR assays can lead to amplification of a single DNA molecule. When the signal is buried in a sea of almost identical nucleic acid molecules additional technical problems need to be addressed to avoid amplifying the background of almost identical nucleic acid molecules instead of the desired target nucleic acid molecules because the background signal is due to molecules that have the advantage of numbers. As a result, a PCR based amplification may simply not readily amplify the nucleic acid variant present at a low frequency—typically present in a tumor cell since the variant is often present at a much lower level than normal tissue in a sample (often at just a fraction of a percent)—when attempting early detection of a cancer or detection and monitoring by relatively non-invasive means. 
         [0005]    Utility of such an assay often depends to a great degree on successfully segregating cancerous tissue from normal tissue to enrich for the target nucleic acid variant before conducting a genotype analysis or on adopting procedures that greatly favor amplification of the target nucleic acid molecule instead of an almost identical sea of background molecules. Examples of such techniques are described by, for instance, Newton et al. in “Analysis of any point mutation in DNA: The amplification refractory mutation system (ARMS)” Nucleic Acids Res. 17:2503-2516 (1989). Sensitivity of such a technique may be expressed in terms of its sensitivity. A sensitivity of 1% indicates an ability to amplify and generate a signal corresponding to a target mutant molecular species present at a level of 1:100 in a sea of almost identical background normal copies. 
         [0006]    A promising technique useful in the context of cancer is Allele-Specific amplification using Real-Time Polymerase Chain Reaction technology (“AS-RT-PCR”) that suppresses the amplification of the wild type sequence using a blocker oligonucleotide that preferentially binds to the wild type sequence to suppress its amplification relative to a variant of the wild type sequence. This technique helps evaluate genetic mutations present at a low frequency in a patient—such as in tumor cells or in a chimera. This technique can help detect genetic variants, single nucleotide polymorphisms (SNP) and genetic mutations present at low frequency—as is demonstrated herein later. 
         [0007]    Example of AS-RT-PCR protocols and their limitations are described in, for instance, Morlan et al. “Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method”, PLoS ONE 4(2): e4584. doi:10.1371/journal.pone.0004584(2009). The Morlan reference describes the use of center-blocker oligonucletides to enhance the detection of point mutations—which is an improvement of the allele specific amplification technique reported by Wu et al. in “Allele-specific enzymatic amplification of f8-globin genomic DNA for diagnosis of sickle cell anemia”, Proc. Natl. Acad. Sci. USA 86:2757-60 (1989). In the Morlan technique a mutant specific primer is used together with a center-blocker oligonucleotide. By center-blocker oligonucleotide is meant an oligonucleotide with a mismatch that is about equidistant from either end. The mutant specific primer is entirely complementary to the mutant sequence. The mutant specific primer of Morlan is further trimmed at its 5′ end to reduce its melting temperature to about 10° C. below the anneal/extend temperature used in the PCR. The center blocker oligonucleotide is complementary to the wild type sequence and spans the site of a point mutation so that the point mutation is about equally spanned by it. The center blocker oligonucleotide is further phosphorylated at its 3′ end to prevent extension during a PCR reaction. 
         [0008]    Other example efforts include Dames et al. “Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays”, Journal of Molecular Diagnostics, Vol. 9, No. 3, July 2007; and Willem Maat and Pieter A. Van der Velden, “Pyrophosphorolysis Detects B-RAF Mutations in Primary Uveal Melanoma”, Investigative Ophthalmology &amp; Visual Science, January 2008, Vol. 49, No. 1. 
         [0009]    With the center-blocker oligonucleotide technique, despite good results with detection of selected point mutations, and significant improvements over prior efforts, it remains a challenge to routinely achieve sensitivities of better than 1% to really utilize the technique to the fullest extent to detect rare mutations. Further, the technique is limited to the detection of point mutations. Sensitive detection of rare alleles that are not solely defined by point mutations continues to be an additional challenge. 
       SUMMARY 
       [0010]    Disclosed is a novel edge-blocker oligonucleotide based Non-Extendable Primer Blocker Allele Specific-Real Time Polymerase Chain Reaction (“AS-NEPB-PCR”) based mutation assay methodology that overcomes the limitations of prior art approaches to enable amplification and detection of nucleic acid variants present at a frequency lower than 1% to achieve selectivity for targets present at levels of less than 1%. The disclosed method enables a universal design of Allele Specific primer and primer blocker that can be used in any of AS-RT-PCR assays to detect SNP or genetic mutations. The method simplifies assay optimization procedures and achieved 0.1% detection sensitivity with close to 100% specificity. 
         [0011]    The disclosed edge-blocker oligonucleotide based AS-NEPB-PCR method amplifies allele specific DNA (or RNA) while dramatically blocking amplification of wild type (WT) DNA (or RNA). The disclosed AS-NEPB-PCR design allows ready modification of an existing PCR reaction setup by introducing an edge-blocker oligonucleotide together with an allele specific primer complementary to the mutant sequence to achieve allele specific amplification. In a preferred embodiment the edge-blocker oligonucleotide and allele specific primer may have the same length and differ only at the 3′ end where the edge-blocker oligonucleotide has a non-complementary base relative to the mutant sequence and a blocked 3′ end while the allele specific primer is preferably entirely complementary to the mutant sequence of interest and has a hydroxyl group at its 3′ end to allow extension during a PCR reaction. This method is not only simpler to implement but also successful in routinely suppressing the amplification of the wild type sequence to almost undetectable levels even when the mutant sequence is present at a frequency of about 0.1% of the wild type sequence. Further, the edge-blocker oligonucleotide based AS-NEPB-PCR method is not limited to just detecting point mutations, but can detect specified insertions or deletions as well. 
         [0012]    The edge-blocker oligonucleotide based AS-NEPB-PCR method was used to detect three different genetic mutations in cancers. The genetic mutations targeted here were in KRAS, BRAF, and EGFR genes, which were detected with the use of three different types of modified edge-blocker oligonucleotides (phosphate, inverted dT and amino-C7). The resulting data were compared to one of the known common blocking methods as a reference. The novel method disclosed herein was able to detect one copy of mutant DNA in 1000-copy of normal DNA background of a heterogeneous sample, and was far more sensitive than the reference blocking method. 
         [0013]    A preferred method for detecting a mutant nucleic acid sequence, defined by one or more mutations due to at least one or more of a substitution, a deletion or an insertion, while suppressing the signal due to the wild type sequence includes several steps. Preferably a primer complementary to the mutant nucleic acid sequence is selected such that its 3′ end matches up with at least one mutated nucleic acid position. A second primer, an edge-blocker wild type oligonucleotide, is also used. The edge-blocker wild type oligonucleotide corresponds to the wild type sequence such that the 3′ end of the edge-blocker wild type primer has at least one mismatch at or about its 3′ end relative to the mutant nucleic acid sequence but has no mismatches relative to the wild type sequence. Further, the 3′ hydroxyl group at end of the edge-blocker wild type primer is blocked whereby making it non-extendable in a polymerase chain reaction. In a preferred embodiment reverse primers are selected as usual although it should be noted that when trying to detect deletions or insertions, it may be advantageous to use reverse primers similar to the one corresponding to the wild type sequence having a blocked 3′ end. The amplification products of a PCR reaction are detected with at least one probe specific for the amplified product in a polymerase chain reaction. Preferably, the polymerase chain reaction is a real-time polymerase chain reaction. 
         [0014]    In a variation, one or more probes may be added after the polymerase chain reaction is initiated. Further, the initial starting materials may be generated using a reverse transcriptase to investigate transcription products for point or other mutations of interest. 
         [0015]    An interesting situation handled by this method with ease is when two point mutations of interest are close to each other including being adjacent. In such a situation this method can use the 3′ end of the edge-blocker wild type oligonucleotide with at least one mismatch at or about its 3′ end relative to the mutant nucleic acid sequence—even two mismatches to cover both the point mutations to even more effectively suppress the amplification of the wild type sequence. It is preferred that the allele specific primer cover both the mutant positions. With such coverage even if there is extension based on the binding of the allele specific primer, the amplification products will correspond to the target mutations rather than the wild type sequence. 
         [0016]    In an embodiment, as many as two out of the three base pairs immediately adjacent to the blocked 3′ end of the edge-blocker wild type oligonucleotide may have a mismatch, but may be counterbalanced by increasing the length of the edge-blocker wild type oligonucleotide to suppress amplification of the wild type sequence by the allele specific primer. 
         [0017]    In a preferred embodiment, the edge-blocker wild type oligonucleotide is equal in length to the allele specific primer with both having 3′ ends that cover similar portions of the mutant or wild type sequence. The method can detect a mutant sequence even when it is present at a level of only about 1 in 1000 or even rarer. 
         [0018]    However, as noted the edge-blocker wild type oligonucleotide may be longer at its 5′ end than the allele specific primer to assist it in competing out the allele specific primer to prevent accidental extension of the wild-type sequence by the allele specific primer. 
         [0019]    To ensure effective competition by the edge-blocker wild type oligonucleotide the melting temperature of the allele specific primer is lower than that for the edge-blocker wild type oligonucleotide relative to the wild type sequence. Preferably, the melting temperature of the allele specific primer is lower, e.g., about 10° C. lower, than that for the edge-blocker wild type oligonucleotide. 
         [0020]    Effective competition by the edge-blocker wild type oligonucleotide for the wild type sequence is helped by ensuring that the edge-blocker wild type oligonucleotide is present at a concentration suitable for suppression of the wild-type sequence while allowing amplification of the mutant sequence. Preferably, this concentration is comparable—while being at least equal—to the level of the wild type sequence concentration. The concentration of the allele specific primer, on the other hand, is in excess of that of the wild type sequence since it is incorporated into the PCR product while the edge-blocker wild type oligonucleotide serves to suppress amplification of the wild-type sequence, the likelihood of which decreases as the allele specific primer levels decrease with amplification of the target PCR product. Thus, preferably, the concentrations of the edge-blocker wild type oligonucleotide and the allele specific primer are comparable. 
         [0021]    Preferably, using, for instance calibration curves, the disclosed method for the detection of rare mutant nucleic acid sequence includes quantitation to estimate a level of the mutant nucleic acid sequence relative to the wild type sequence. Such a calibration curve may be generated by spiking the samples for a polymerase chain reaction. 
         [0022]    Early detection of cancer to better provide therapeutic intervention is made possible when the disclosed method is used to detect tumor cells by the mutant nucleic acid sequence corresponding to a tumor cell type in a tissue, CTCs or other sample collected from a patient. If the rare target allele corresponds to metastatic cell disease, intervention before the tumor cells become noticeable becomes a more realistic possibility. 
         [0023]    Thus, the disclosed method is a diagnostic method suitable for early detection of cancer by way of detecting the presence of one or more target cells in a sample derived from a patient, which cells harbor a mutant nucleic acid sequence, and the presence of which cells likely leads to malignancy or recurrence. The method comprises selecting an allele specific primer corresponding to a portion of the mutant nucleic acid sequence such that the 3′ end of the allele specific primer does not have a mismatch while being aligned with at least one mutated nucleic acid position in a target. Also used is an edge-blocker wild type oligonucleotide corresponding to the wild type sequence such that the 3′ end of the edge-blocker wild type oligonucleotide has at least one mismatch at or about its 3′ end, and, wherein, furthermore, the 3′ end of the edge-blocker wild type oligonucleotide is blocked whereby making it non-extendable competitive inhibitor in a polymerase chain reaction. Together with other ingredients and one or more probes to detect the desired amplification products, a polymerase chain reaction, preferably a real time polymerase chain reaction is carried out. The method not only detects cancer usefully early, but also can guide one to therapies best suited for treating the patient. 
         [0024]    As a check on spurious signals, the mutant nucleic acid sequence is detected by detecting the reaction products less than a pre-specified number of amplification cycles. 
         [0025]    As a further safeguard against spurious signals, mutant nucleic acid sequence&#39;s presence is detected if amplification products corresponding to it are detected but a reference sequence, treated like the mutant sequence is not detected in the same sample. The target sequence, with or without its corresponding wild-type like sequence, may be used to spike the sample. This can determine sensitivity and other parameters of interest. 
         [0026]    The edge-blocker wild type oligonucleotide is blocked by derivatizing or replacing its 3′ hydroxyl group with one or more selected from the group consisting of phosphate, inverted dT and amino-C7. 
         [0027]    These and other benefits of the disclosed novel AS-NEPB-PCR method and variations thereof are described next with the aid of the included Figures. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0028]      FIG. 1 . General diagram for edge-blocker oligonucleotide based AS-NEPB-PCR design: Primer with 3′ end modification (phosphate or inverted dT) functions as a blocking group to prevent polymerase extension on wild type sequence.—PCR amplifies only AS primed mutation while WT strain is blocked by a modified non-extendable primer (edge-blocker oligonucleotide based AS-NEPB-PCR). 
           [0029]      FIG. 2 . Mapping of edge-blocker oligonucleotide designs for V600E. AS Primer-1 and 2 indicate BRAF-AS-Forward Primer-1 and 2 (AS Primer-1 is 6 bases longer than AS Primer-2 at 5′) corresponding to Seq. Id. 1 and Seq. Id. 2 respectively. Edge-blocker oligonucleotide based AS-NEPB-PCR: WT-1 and 2 stand for edge-blocker oligonucleotide-1 and edge-blocker oligonucleotide-2 and they correspond to Seq Id. 5 and Seq. Id. 6 respectively. Probe-dye (Dye labeled probe—Seq. Id. 4) and reverse primer (Seq. Id. 3) are common for both assays. AS primer-1, Seq. Id. 1, runs with edge-blocker oligonucleotide-WT-1 (NEPB-WT-1), Seq. Id. 5, and AS primer-2, Seq. Id. 2, with edge-blocker oligonucleotide-WT-2 (NEPB-WT-2), Seq. Id. 6. Bold letters in legends and in the figures represent positions for mismatch, such as ‘A-BRAF mutation (V600E; T&gt;A)’ in AS Primer 1 and 2; ‘*’ represents 3′ modifications here and in the figures. 
           [0030]      FIG. 3 . Mapping of edge-blocker oligonucleotide designs for two K-ras gene mutations. AS Primer-KrasP4 (Seq. Id. 17) and KrasP7 (Seq. Id. 21) indicate G12V-AS-Forward Primer and G13D-AS-Forward Primer. NEPB-WT-KrasP4B (Seq. Id. 18) and P7B (Seq. Id. 22) stand for G12V-NE edge-blocker oligonucleotide and G13D-NE edge-blocker oligonucleotide. KProbe1 (Seq. Id. 20) and KProbe2 (Seq. Id. 24) are Dye labeled probes, and reverse primer is common for both assays. AS Primer-KrasP4 (Seq. Id. 17) runs with edge-blocker oligonucleotide-WT-KrasP4B (Seq. Id. 18) and KProbe1 (Seq. Id. 20). AS Primer-KrasP7 (Seq. Id. 21) runs with center-blocker oligonucleotide-WT-KrasP7B (Seq. Id. 22) and KProbe2 (Seq. Id. 24). Red “t” indicated K-ras p.G12V; mutation (c.G&gt;T) and Red “a” indicated K-ras p.G13D; mutation (c.G&gt;A) in AS Primer-KrasP4 and P7 and as usual ‘*’ represents -3′ modifications. 
           [0031]      FIG. 4 . BRAF V600E mutant detection: 8 out of 20 ul PCR products from the 20 ng DNA reaction were loaded on the gel. The single sharp bands were observed from 5% to 0.1% mutant reactions and no PCR products were observed from SKBR3 WT AS-NEPB2-PCR-2 reaction, except Actin-PCR products. 
           [0032]      FIG. 5 a   . KrasP4 (G12V; G&gt;T) NEPB or CBO blocker PCR on SW480 Cell line DNA: 8 out of 20 ul PCR products from the 20 ng DNA reaction were loaded on the gel. Clean PCR products were visualized on a 4% agarose gel from 0.1% mutant reactions and no PCR products were observed from WT reaction, except Actin-PCR products. The PCR product was also visualized from WT amplification without adding any Blockers. All of NTC was not undetermined. 
           [0033]      FIG. 5 b   . KrasP7 (G13D; 13G&gt;A) NEPB or CBO blocker PCR on HCT116 Cell line DNA: 8 ul PCR products were loaded on the gel. Clean PCR products were visualized from 0.1% mutant reactions and no PCR products were observed from WT reaction. The PCR product was visualized as well from WT amplification without adding any Blockers. 
           [0034]      FIG. 6 . EGFR Exon 21 L858R (2573 T&gt;G) AS-NEPB-PCR on NCI-H1975 Cell line DNA: 8 ul PCR products were loaded on the gel. Clean PCR products were visualized from 0.1% mutant reactions and no PCR products were observed from WT reaction. The PCR product was visualized as well from WT amplification without adding any Blockers. 
       
    
    
     DETAILED DESCRIPTION 
       [0035]    With the aid of examples and exemplary discussions, disclosed herein is a novel edge-blocker oligonucleotide based Non-Extendable Primer Blocker Allele Specific-Real Time Polymerase Chain Reaction (“AS-NEPB-PCR”) based mutation assay methodology that overcomes the limitations of prior art approaches to enable amplification and detection of nucleic acid variants present at a frequency lower than 1% to achieve selectivity for targets present at levels of less than 1%. The disclosed method enables a universal design of Allele Specific primer and primer blocker that can be used in any of AS-RT-PCR assays to detect SNP or genetic mutations. The method simplifies assay optimization procedures and achieved 0.1% detection sensitivity with close to 100% specificity. The description starts with a detailed outline of experiments demonstrating the effectiveness of the technique. 
       Materials and Methods 
       [0036]    Cell Line and FFPE Tissue Samples 
         [0037]    Cancer cell lines were ordered from American Type Culture Collection (ATCC, Manassas, Va. US) and cultured according ATCC protocols. The following cell lines containing specific allele sequences were used in this study: HT29 cell line (ATCC# HTB-38D) is heterozygous and SK-MEL28 cell line (ATCC# CRL-5908) is homozygous in BRAF mutation with predicted mutation effect of p.V600E (c.1799T&gt;A). Characterization of BRAF mutation was described as likely oncogenic mutation (11). HCT 116 cell line (ATCC# CCL-247) has a mutation in codon 13 (p.G13D; c.G&gt;A) and SW480 cell line (ATCC# CCL-228) has a mutation in codon 12 (p.G12V; c.G&gt;T) of K-ras protooncogene. NCI-H1975 cell line (ATCC# CRL-5908) carries EGFR Exon 21 recurrent heterozygous missense mutation of L858R-2573T&gt;G (12). SKBR3 cell line (ATCC# HTB-30) was used as wild type control for BRAF, K-ras and NCI-H358 cell line (ATCC# CRL-5807) as wild type control for EGFR mutation detection assays. 
         [0000]    Melanoma and Colon tissue samples were purchased from ProteoGenex (Culver City, Calif. US), one of the providers of biological specimens. 
         [0038]    Patient Samples 
         [0039]    From 42 patients with metastatic colorectal cancer, 2×30 mL blood samples were taken for circulating tumor cells (CTC) enumeration and characterization by way of vena puncture before liver metastasis resection and prior to tumor manipulation. CTC enrichment and enumeration were processed by CellSearch system (Veridex LLC, Raritan, N.J.). All patients were included in the Erasmus Medical Center, Rotterdam, Netherlands after written informed consent was obtained. 
         [0040]    DNA Extraction 
         [0041]    Cell line DNA was extracted by using AllPrep™ DNA/RNA Micro Kit and FFPE tissue DNA was extracted by using RNeasy FFPE kit from Qiagen (Valencia Calif. US Cat#80284 and 74404) according to the manufacturer&#39;s instructions. Then, extracted DNA was quantified on Nanodrop-2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, Del. US) following the User Manual and stored at −20° C. until later use. 
         [0042]    Oligo Design 
         [0043]    A general diagram for NEPB is demonstrated in  FIG. 1 . For an allele that was analyzed, allele-specific primers (ASP) can be designed to either positive or negative DNA (or RNA) strand. Either for the forward or the reverse primer, 3′-end is anchored on the variant base. The melting temperatures (Tm) of ASP should be close to the PCR extension temperature. 
         [0044]    Edge-blocker oligonucleotide based AS-NEPB-PCR method was designed to the same strand and length as the allele-specific primer except the forward or reverse primer 3′-end is anchored on the WT base and not extendable by polymerases with 3′ end modification (phosphate or inverted dT or amino-C7). Center-blocker oligonucleotide design was based on the criteria listed in the paper (7) and used for comparison to edge-blocker oligonucleotide based AS-NEPB-PCR method. The design sequences were assembled by SeqMan II expert sequence analysis software (DNASTAR Inc, WI, US);  FIG. 2  for the design of BRAF gene and  FIG. 3  for two K-ras genes. 
         [0045]    All of the designs including non-AS forward or reverse primers and probe used Oligo Primer Analysis Software from Molecular Biology Insights (Cascade, Colo. US). Tm was calculated by Oligo software based on PCR condition of 0.2 uM primers, 100 mM [Monovalent Cation] and 3 mM free Mg [2+]. 
         [0046]    All of the oligonucleotides, including primers, probe and blocker, were purchased from Biosearch Technologies, Inc (Novato, Calif. US), except MGB probes. Modified oligonucleotides including Fluorophore dye (FAM and CAL Fluor Orange) labeled at 5′ ends and BHQ or Phosphate at 3′ ends (Table 1a, b and C) were synthesized according to the manufacturer&#39;s instructions. Two MGB probes for K-ras assay were purchased from Applied Biosystems (Foster City, Calif.). 
       AS-NEPB-PCR Amplification 
       [0047]    The AS-NEPB-PCR assay for allele analysis of B-Raf, K-ras and EGFR included one ASP on the positive strand, one NEPB, one fluorescence labeled sequence specific TaqMan probe with BHQ or MGB at 3′ end and one non-AS reverse primer (RP) on the negative strand. The sequences of primers, probe and oligonucleotide blockers are listed in Table1a, Table1b and Table 1c below for the BRAF gene, Kras gene and EGFR gene respectively. All Tables are provided in the section titled ‘Tables’, which follows the ‘References’ section. 
         [0048]    The assay was run as singlex or duplex AS-RT-PCR format, AS gene with Internal Control gene in two individual reactions or in one reaction, on Applied Biosystems 7500 (or 7900) Real-Time PCR System (Foster City, Calif.). 
         [0049]    For BRAF p.V600E (c.1799T&gt;A) detection, the final concentrations of each primer, blocker and probe of AS-NEPB-PCR assay are listed in Tables 2a. The assay was set up as follows: 10 ng to 50 ng of DNA heterogeneous mixture was used and was carried out in a final volume of 20 ul in reaction. The AS-NEPB-PCR was carried out using TaqMan® Gene Expression Master Kit (Applied Biosystems, Part #4368814). Each reaction consisted of 10.0 ul of 2×PCR Master Mix, 1 ul of 20× primer/blocker/probe mix, and 1-5 ul of 10 ng/ul total DNA sample. The AS-NEPB-PCR assays were run as follows: 1 cycle of denaturation at 95° C. for 10 min, 40 cycles of 95° C. for 20 seconds denaturation and 64° C. in favor of BRAF-NEPB1-PCR-1 or 58° C. in favor of BRAF-NEPB2-PCR-2 for 45 seconds annealing and extension under run Standard Mode. 
         [0050]    To compare with the center-blocker oligonucleotide based AS-NEPB-PCR method, two BRAF center-blocker oligonucleotides (Table1a) designed based on the criteria listed in the publication (7) were tested. The final concentrations of center-blocker oligonucleotides were tested with 1×, 2× of the AS primer concentrations. The AS primer concentrations are 0.9 um with 0.9 um of reverse primers and 0.2 um of probe in a final 20 ul reaction. The PCR reagents and conditions are the same as the BRAF-NEPB1-PCR-1 assay. The concentrations of each primer, blocker and probe are listed in Table 2a. 
         [0051]    For two K-ras mutations, the final concentrations of each primer, blocker oligonucleotides and probes of AS-NEPB-PCR assay are listed in Tables 1b and 2b. The assay was set up as follows: 20 ng of DNA heterogeneous mixture was used and was carried out in a final volume of 20 ul in reaction. The AS-NEPB-PCR was carried out using TaqMan® Gene Expression Master Kit. Each reaction consisted of 10.0 ul of 2×PCR Master Mix, 2 ul of 10× primer/blocker/probe mix, and 2 ul of 10 ng/ul total DNA sample. The AS-NEPB-PCR assays were run as follows: 1 cycle of denaturation at 95° C. for 10 min, 40 cycles of 95° C. for 20 seconds denaturation and 60° C. for 45 seconds annealing and extension under run Standard Mode. 
         [0052]    The center-blocker oligonucleotide based AS-PCR method for K-ras was run at the same PCR condition as the edge-blocker oligonucleotide based AS-NEPB-PCR method except for using 4× center-blocker oligonucleotide concentration as corresponding ASP concentration, which was suggested in the publication (7). 
         [0053]    For EGFR mutation, the final concentrations of each primer, blocker oligonucleotides and probes of AS-NEPB-PCR assay are listed in Tables 1c and 2c. The assay set-up was the same as K-ras mutation assay except DNA template. DNA samples were from NCI-H1975 and NCI-H358 heterogeneous mixture. The AS-NEPB-PCR assays were run as follows: 1 cycle of denaturation at 95° C. for 10 min, 40 cycles of 95° C. for 20 seconds denaturation and 63° C. for 45 seconds annealing and extension under run Standard Mode. 
       Data Analysis 
       [0054]    Edge-blocker oligonucleotide based AS-NEPB-PCR detection sensitivity/specificity of BRAF (V600E) and K-ras (G12V or G13D) were estimated by using dilutions of the related mutant cell line DNA (describe the above Cell Line Sample section) in wild-type DNA of the cell lines SKBR3. Dilutions were made at 5%, 1%, 0.5% and 0.1% mutant DNA and data were collected and analyzed by ABI 7500 fast System SDS software (Applied Biosystems). The same analysis method was used for both center-blocker oligonucleotide based AS-NEPB-PCR and edge-blocker oligonucleotide based AS-NEPB-PCR methods. Data was analyzed by manual threshold of 0.1 and baseline from 5 to 15 to obtain C T  value for both FAM and VIC channels. Assay was considered valid when Actin C T  value was less than or equal to 27, specific mutant gene C T  was less than or equal to 37 (˜3 copies) and all No Template Control (NTC) had undetectable C T . PCR aliquots were also analyzed by agrose gel electrophoresis with 100 bases molecular marker (Invitrogen, Carlsbad, Calif.). One specific PCR product from a corresponding positive sample should be present after amplification. 
       Sequence Analysis 
       [0055]    Mutations detected by AS-NEPB-PCR in BRAF V600E were confirmed by direct sequencing using Rhodamine dye terminator cycle sequencing kit (Big Dye; Applied Biosystems). Cell line (20 ng) and FFPE (50 ng). DNA samples containing mutations were amplified by non-AS-PCR using sequence primers (Table1a) under the same PCR condition as AS-NEPB2-PCR-2. To verify the sequences, PCR amplified products were sent to GENEWIZ (South Plainfield, N.J., US). Sequencing was done on ABI 3730xl DNA Analyzer and analyzed using ABI PRISM DNA Sequencing Analysis Software (Applied Biosystems) according to the manufacturer&#39;s instructions. 
       Results and Discussions 
       [0056]    For BRAF V600E gene mutation detection, center-blocker Oligo (CBO) method was first adapted from the publication of K-ras mutation detection (7), the ASP and blocker designs were followed the criteria listed in the paper. Several assay conditions were tested in order to reach 0.1% detection sensitivity of BRAF mutation gene. We have tried to optimize assay conditions by titrated various annealing temperature (58, 60, 62, 64 and 65° C.) and ratio of ASP:PB (1:4, 1:2 and 1:1). However, none of conditions could reach 0.1% mutant detection sensitivity and without non-specific amplification on WT template. The results were observed under one of conditions for each CBO; 0.5% detection sensitivity was obtained without non-specific amplification from CBO-1 (64° C. and 1:1 ratio), however, the C T  in 0.5% has been shown great than 36. CBO-2 gave constantly non-specific amplification (64° C. and 1:2 ratio) if having 0.1% detection sensitivity (Table 3). 
         [0057]    Under other conditions, AS-PCR reaction was blocked by increased concentration of the blocker or annealing temperature; and more non-specific amplification occurred when reducing the concentration of the blocker or annealing temperature (data not showed). In addition, CBO method required that the sequences of Primer, Blocker and Probe have to be partial-overlapping, blocker discriminating base in the middle of the oligonucleotide and different Tm (length), which bring about challenges for BRAF gene Oligo selection and assay condition optimization although the method was successful in the K-ras mutation assay. 
         [0058]    Edge-blocker oligonucleotide (EBO) based AS-NEPB-PCR method was developed to improve detection sensitivity and remove non-specific amplification for BRAF gene mutation detection assay. Two sets of EBO, EBO-1 and EBO-2, with the corresponding forward AS primers, BRAF-AS-Forward Primer-1 and -2, were designed and evaluated with the BRAF V600E allelic variant. A common reverse primer and probe were designed downstream of the polymorphic site and used in AS-NEPB-PCR. A few of assay conditions were needed to be tested due to the same Tm for both ASP and NEPB; annealing temperature (64 and 65° C.) and ratio of ASP:EBO (1:1 or 1:2) for NE primer blocker-1 and annealing temperature (56, 58 and 60° C.) and ratio of ASP:EBO (7:1 or 3:1) for NE primer blocker-2. The annealing temperature screening was selected to be close to Tm of ASP (Table 1a). The ratio of ASP:EBO screening was decided based on the data generated from AS-PCR without adding up edge-blocker oligonucleotide (Table 3). BRAF-AS-Forward Primer-2 without edge-blocker oligonucleotide gave non-specific amplification when WT DNA was greater 50 ng input (data not shown), so less EBO was needed. 
         [0059]    The results demonstrated that incorporation of edge-blocker oligonucleotide based AS-NEPB-PCR enhanced the sensitivity of the AS-PCR, without non-specific amplification on WT DNA. It performed better than CBO method (Table 3). Edge-blocker oligonucleotide based AS-NEPB-PCR method also showed strong allele specific amplifications, detected one copy of mutant DNA in 1000-copy normal DNA background of heterogeneous mixture (0.1% mutation frequency and 2-3 mutant copies) in both AS-NEPB-PCR assays. BRAF-AS-Forward Primer-2 with EBO-2 (AS-NEPB2-PCR-2) gave the best result to discriminate the wild type and mutant alleles, in which delta C T  is calculated as the difference between SKBR3 WT cell line C T  and the HT29 mutant/SKBR3 WT mixtures cell line C T . Repeatable 0.1% mutant detection sensitivity (down to 3-5 copies of mutant) and undetectable WT specificity (up to 50 ng WT cell line DNA) were obtained by using AS-NEPB2-PCR-2 (Table 4). Undetectable WT specificity was also observed with 175 ng WT tissue DNA (data not shown). On gel image, single sharp bands were observed from 5% to 0.1% mutant reactions and no PCR products were observed from SKBR3 WT AS-NEPB2-PCR-2 reaction, except Actin-PCR products ( FIG. 4 ). 
         [0060]    Direct sequencing, as the gold standard, was used to verify the AS-NEPB2-PCR-2 method in both cell line and FFPE tissue DNA samples. The 100% sensitivity and specificity was obtained by using AS-NEPB-PCR method based on the sequence data (Table 5). 
         [0061]    The edge-blocker oligonucleotide based AS-NEPB-PCR method was also verified on two KRAS gene mutants (p.G12V; G&gt;T and p.G13D; 13G&gt;A) and compared to the center-blocker oligonucleotide based AS-PCR method. A small number of ASP vs. edge-blocker oligonucleotide ratios were tested to obtain the best concentration of edge-blocker oligonucleotides. The same AS primers described in the paper were used under the annealing temperature 60° C. suggested by the paper (7). The best result was observed with 1:1 ratio of ASP to edge-blocker oligonucleotide for both Kras G12V and G13D mutant gene detection assays; 0.1% detection sensitivity (˜5 copies) without non-specific amplification on WT DNA (Tables 6a, 6b, and 6c). We have tested the edge-blocker oligonucleotide based AS-NEPB-PCR and center-blocker oligonucleotide based AS-PCR methods under the same reaction condition. Equivalent assay performances were obtained (Table 7 and  FIGS. 5 a  and 5 b   ). Good assay precision was obtained from the edge-blocker oligonucleotide based AS-NEPB-PCR method with &lt;3% CV in three individual runs for two K-ras 0.1% (˜5 copies) mutants. 
         [0062]    Edge-blocker oligonucleotides modified by inverted dT or amino-C7 were also evaluated. The equivalent assay performances were obtained as 3′ end modified by Phosphate (Table 8). 
         [0063]    We have tested edge-blocker oligonucleotide based AS-NEPB-PCR method on 42 clinical samples, circulating colorectal tumor cells. BRAF (V600E) mutations were detected in two tissue samples and one CTC sample, which were matched with the sequencing data. Non-specific amplification was not observed in both tissue and CTC samples which confirmed by sequencing data (Table 9). 
         [0064]    Edge-blocker oligonucleotide based AS-NEPB-PCR method was also evaluated on EGFR gene (exon 21_L858R) mutation detection. The results showed 0.1% of mutations (˜5 copies) were detected without non-specific amplification at 1:1 ratio of ASP to edge-blocker oligonucleotide and Annealing Temp 63° C. (Table 10 and  FIG. 6 ). Good assay precision, &lt;2% CV, was obtained from 5%, 1% and 0.1% in the triplicates. 
         [0065]    Edge-blocker oligonucleotide based AS-NEPB-PCR method has been employed on the detection of 3 different genes (B-Raf, K-Ras, and EGFR) and 4 mutants (V600E, G12V, G13D and L858R) effectively. Optimal assay conditions were determined easily for each of the assays due to the advantage of edge-blocker oligonucleotide design, which has the same strand and length as the allele-specific primer producing almost the same melting temperatures (Tm) as ASP. We found that (1) normally 2 annealing temperatures are only needed beside Tm, one degree below and one degree up of ASP&#39;s Tm and (2) 1:1 ratio of ASP:blocker is suitable for most cases to get an optimal assay condition. 
         [0000]    In conclusion, edge-blocker oligonucleotide based AS-NEPB-PCR method is a highly sensitive and specific method for mutation detection in highly heterogeneous samples. Also, the edge-blocker oligonucleotide based AS-NEPB-PCR method provides great advantages in simplifying assay design and assay optimization over the other blocking method. Edge-blocker oligonucleotide based AS-NEPB-PCR method allows an efficient workflow when a number of different mutation assays need to be developed. 
       REFERENCES 
       [0000]    
       
         1. Guttmacher, A. E. and F. S. Collins. 2002. Genomic medicine—a primer. N. Engl. J. Med. 347:1512-1520. 
         2. Phillips, K. A., D. L. Veenstra, E. Oren, J. K. Lee, and W. Sadee. 2001. Potential role of pharmacogenomics in reducing adverse drug reactions: a systematic review. JAMA 286: 2270-2279. 
         3. Newton, C. R., A. Graham, L. E. Heptinstall, S. J. Powell, C. Summers, N. Kalsheker, J. C. Smith, and A. F. Markham. 1989. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17:2503-2516. 
         4. Livak, K. J., S. J. A. Flood, and J. A. Todd. 1995. Towards fully automated genome-wide polymorphism screening. Nat. Genet. 9:341-342. 
         5. Shale Dames and Karl V. Voelkerding at el, Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays, Journal of Molecular Diagnostics, Vol. 9, No. 3, July 2007 
         6. Willem Maat and Pieter A. Van der Velden, Pyrophosphorolysis Detects B-RAF Mutations in Primary Uveal Melanoma, Investigative Ophthalmology &amp; Visual Science, January 2008, Vol. 49, No. 1 
         7. Morlan J, Baker J, Sinicropi D, Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method. PLoS ONE 4(2): e4584. doi:10.1371/journal.pone.0004584(2009) 
         8. Methods, Compositions, and Kits for Detecting Allelic Variants, Patent by life technologies corporation, International Application No.: PCT/US2010/028963 
         9. Susana Benlloch, et al. Detection of BRAF V600E Mutation in Colorectal Cancer. JMD November 2006, Vol. 8, No. 5 
         10. Tomoaki Tanaka at el, Frequency of and variables associated with the EGFR mutation and its subtypes. Int. J. Cancer: 126, 651-655 (2010) VC 2009 
         11. Ikediobi, O. N et al. Mutation Analysis of 24 Known Cancer Genes in the NCI-60 Cell Line Set Mol. Cancer Ther., 5(11):2606-2612, (2006) 
         12. Raffaella Sordella at el, Gefitinib-Sensitizing EGFR Mutations in Lung Cancer Activate Anti-Apoptotic Pathways. Science 305, 1163 (2004) 
       
     
       TABLES 
       [0078]      
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1a 
               
             
             
               
                   
               
               
                 Primer/Probe/blocker Sequences and Labeling 
               
               
                 for BRAF gene: for probes 5′ Modification 
               
               
                 of FAM and CAL Fluor orange 560 and 3′ 
               
               
                 Modification of BHQ; blockers have 3′ end 
               
               
                 phosphate modification. 
               
             
          
           
               
                   
                 BRAF 
                   
                   
                   
               
               
                 Seq. 
                 V600E 
                 Sequence 
                 Sequence 
                 Tm 
               
               
                 ID 
                 Assay 
                 Name 
                 (5′-3″) 
                 [° C.] 
               
               
                   
               
             
          
           
               
                  1 
                 BRAF-AS- 
                 BRAF- 
                 AGGTGATTTTGG 
                 68.2 
               
               
                   
                 Forward 
                 268DF-AS 
                 TCTAGCTACAG A   
                   
               
               
                   
                 Primer-1 
                   
                   
                   
               
               
                   
               
               
                  2 
                 BRAF-AS- 
                 BRAF- 
                 TTTTGGTCTAGC 
                 58.1 
               
               
                   
                 Forward 
                 274DF-AS 
                 TACAG A   
                   
               
               
                   
                 Primer-2 
                   
                   
                   
               
               
                   
               
               
                  3 
                 Common 
                 BRAF- 
                 AGCCTCAATTCT 
                 71.5 
               
               
                   
                 Reverse 
                 347SR 
                 TACCATCCA 
                   
               
               
                   
                 Primer 
                   
                   
                   
               
               
                   
               
               
                  4 
                 Common 
                 BRAF- 
                 FAM-AGTGGGTC 
                 79.0 
               
               
                   
                 Probe 
                 305DP 
                 CCATCAGTTTGA 
                   
               
               
                   
                   
                   
                 ACAGT-BHQ 
                   
               
               
                   
               
               
                  5 
                 EBO NE 
                 BRAF- 
                 AGGTGATTTTGG 
                 68.2 
               
               
                   
                 Primer 
                 268DF-WTB 
                 TCTAGCTACAG 
                   
               
               
                   
                 blocker-1 
                   
                 T_PO 4   
                   
               
               
                   
               
               
                  6 
                 EBO NE 
                 BRAF- 
                 TTTTGGTCTAGC 
                 58.1 
               
               
                   
                 Primer 
                 274DF-WTB 
                 TACAGT_PO 4   
                   
               
               
                   
                 blocker-2 
                   
                   
                   
               
               
                   
               
               
                  7 
                 CBO 
                 BRAF- 
                 TCTAGCTACAGT 
                 83.4 
               
               
                   
                 blocker-1 
                 280ASB32 
                 GAAATCTCGATG 
                   
               
               
                   
                   
                   
                 GAGTGGGT-PO 4   
                   
               
               
                   
               
               
                  8 
                 CBO 
                 BRAF- 
                 TCTAGCTACAGT 
                 68.3 
               
               
                   
                 blocker-2 
                 280ASB23 
                 GAAATCTCGAT- 
                   
               
               
                   
                   
                   
                 PO 4   
                   
               
               
                   
               
               
                  9 
                 Actin 
                 B-actin 
                 AAGCCACCCCAC 
                 71.6 
               
               
                   
                 (Internal 
                 3295U20 
                 TTCTCTCT 
                   
               
               
                 10 
                 Control) 
                 B-actin 
                 AATGCTATCACC 
                 71.4 
               
               
                   
                   
                 3346L20 
                 TCCCCTGT 
                   
               
               
                 11 
                   
                 B-actin 
                 Orange-AGAAT 
                 84.0 
               
               
                   
                   
                 3319P26 
                 GGCCCAGTCCTC 
                   
               
               
                   
                   
                   
                 TCCCAAGTC- 
                   
               
               
                   
                   
                   
                 BHQ 
                   
               
               
                   
               
               
                 12 
                 BRAF 
                 BRAF- 
                 TGATAGGAAAAT 
                 66.9 
               
               
                   
                 Outer 
                 186DF 
                 GAGATCTACTGT 
                   
               
               
                 13 
                 Primer 
                 BRAF- 
                 TTTACATAAAAA 
                 67.0 
               
               
                   
                 (Nest- 
                 472DR 
                 ATAAGAACACTG 
                   
               
               
                   
                 PCR) 
                   
                 ATT 
                   
               
               
                   
               
               
                 14 
                 Sequence 
                 BRAF- 
                 GTGATTTTGGTC 
                 58.5 
               
               
                   
                 PCR 
                 270DF 
                 TAGCTA 
                   
               
               
                 15 
                 Primer 
                 BRAF- 
                 AGCCTCAATTCT 
                 71.5 
               
               
                   
                   
                 347SR 
                 TACCATCCA 
                   
               
               
                   
               
               
                 16 
                 Sequence 
                 BRAF- 
                 CTCAATTCTTAC 
                 69.6 
               
               
                   
                 Primer 
                 343SR 
                 CATCCACAAA 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1b 
               
             
             
               
                   
               
               
                 Primer/Probe/blocker Sequences and Labeling 
               
               
                 for K-ras gene: for probes 5′ Modification 
               
               
                 of FAM and 3′ Modification of MGB; blockers 
               
               
                 have 3′ end phosphate modification. Actin 
               
               
                 primers and probe are the same as BRAF assay 
               
               
                 in Table 1a. The melting temperatures (Tm) 
               
               
                 were calculated by Oligo software as described 
               
               
                 in “Oligo Design” for Tables 1a, 1b and 1c. 
               
             
          
           
               
                   
                 K-ras 
                   
                   
                   
               
               
                 Seq 
                 Mutation 
                 Sequence 
                 Sequence 
                 Tm 
               
               
                 ID 
                 Assay 
                 Name 
                 (5′-3″) 
                 [° C.] 
               
               
                   
               
             
          
           
               
                 17 
                 G12V-AS 
                 KrasP4 
                 TTGTGGTAGTTG 
                 66.3 
               
               
                   
                 Forward Primer 
                   
                 GAGCTGT 
                   
               
               
                   
               
               
                 18 
                 G12V-EBO NE 
                 KrasP4B 
                 TTGTGGTAGTTG 
                 ~66.3 
               
               
                   
                 Primer blocker 
                   
                 GAGCTGG-P04 
                   
               
               
                   
               
               
                 19 
                 G12V-CBO 
                 ASB1 
                 TTGGAGCTGGTG 
                 77.9 
               
               
                   
                 blocker 
                   
                 GCGTAGG-P04 
                   
               
               
                   
               
               
                 20 
                 G12V-Probe 
                 KProbe1 
                 FAM-CACTCTTG 
                 65.9 
               
               
                   
                   
                   
                 CCTACGC-MGB 
                   
               
               
                   
               
               
                 21 
                 G13D-AS Forward 
                 KrasP7 
                 GTAGTTGGAGCT 
                 60.2 
               
               
                   
                 Primer 
                   
                 GGTGA 
                   
               
               
                   
               
               
                 22 
                 G13D-EBO NE 
                 KrasP7B 
                 GTAGTTGGAGCT 
                 ~60.2 
               
               
                   
                 Primer blocker 
                   
                 GGTGG-P04 
                   
               
               
                   
               
               
                 23 
                 G13D-CBO 
                 ASB2 
                 GCTGGTGGCGTA 
                 74.8 
               
               
                   
                 blocker 
                   
                 GGC-P04 
                   
               
               
                   
               
               
                 24 
                 G13D-Probe 
                 KProbe2 
                 FAM-CACTCTTG 
                 59.9 
               
               
                   
                   
                   
                 CCTACG-MGB 
                   
               
               
                   
               
               
                 25 
                 Common 
                 KrasR 
                 TGATTCTGAATT 
                 74.3 
               
               
                   
                 Reverse 
                   
                 AGCTGTATCGTC 
                   
               
               
                   
                 Primer 
                   
                 AA 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1C 
               
             
             
               
                   
               
               
                 Primer/Probe/blocker Sequences and Labeling 
               
               
                 for EGFR gene (L858R; 2573 T &gt; G): 
               
               
                 5′ Modification of FAM and 3′ Modification 
               
               
                 of BHQ for probes; 3′ end phosphate 
               
               
                 modification for blockers. Actin primer/probe 
               
               
                 are the same as BRAF assay in Table 1a. 
               
             
          
           
               
                   
                 EGFR 
                   
                   
                   
               
               
                 Seq 
                 Mutation 
                 Sequence 
                 Sequence 
                 Tm 
               
               
                 ID 
                 Assay 
                 Name 
                 (5′-3″) 
                 (° C.) 
               
               
                   
               
             
          
           
               
                 26 
                 Ex21_L858R 
                 Ex21-248FAS 
                 ATCACAGATTTT 
                 64.3 
               
               
                   
                   
                   
                 GGGCG 
                   
               
               
                 27 
                   
                 Ex21-248FWTB 
                 ATCACAGATTTT 
                 ~63.0 
               
               
                   
                   
                   
                 GGGCT-P04 
                   
               
               
                 28 
                   
                 Ex21-330SR 
                 GAAAATGCTGGC 
                 72.5 
               
               
                   
                   
                   
                 TGACCTAAA 
                   
               
               
                 29 
                   
                 Ex21-271P 
                 FAM-TGGGTGCG 
                 79.6 
               
               
                   
                   
                   
                 GAAGAGAAAGAA 
                   
               
               
                   
                   
                   
                 TACC-BHQ 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2a 
               
             
             
               
                   
               
               
                 Concentration of primer/blocker/probe in 
               
               
                 AS-NEPB-PCR BRAF V600E duplex assay 
               
             
          
           
               
                   
                   
                 Final 
               
               
                 BRAF V600E NEPB 
                   
                 Conc. 
               
               
                 Assay 
                 Name(Primer/Blocker/Probe) 
                 (uM) 
               
               
                   
               
             
          
           
               
                 BRAF-AS-NEPB1-PCR-1 
                 BRAF-268DF-AS (SEQ ID 1) 
                 0.450 
               
               
                   
                 BRAF-347SR (SEQ ID 3) 
                 0.450 
               
               
                   
                 BRAF-268DF-WTB (SEQ ID 5) 
                 0.900 
               
               
                   
                 BRAF-305DP (SEQ ID 4) 
                 0.250 
               
               
                 BRAF-AS-NEPB2-PCR-2 
                 BRAF-274DF-AS (SEQ ID 2) 
                 0.900 
               
               
                   
                 BRAF-347SR (SEQ ID 15) 
                 0.900 
               
               
                   
                 BRAF-274DF-WTB (SEQ ID 6) 
                 0.125 
               
               
                   
                 BRAF-305DP (SEQ ID 4) 
                 0.250 
               
               
                 Actin 
                 B-actin 3295U20 (SEQ ID 9) 
                 0.030 
               
               
                   
                 B-actin 3346L20 (SEQ ID 10) 
                 0.030 
               
               
                   
                 B-actin 3319P26_Ora (SEQ ID 
                 0.030 
               
               
                   
                 11) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2b 
               
             
             
               
                   
               
               
                 Concentration of primer/blocker/probe in AS-NEPB-PCR K-ras singlex 
               
               
                 assay 
               
             
          
           
               
                   
                   
                 Final 
               
               
                   
                   
                 Conc. 
               
               
                 K-ras Mutation Assay 
                 Name(Primer/Blocker/Probe) 
                 (uM) 
               
               
                   
               
             
          
           
               
                 G12V-NE Primer Blocker 
                 KrasP4 (SEQ ID 17) 
                 0.90 
               
               
                   
                 KrasR (SEQ ID 25) 
                 0.90 
               
               
                   
                 KrasP4B (SEQ ID 18) 
                 0.90 
               
               
                   
                 KProbe1 (SEQ ID 20) 
                 0.20 
               
               
                 G13D-NE Primer Blocker 
                 KrasP7 (SEQ ID 21) 
                 0.90 
               
               
                   
                 KrasR (SEQ ID 25) 
                 0.90 
               
               
                   
                 KrasP7B (SEQ ID 22) 
                 0.90 
               
               
                   
                 KProbe2 (SEQ ID 24) 
                 0.20 
               
               
                 Actin 
                 B-actin 3295U20 (SEQ ID 9) 
                 0.40 
               
               
                   
                 B-actin 3346L20 (SEQ ID 10) 
                 0.40 
               
               
                   
                 B-actin 3319P26_Ora (SEQ ID 11) 
                 0.20 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2C 
               
             
             
               
                   
               
               
                 Concentration of primer/blocker/probe in 
               
               
                 AS-NEPB-PCR EGFR singlex assay 
               
             
          
           
               
                 EGFR 
                   
                   
               
               
                 Mutation Assay 
                 Name(Primer/Blocker/Probe) 
                 Final Conc. (uM) 
               
               
                   
               
             
          
           
               
                 Ex21_L858R 
                 Ex21_248FAS (SEQ ID 26) 
                 0.90 
               
               
                   
                 Ex21-248FWTB (SEQ ID 27) 
                 0.90 
               
               
                   
                 Ex21-330SR (SEQ ID 28) 
                 0.90 
               
               
                   
                 Ex21-271P (SEQ ID 29) 
                 0.20 
               
               
                 Actin 
                 B-actin 3295U20 (SEQ ID 9) 
                 0.40 
               
               
                   
                 B-actin 3346L20 (SEQ ID 10) 
                 0.40 
               
               
                   
                 B-actin 3319P26_Ora (SEQ ID 11) 
                 0.20 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 10 ng DNA of HT29 mutant/SKBR3 WT mixtures at various ratios 
               
               
                 were input into the PCR reactions for BRAF V600E mutant 
               
               
                 detection, 0.1% of mixtures equivalent to ~3 copies of mutant. 
               
               
                 Undetermined in PCR was considered as C T  40 during delta 
               
               
                 C T  calculations. Both NE Primer blocker, EBO, method 
               
               
                 reached 0.1% detection sensitivity without non-specific amplification. 
               
               
                 NE Primer blocker-2 showed the best results to discriminate alleles 
               
               
                 (bigger delta C T  between WT and mutant) compared to 
               
               
                 CBO blocker method. CBO blocker-1 showed 0.5% detection 
               
               
                 sensitivity and non-specific amplification on WT DNA. However, the 
               
               
                 delta C T  between WT and mutant was ~3Ct less than NEPB 
               
               
                 method. CBO blocker-2 gave constantly non-specific amplification if 
               
               
                 having 0.1% detection sensitivity. All the final reaction conditions 
               
               
                 were described in the Method of “AS-NEPB-PCR Amplification”. 
               
             
          
           
               
                   
                 No 
                 NE Primer 
                 NE Primer 
                 CBO 
                 CBO 
               
               
                   
                 blocker 
                 blocker-1 
                 blocker-2 
                 blocker-1 
                 blocker-2 
               
               
                   
                   
               
             
          
           
               
                 % Mutant/ 
                   
                   
                   
                   
                   
               
               
                 PCR 
               
               
                 Result 
               
               
                   5% 
                 28.9 
                 31.2 
                 32.6 
                 33.5 
                 34.2 
               
               
                   1% 
                 28.7 
                 31.1 
                 32.3 
                 34.6 
                 39.9 
               
               
                 0.5% 
                 29.0 
                 30.9 
                 32.1 
                 34.6 
                 Undetermined 
               
               
                 0.1% 
                 29.3 
                 31.3 
                 33.3 
                 34.4 
                 Undetermined 
               
               
                 Mutant 
               
               
                 Delta 
               
               
                 C T  to WT 
               
               
                   5% 
                 2.9 
                 7.4 
                 8.5 
                 5.5 
                 3.3 
               
               
                   1% 
                 1.6 
                 3.8 
                 5.9 
                 3.6 
                 3.3 
               
               
                 0.5% 
                 0.7 
                 3.6 
                 6.1 
                 2.6 
                 2.2 
               
               
                 0.1% 
                 0.5 
                 2.2 
                 3.1 
                 −1.0 
                 1.2 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 10-50 ng DNA of HT29 mutant/SKBR3 WT mixtures at various ratios 
               
               
                 were input into AS-NEPB2-PCR-2 of BRAF V600E mutant detection. 
               
               
                 The method detected constantly 0.1% of mutation rate without 
               
               
                 non-specific amplification. All of Actin C T s were &lt;25 and 
               
               
                 NTC was not undetermined. 
               
             
          
           
               
                 % Mutant/PCR 
                   
                   
                   
               
               
                 Result (C T ) 
                 10 ng 
                 20 ng 
                 50 ng 
               
               
                   
               
               
                 5.0% 
                 31.5 
                 28.5 
                 27.5 
               
               
                 1.0% 
                 34.1 
                 31.9 
                 30.2 
               
               
                 0.5% 
                 33.9 
                 32.1 
                 31.7 
               
               
                 0.1% 
                 36.9 
                 33.6 
                 33.6 
               
               
                 WT 
                 Undetermined 
                 Undetermined 
                 Undetermined 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 The assay gave consistent performance on detecting the mutated 
               
               
                 allele without non-specific amplification of DNA samples, which 
               
               
                 were confirmed by sequencing. NVD-No variants detected. 
               
             
          
           
               
                   
                   
                   
                 Sequence Data 
               
               
                   
                   
                   
                 (p.V600E 
               
               
                   
                 BRAF (C T ) 
                 ACTIN (C T ) 
                 GTG &gt; GAG) 
               
               
                   
                   
               
             
          
           
               
                 Melanoma FFPE 
                   
                   
                   
               
               
                 13820T2 
                 26.6 
                 24.5 
                 1. T &gt; A(50%) 
               
               
                 13819T2 
                 25.5 
                 24.1 
                 2. T &gt; A(50%) 
               
               
                 13788T2 
                 28.5 
                 24.9 
                 3. T &gt; A(50%) 
               
               
                 13724T2 
                 38.6 
                 26.6 
                 4. p.V600K 
               
               
                   
                   
                   
                 c.1798_1799 
               
               
                   
                   
                   
                 GT &gt; AA  
               
               
                   
                   
                   
                 (Complex60%) 
               
               
                 Colon FFPE 
               
               
                 02671T1 
                 Undetermined 
                 22.7 
                 5. NVD 
               
               
                 02671T2 
                 Undetermined 
                 24.1 
                 6. NVD 
               
               
                 02973T1 
                 Undetermined 
                 24.8 
                 7. NVD 
               
               
                 02973T2 
                 Undetermined 
                 25.7 
                 8. NVD 
               
               
                 Cell Line DNA 
               
               
                 H29_40% 
                 26.7 
                 24.4 
                 9. T &gt; A(40%) 
               
               
                 H29_20% 
                 N/A 
                 N/A 
                 10. T &gt; A(20%) 
               
               
                 H29_10% 
                 27.2 
                 23.2 
                 11. T &gt; A(10%) 
               
               
                 WT_SKBR3 
                 Undetermined 
                 24.9 
                 12. NVD 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6a 
               
             
             
               
                   
               
               
                 KrasP4 (G12V; G &gt; T) AS-NEPB-PCR on SW480 Cell line 
               
               
                 DNA: 20 ng DNA of SW480 mutant/SKBR3 WT mixtures at three 
               
               
                 ASP:NEPB ratios were input into the PCR reactions, 0.1% of 
               
               
                 mixtures equivalent to ~5 copies of mutant. PCR conditions 
               
               
                 were as stated previously. The ratio of 1:1 gave the best result that 
               
               
                 reached 0.1% detection sensitivity without non-specific 
               
               
                 amplification. All of NTC was not undetermined. 
               
             
          
           
               
                 NEPB 
                   
                   
                   
                   
                   
               
               
                 Titration/ 
               
               
                 AS-PCR (C T ) 
                 5% 
                 1% 
                 0.5% 
                 0.1% 
                 WT 
               
               
                   
               
             
          
           
               
                 No Blocker 
                 28.9 
                 31.2 
                 32.6 
                 33.5 
                 34.2 
               
               
                 2:1-ASP:NEPB 
                 28.7 
                 31.1 
                 32.3 
                 34.6 
                 39.9 
               
               
                 1:1-ASP:NEPB 
                 29.0 
                 30.9 
                 32.1 
                 34.6 
                 Undetermined 
               
               
                 1:2-ASP:NEPB 
                 29.3 
                 31.3 
                 33.3 
                 34.4 
                 Undetermined 
               
               
                 Actin (Ctrl) 
                 22.8 
                 22.8 
                 22.5 
                 23.1 
                 22.8 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6b 
               
             
             
               
                   
               
               
                 KrasP7 (G13D; 13G &gt; A) AS-NEPB-PCR on HCT116 Cell line 
               
               
                 DNA: 20 ng DNA of HCT116 mutant/SKBR3 WT mixtures with three 
               
               
                 ASP:NEPB ratios were tested. PCR condition was the same as 
               
               
                 KrasP4 assay. The ratio of 2:1 or 1:1 reached 0.1% detection 
               
               
                 sensitivity without non-specific amplification (ratio of 1:1 showed 
               
               
                 the similar C T  value as ratio of 2:1, 1:1 ratio was selected 
               
               
                 for the further study). All of NTC was not undetermined. 
               
             
          
           
               
                 NEPB 
                   
                   
                   
                   
                   
               
               
                 Titration/ 
               
               
                 AS-PCR (C T ) 
                 5% 
                 1% 
                 0.5% 
                 0.1% 
                 WT 
               
               
                   
               
             
          
           
               
                 No Blocker 
                 30.6 
                 34.1 
                 33.8 
                 35.0 
                 38.7 
               
               
                 2:1-ASP:NEPB 
                 31.4 
                 32.9 
                 35.4 
                 37.6 
                 Undetermined 
               
               
                 1:1-ASP:NEPB 
                 31.6 
                 33.9 
                 34.0 
                 36.5 
                 Undetermined 
               
               
                 1:2-ASP:NEPB 
                 32.2 
                 33.4 
                 33.8 
                 38.2 
                 Undetermined 
               
               
                 Actin (Ctrl) 
                 22.4 
                 22.8 
                 22.7 
                 22.6 
                 22.9 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
             
             
               
                 NEPB and CBO Blocker methods were tested on SW480 and HCT116 Cell 
               
               
                 line DNA with ASP of KrasP4 and KrasP7: 20 ng DNA of each mutant/SKBR3 WT 
               
               
                 mixtures with 1:1 ratio of ASP:NEPB or 1:4 ratio of ASP:CBO blocker was used 
               
               
                 the PCR reactions. PCR conditions were as stated previously. NEPB method 
               
               
                 showed the equivalent, if not better, assay performances as CBO Blocker 
               
               
                 method; reached 0.1% detection sensitivity without non-specific amplification on 
               
               
                 WT DNA. All of NTC was not undetermined. 
               
             
          
           
               
                 K-rasP4 (G12V; G &gt; T) 
                 5% 
                 1% 
                 0.5% 
                 0.1% 
                   
               
               
                 AS-PCR (C T ) 
                 SW480 
                 SW480 
                 SW480 
                 SW480 
                 WT 
               
               
                   
               
               
                 No Blocker 
                 28.9 
                 31.2 
                 32.6 
                 33.0 
                 34.2 
               
               
                 1:1-ASP:NEPB 
                 28.6 
                 30.8 
                 32.7 
                 34.2 
                 Undetermined 
               
               
                 1:4-ASP:CBO 
                 29.3 
                 31.3 
                 33.3 
                 22.5 
                 Undetermined 
               
               
                 blocker 
               
               
                 Actin (Ctrl) 
                 23.1 
                 22.7 
                 22.7 
                 22.8 
                 22.9 
               
               
                   
               
               
                 K-rasP7 (G13V; 
               
               
                 13G &gt; A) 
                 5% 
                 1% 
                 0.5% 
                 0.1% 
               
               
                 AS-PCR (C T ) 
                 HCT116 
                 HCT116 
                 HCT116 
                 HCT116 
                 WT (SKBR3) 
               
               
                   
               
               
                 No Blocker 
                 30.6 
                 34.1 
                 33.8 
                 35.0 
                 38.7 
               
               
                 1:1-ASP:NEPB 
                 30.6 
                 33.2 
                 35.0 
                 36.1 
                 Undetermined 
               
               
                 1:4-ASP:CBO 
                 30.6 
                 33.2 
                 34.1 
                 37.2 
                 Undetermined 
               
               
                 blocker 
               
               
                 Actin (Ctrl) 
                 23.1 
                 22.7 
                 22.7 
                 22.8 
                 22.9 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 20 ng DNA of HT29 mutant (BRAF V600E)/SKBR3 WT mixtures 
               
               
                 at various ratios were input into AS-NEPB-PCR. Three NEPB 
               
               
                 modifications, Phosphate or inverted dT or amino-C7, gave 
               
               
                 equivalent results. The method detected 0.1% of mutation rate 
               
               
                 without non-specific amplification. All of Actin Cts were &lt;25 
               
               
                 and NTC was not undetermined. 
               
             
          
           
               
                 % 
                   
                   
                   
                   
               
               
                 Mutant/PCR 
                 No 
               
               
                 Result (C T ) 
                 Blocker 
                 Phosphate 
                 Inverted dT 
                 Amino-C7 
               
               
                   
               
               
                   5% 
                 28.9 
                 29.2 
                 28.7 
                 29.0 
               
               
                   1% 
                 31.7 
                 31.4 
                 30.7 
                 32.1 
               
               
                 0.5% 
                 32.6 
                 33.9 
                 33.2 
                 33.3 
               
               
                 0.1% 
                 35.6 
                 35.7 
                 36.7 
                 36.8 
               
               
                 WT 
                 36.1 
                 Undetermined 
                 Undetermined 
                 Undetermined 
               
               
                 (SKBR3) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Using AS-NEPB-PCR method, BRAF (V600E) mutations were 
               
               
                 detected in the two clinical tissue samples (2/42) and one CTC 
               
               
                 sample (1/42), which were 100% (tissues) or 50% (CTCs), 
               
               
                 matched with sequencing data. For CTC samples, the mutation 
               
               
                 only was detected for CTC-196 which had 5 CTCs, but not 
               
               
                 detected for CTC-220 which had no CTC account based on the 
               
               
                 analysis of CellSearch system. Non-specific amplification was 
               
               
                 not observed in both tissue and CTC samples which were 
               
               
                 confirmed by their sequence data. All of Actin C T s 
               
               
                 were &lt;25 (data not shown). 
               
             
          
           
               
                 Sample 
                 Matched 
                 Tissue 
                 Tissue 
                 Tissue PCR 
                 CTC PCR 
               
               
                 # 
                 CTC ID 
                 ID 
                 Sequence Data 
                 data (Ct) 
                 data (Ct) 
               
               
                   
               
             
          
           
               
                 1 
                 CTC207 
                 8090 
                 ND 
                 ND 
                 ND 
               
               
                 2 
                 CTC249 
                 8110 
                 ND 
                 ND 
                 ND 
               
               
                 3 
                 CTC253 
                 5439 
                 ND 
                 ND 
                 ND 
               
               
                 4 
                 CTC209 
                 8086 
                 ND 
                 ND 
                 ND 
               
               
                 5 
                 CTC252 
                 5426 
                 ND 
                 ND 
                 ND 
               
               
                 6 
                 CTC195 
                 5419 
                 ND 
                 ND 
                 ND 
               
               
                 7 
                 CTC202 
                 8085 
                 ND 
                 ND 
                 ND 
               
               
                 8 
                 CTC194 
                 5423 
                 ND 
                 ND 
                 ND 
               
               
                 9 
                 CTC192 
                 8109 
                 ND 
                 ND 
                 ND 
               
               
                 10 
                 CTC218 
                 8094 
                 ND 
                 ND 
                 ND 
               
               
                 11 
                 CTC211 
                 8101 
                 ND 
                 ND 
                 ND 
               
               
                 12 
                 CTC220 
                 8095 
                 c.T &gt; A; 
                 26.3 
                 ND 
               
               
                   
                   
                   
                 p.V600E, 10% 
               
               
                 13 
                 CTC247 
                 8103 
                 ND 
                 ND 
                 ND 
               
               
                 14 
                 CTC191 
                 5416 
                 ND 
                 ND 
                 ND 
               
               
                 15 
                 CTC216 
                 8093 
                 ND 
                 ND 
                 ND 
               
               
                 16 
                 CTC198 
                 8082 
                 ND 
                 ND 
                 ND 
               
               
                 17 
                 CTC246 
                 8106 
                 ND 
                 ND 
                 ND 
               
               
                 18 
                 CTC201 
                 8084 
                 ND 
                 ND 
                 ND 
               
               
                 19 
                 CTC189 
                 8108 
                 ND 
                 ND 
                 ND 
               
               
                 20 
                 CTC190 
                 5454 
                 ND 
                 ND 
                 ND 
               
               
                 21 
                 CTC215 
                 8102 
                 ND 
                 ND 
                 ND 
               
               
                 22 
                 CTC227 
                 8100 
                 ND 
                 ND 
                 ND 
               
               
                 23 
                 CTC243 
                 5445 
                 ND 
                 ND 
                 ND 
               
               
                 24 
                 CTC196 
                 5414 
                 c.T &gt; A; 
                 30.3 
                 34.6 
               
               
                   
                   
                   
                 p.V600E, 80% 
               
               
                 25 
                 CTC200 
                 8083 
                 ND 
                 ND 
                 N/A 
               
               
                 26 
                 CTC254 
                 8118 
                 ND 
                 ND 
                 ND 
               
               
                 27 
                 CTC221 
                 8097 
                 ND 
                 ND 
                 ND 
               
               
                 28 
                 CTC204 
                 8089 
                 ND 
                 ND 
                 ND 
               
               
                 29 
                 CTC219 
                 8096 
                 ND 
                 ND 
                 ND 
               
               
                 30 
                 CTC250 
                 8112 
                 ND 
                 ND 
                 ND 
               
               
                 31 
                 CTC251 
                 8114 
                 ND 
                 ND 
                 ND 
               
               
                 32 
                 CTC244 
                 5512 
                 ND 
                 ND 
                 ND 
               
               
                 33 
                 CTC210 
                 8087 
                 ND 
                 ND 
                 ND 
               
               
                 34 
                 CTC248 
                 8107 
                 ND 
                 ND 
                 ND 
               
               
                 35 
                 CTC222 
                 8098 
                 ND 
                 ND 
                 ND 
               
               
                 36 
                 CTC226 
                 5438 
                 ND 
                 ND 
                 ND 
               
               
                 37 
                 CTC217 
                 5431 
                 Seq Fail 
                 ND 
                 ND 
               
               
                 38 
                 CTC245 
                 8105 
                 ND 
                 39.5 
                 ND 
               
               
                 39 
                 CTC208 
                 8091 
                 ND 
                 ND 
                 N/A 
               
               
                 40 
                 CTC284 
                 8116 
                 ND 
                 ND 
                 ND 
               
               
                 41 
                 CTC203 
                 8078 
                 ND 
                 ND 
                 ND 
               
               
                 42 
                 CTC225 
                 8099 
                 ND 
                 39.4 
                 ND 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 EGFR Exon 21 L858R (2573 T &gt; G) AS-NEPB-PCR on NCI-H1975 
               
               
                 Cell line DNA: 20 ng DNA of NCI-H1975 mutant/NCI-H358 DNA WT 
               
               
                 mixtures at 1:1 ASP:NEPB ratios were input into the PCR reactions, 
               
               
                 0.1% of mixtures equivalent to ~5 copies of mutant. PCR conditions 
               
               
                 were as stated previously. 0.1% detection sensitivity was reached 
               
               
                 without non-specific amplification. All of NTC was not undetermined. 
               
             
          
           
               
                 % Mutant/PCR 
                   
                   
                   
                   
                   
               
               
                 Result (C T ) 
                 5% 
                 1% 
                 0.5% 
                 0.1% 
                 WT 
               
               
                   
               
             
          
           
               
                 No Blocker 
                 29.2 
                 29.5 
                 31.0 
                 33.0 
                 35.5 
               
               
                 AS-NEPB-PCR 
                 28.3 
                 29.8 
                 31.3 
                 34.2 
                 Undetermined 
               
               
                 Actin (Ctrl) 
                 22.4 
                 22.9 
                 22.4 
                 22.5 
                 22.3