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EP-4739792-A1 - PRIMERS FOR SELECTIVE AMPLIFICATION OF RARE TARGET SEQUENCES

EP4739792A1EP 4739792 A1EP4739792 A1EP 4739792A1EP-4739792-A1

Abstract

The present invention discloses multi-part amplification primers that distinguish between a mutant DNA target sequence and a closely related wild-type DNA target sequence. Further disclosed are methods for the detection of low copy targets in qPCR reactions using discloses primers.

Inventors

  • PELEG, OFER

Assignees

  • Infiniplex Ltd.

Dates

Publication Date
20260513
Application Date
20240703

Claims (15)

  1. 1. A multi-part amplification primer that distinguishes between a mutant DNA target sequence and a closely related wild-type DNA target sequence characterized in that it consists of or comprises, in the 5' to 3' direction, the following five contiguous DNA sequences, (i) a docking sequence, capable of forming a hybrid 15-40 nucleotides in length with the mutant DNA target sequence and with the wildtype DNA target sequence, (ii) a hairpin sequence incapable of forming a hybrid with the mutant DNA target sequence and with the wildtype DNA target sequence, (iii) a spacer sequence, (iv) a palm sequence complementary to the mutant DNA target sequence and wildtype DNA target sequence capable of forming a hybrid 4 to 10 nucleotides in length with the hairpin sequence and (v) a mutation-recognizing sequence, comprising a mutation-specific nucleotide at the first or second 5'position of the sequence, wherein the contiguous hairpin sequence, spacer sequence, and palm sequences form a stemloop structure, the mutant target DNA sequence is perfectly complementary to the contiguous sequence formed by the palm sequence and the mutation-recognizing sequence, the wild-type DNA target sequence is mismatched to said contiguous sequence with the mutation-specific nucleotide of the mutation-recognizing sequence, and the docking sequence further comprises at the ultimate 3'position a dimer-preventing nucleotide that does not pair with the mutation-specific nucleotide and/or with any other nucleotide at the 3' end position of the multi-part amplification primer.
  2. 2. The multi-part amplification primer of claim 1, wherein said dimer-preventing nucleotide is the same nucleotide as the first 5' nucleotide of the mutation-recognizing sequence.
  3. 3. The multi-part amplification primer of claim 1 or 2, wherein said dimer-preventing nucleotide is a locked nucleic acid (LNA).
  4. 4. The multi-part amplification primer of any of claims 1 to 3, further comprising at least one ribonucleotide.
  5. 5. The multi-part amplification primer of claim 4, wherein said the at least one ribonucleotide is placed within the last 10 nucleotides of the 3' terminus of the primer, no two ribonucleotides are adjacent to one another, and the 3' terminal base is a deoxyribonucleotide.
  6. 6. The multi-part amplification primer of any of claims 1 to 4, wherein the primer comprises or consists of a nucleotide sequence selected from the group of polynucleotides according to SEQ ID NOs 9-22, 32-35, 38, 39, 44, and 46-50.
  7. 7. A primer-dependent amplification and detection method that is capable of amplifying and detecting in a sample at least one mutant DNA target sequence in a mixture containing, for each mutant DNA target sequence, a closely related wild-type DNA target sequence that differs from the mutant DNA target sequence by as little as one or two base pairs, comprising consisting or essentially consisting of the following steps: (a) providing a sample comprising the at least one mutant and/or wild-type DNA target sequence, and for each mutant DNA target sequence a pair of a forward primer and a reverse primer, (b) preparing a primer-dependent amplification reaction mixture comprising or consisting of a DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, other reagents required for amplification, the sample and the pair of the forward primer and reverse primer for each mutant DNA target sequence of step (a), (c) repeatedly cycling the mixture thus obtained in a primer-dependent amplification reaction, under primer annealing conditions, having a primerannealing temperature, to amplify each mutant DNA target sequence present in the sample, and (d) detecting said at least one mutant DNA target sequence by measuring the quantity of the amplification products thus obtained; wherein the forward and/or reverse primer of the primer pair for each mutant DNA target sequence is a multi-part amplification primer according to any of the preceding claims characterized in that it is specific for the mutated DNA target sequence but mismatched to the wild-type DNA target sequence, the docking sequence hybridizes at the primer-annealing temperature with the mutant DNA target sequence and with its closely related wild-type DNA target sequence.
  8. 8. The method of claim 7, wherein either the forward or reverse primer of the primer pair for each mutant DNA target sequence is a conventional primer.
  9. 9. The method of claim 7 or 8, wherein if the docking sequence and the palm sequence of the primer are hybridized either to the mutant DNA target sequence or to the wildtype DNA target sequence, and wherein the probability that during said cycling a multi-part amplification primer/wild-type DNA target sequence hybrid will be extended is at least 1,000 times lower than the probability that during said cycling a multi-part amplification primer/mutant DNA target sequence hybrid will be extended as evidenced by a difference in cycle numbers (ACq) of at least ten thermal cycles.
  10. 10. The method according to any of claims 7 to 9, wherein the forward and reverse primers of said pair of forward primer and reverse primer are multi-part amplification primers according to any of claims 1 to 6, the forward primer has a mutation-specific nucleotide that is complementary to the mutation site in one of the two strands of the mutant DNA target sequence, the reverse primer has a mutation-specific nucleotide that is complementary to the other mutant DNA target sequence of that base pair in the other of the two strands of the mutant DNA target sequence, whereby one primer binds to a target strand, and the other primer binds to the complementary target strand.
  11. 11. The method according to any of claims 7 to 10, wherein the closely related wild-type DNA target sequence differs from the mutant DNA target sequence by one base.
  12. 12. The method of any of claims 7 to 11, wherein the docking sequence hybridizes with the mutant and wild-type DNA target sequence at the primer annealing temperature.
  13. 13. The method of any of claims 7 to 12, wherein the primer-dependent amplification mixture of step (b) further comprises homogeneous fluorescence detection means for detecting amplification products, and step (d) comprises detecting said at least one mutant DNA target sequence by measuring the intensity of fluorescence from said homogeneous fluorescence detection means.
  14. 14. The method of claim 13, wherein the primer-dependent amplification and detection method is a polymerase chain reaction (PCR), and detection is real-time (RT) detection.
  15. 15. The method of any of claims 7 to 14, wherein method is capable of amplifying and detecting in a sample as few as 10 copies of at least one mutant DNA target sequence in a mixture containing, for each mutant DNA target sequence, 10,000 copies of a closely related wild-type DNA target sequence that differs from the mutant DNA target sequence by as little as one or two base pairs.

Description

PRIMERS FOR SELECTIVE AMPLIFICATION OF RARE TARGET SEQUENCES FIELD OF THE INVENTION [0001] The present invention relates to nucleic acid amplification primers useful for the selective amplification and/or detection of mutant DNA target sequences with exceptional low copy number comprising samples such as liquid biopsies with excess wildtype target DNA sequences for diagnostic purposes. Said primers are particularly useful for the specific and selective amplification and/or detection of rare target sequences differing from highly abundant sequences in as little as one nucleotide. Further disclosed are methods for the amplification and/or detection of rare nucleic acid targets in qPCR reactions using said primers as well as primer designs and kits. BACKGROUND OF THE INVENTION [0002] Confident detection and quantification of target nucleic acid sequences in samples comprising low concentrations of the target may require the selective amplification of said target using Polymerase Chain Reaction (PCR) and real-time detection (RT) of the generated amplification products (amplicons) in a method known as RT-qPCR. The selection of specific primers comprising short DNA sequences complementary to the target sequences is critical to ensure the specific amplification of the target, while preventing unspecific amplification of closely related sequences present in the sample. Accordingly, primer sequences are designed to be complementary to the target in order to allow hybridization of the primers to the target DNA sequence and its amplification products. The selection of a specific annealing temperatures may prevent the annealing of the specific primer to closely related target sequences by favoring annealing to the (near-) perfectly complementary target sequence. However, specific amplification of highly similar sequences differing in as little as one or two nucleotides can be challenging using conventional DNA primers. [0003] This is particularly challenging when distinguishing between mutant and wild-type sequences differing only by single-nucleotide polymorphisms (SNPs). While designing amplification primers with complete complementarity to a mutant DNA target sequences can favor the amplification of the corresponding target, wild-type sequences may be also amplified due to highly similar properties such as annealing temperatures and extensive base complementarity. The undesired amplification of the wild-type DNA target sequence may be less efficient compared to the intended mutant DNA target sequence. However, detection and quantification of mutant and wild-type target sequences becomes unreliable. [0004] This is particularly problematic in diagnostics, where the detection of mutant DNA target sequences in samples comprising predominantly wild-type DNA target sequences is critical. The presence of the wild-type DNA sequence masks the low copy numbers of the mutant DNA target sequence due to insufficient selectivity of amplification caused by the conventional primer design. [0005] One example of these challenges posed by the low selectivity of conventional primer designs is the identification of mutant DNA target sequences in circulating cell-free DNA. Circulating cell-free DNA (cfDNA) is recognized to have reasonable prognostic and diagnostic potential in several pathological diseases including cancer, sepsis, and autoimmune diseases such as systemic lupus erythematosus (SLE). Cancer is one of the deadliest diseases and projected to be responsible for a significant proportion of deaths in our aging society. The detection of cancers in early stages is associated with improved outlook on health and survival rates. While cancer symptoms can arise at late stages of the disease, multiple approaches are available to detect cancers before symptoms are present. For instance, extensive screenings are common at certain ages in order to identify cancerous lesions early and start treatment. However, current diagnostics fail short in providing a cost-effective and rapid identification of early stages of cancer. [0006] Cancer patients usually have a high level of cfDNA in their serum or plasma as a result of cellular necrosis or apoptosis, since tumor cells divide faster than normal cells, and cfDNAs are released in a high proportion (Sorenson et al., 1994; Vasioukhin et al., 1994; Raja et al., 2018). The fraction of cfDNA derived from tumor cells is named circulating tumor DNA (ctDNA) (Leon et al., 1977; Shu et al., 2017). In recent years, both cfDNA and ctDNA have received increased attention as novel blood biomarkers, as quantification and kinetic analysis of cfDNA (Diehl et al., 2008), and molecular profiling of ctDNA have suggested their predictive and prognostic values (lizuka et al., 2006; Tokuhisa et al., 2007). Several liquid biopsy tests, designed for the identification of cancer-specific mutations, have been recommended as companion diagnostic (CDx) tests, by the European Medicines Agency (EMA) and Food and Drug Admi