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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6858—Allele-specific amplification

Definitions

• Nucleic acid-based diagnostic tests are widely used in medicine, forensics and environmental applications. Detecting variations in a particular nucleic acid sequence provides information about polymorphisms and mutations, including disease-causing mutations. For example, detecting an individual's mutant genotype provides disease carrier status for genetic counseling. A more challenging task is detecting somatic mutations that arise in tissues and cause disease or disease progression. For example, many cancers are caused by a particular mutation. Later, additional mutations accumulate in cancer cells during tumor progression. See Lea et al. (2007) Genetic pathways and mutation profiles of human cancers: site and exposure-specific patterns, Carcinogenesis, 28(9):1851-1858. Downward, J. (2003) Targeting RAS signaling pathways in cancer therapy (2005), Nature Rev. Cancer, 3:11-22.
• “Conditions suitable for primer extension” refer to conditions under which primers that hybridize to a template nucleic acid are extended by a nucleotide-incorporating biocatalyst, such as a polymerase. For example, such conditions occur during a polymerase chain reaction (PCR) annealing and extension step.
• PCR polymerase chain reaction
• PCR polymerase chain reaction
• Those of skill in the art will appreciate that such conditions can vary, and are generally influenced by ionic strength of the solution, temperature and sequence of the particular template nucleic acid and primers.
• PCR polymerase chain reaction
• a nucleic acid is "complementary" in relation to another nucleic acid when at least a subsequence of the nucleic acid can combine in an antiparallel association with at least a subsequence of the other nucleic acid to form a duplex.
• each base of the oligonucleotide in an oligonucleotide that is "fully complementary” to a particular nucleic acid sequence, each base of the oligonucleotide is complementary to the corresponding base in the particular sequence.
• An oligonucleotide is "partially complementary" to a particular nucleic acid sequence when one or more of the bases in the oligonucleotide are not complementary (“mismatched") with the corresponding bases in the other nucleic acid!
• the improvement of the present invention is based on the discovery that the relative proximity of the primer and the blocker oligonucleotide, as well as certain chemical modifications of the primer and the polymerase, greatly improve selective amplification.
• the amplification products may be detected after the amplification has been completed, for example, by gel electrophoresis of the unlabeled products and staining of the gel with a nucleic acid-binding dye.
• the amplification products may carry a radioactive or a chemical label, either by virtue of incorporation during synthesis or by virtue of having a labeled primer.
• the labeled amplification products may be detected with suitable radiological or chemical tools known in the art.
• the product After electrophoresis, the product may also be detected with a target- specific probe labeled by any one of the methods known in the art.
• the labeled probe may also be applied to the target without electrophoresis, i.e. in a "dot blot" assay or the like.
• the present invention involves detection of disease-related mutations, including cancer-related mutations in the presence of the wild-type, i.e. non- mutated nucleic acid sequences. It is generally known that during cancer progression, the tumor cells accumulate mutations that confer selective advantages to the mutant cells, see Downward, J. (2003) Targeting RAS signaling pathways in cancer therapy (2005), Nature Rev. Cancer, 3:11-22. Often the mutations confer resistance to anti-tumor agents used in therapy, see Pao et al. (2005) KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib and or erlotinib, PLoS Medicine, 2(1), el7. Detecting such mutations will spare the patients the trouble and unnecessary risk associated with taking an ineffective drug with unpleasant side effects. More broadly, detecting the cancer-related mutations is informative for prognosis of the existing disease, as well as for initial cancer screening.
• the kit of the present invention typically includes one or more of nucleic acid precursors, such as nucleoside triphosphates (deoxyribonucleoside triphosphates or ribonucleoside triphosphates), optionally, a pyrophosphatase, for minimizing pyrophosphorolysis of nucleic acids, a uracil N- glycosylase (UNG) for protection against carry-over contamination of amplification reactions, pre-made reagents and buffers necessary for the amplification reaction and optionally, detection, and a set of instructions for conducting allele-specific amplification of the present invention.
• nucleic acid precursors such as nucleoside triphosphates (deoxyribonucleoside triphosphates or ribonucleoside triphosphates)
• a pyrophosphatase for minimizing pyrophosphorolysis of nucleic acids
• UNG uracil N- glycosylase
• exon 3 sequences were co-amplified in the same reaction with the KRAS exon 2 sequences using the upstream primer SEQ ID NO. 6, downstream primer SEQ ID NO. 7 and detection probe SEQ ID NO. 8. For simplicity, the results of amplification of exon 3 sequences, detected in a separate wavelength channel, are not shown.
• the primer and probe sequences are shown in Table 1. Table 1
• the suppressive conditions are: the limiting primer is a mixture of SEQ ID NO: 3 and 4 and the enzyme has a hot-start capability ( ⁇ Z05 GOLD).
• the control conditions are: the limiting primer is only SEQ ID NO: 3 (without the 3'-terminal chemical modification) and the enzyme has no hot-start capability ( ⁇ Z05).
• the pH was adjusted to 8.3, and the polymerase activation step was removed from the cycling profile.
• Figure 4 shows the results of an experiment identical to that on Figure 3, except both suppressive and control conditions employ the use of the hot-start enzyme ⁇ Z05 GOLD.
• the mixture of SEQ ID NO: 3 and 4 was used for the suppressive conditions, and SEQ ID NO: 3 only (no chemical modification) for the control conditions.
• Figure 6 shows the results of an experiment identical to that on Figure 3, except both suppressive and control conditions employ the use of the mixture of SEQ NO: 3 and 4.
• the suppressive conditions use a hot-start enzyme ⁇ Z05 GOLD, while the control conditions use a non-hot-start enzyme ⁇ Z05.
• Figure 7 shows the results of an experiment identical to that on Figure 3, except both suppressive and control conditions employ the use of SEQ NO: 4 only (with a chemical modification).
• the suppressive conditions use a hot-start enzyme ⁇ Z05 GOLD, while the control conditions use a non-hot-start enzyme ⁇ Z05.
• the mutant targets are identified by a lower melt peak maximum (T m ) than the wild type targets.
• T m melt peak maximum
• the dashed lines show wild-type sequences while the solid lines show patient samples where mutant sequences are present. Some patient samples contain both the wild-type and the mutant sequences.

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• 2010
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