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US-12624387-B2 - Method for reducing primer dimer formation and increasing amplification efficiency

US12624387B2US 12624387 B2US12624387 B2US 12624387B2US-12624387-B2

Abstract

The present invention relates to a method for amplifying at least three target nucleic acid molecules with reduced primer dimer formation in a multiplex amplification reaction. The method of present invention can inhibit primer dimer formation and hence generation of nonspecific amplification products in an effective manner in a multiplex amplification reaction for at least three target nucleic acid molecules.

Inventors

  • Young Jo Lee
  • Dae Young Kim

Assignees

  • SEEGENE, INC.

Dates

Publication Date
20260512
Application Date
20171222
Priority Date
20161229

Claims (15)

  1. 1 . A method for amplifying at least three target nucleic acid molecules with reduced primer dimer formation and determining the presence of them in a multiplex real-time amplification reaction, comprising the steps of: (a) preparing at least three primer pairs and at least three probes, each primer pair comprising a forward primer and a reverse primer; wherein each primer comprises a hybridizing nucleotide sequence to a corresponding target nucleic acid molecule; wherein at least 50% of primers in said at least three primer pairs are universal base primers (UBPs); wherein the primers in said at least three primer pairs are different from each other; wherein the UBP has 1-3 universal base nucleotides; wherein one or two of the universal base nucleotides are located in a core region ranging from 3 rd nucleotide to 6 th nucleotide at the 3′-end of the UBP, and the remainder is located in a region ranging from 4 th nucleotide at the 5′-end of the UBP to 7 th nucleotide at the 3′-end of the UBP such that a multiplex real-time amplification reaction using the at least three primer pairs generates amplification products of the target nucleic acid molecules with reduced primer dimer formation; wherein the nucleotide sequence in the 5′ direction from the core region comprises a hybridizing nucleotide sequence to the corresponding target nucleic acid molecule; wherein when the UBP has 2 or 3 universal base nucleotides, the universal base nucleotides are nonconsecutive in the UBP; wherein each of the UBPs is at least 12 nucleotides in length; wherein each of the at least three probes comprises a hybridizing nucleotide sequence to the target nucleic acid molecule to be amplified and is located between the forward primer and the reverse primer; and (b) performing a multiplex real-time amplification reaction comprising at least two cycles of primer annealing, primer extension and denaturation by using the at least three primer pairs and the at least three probes; wherein the multiplex real-time amplification reaction generates amplification products of the target nucleic acid molecules with reduced primer dimer formation; and (c) determining the presence of the at least three target nucleic acid molecules by detecting the amplification products.
  2. 2 . The method of claim 1 , wherein the core region ranges from 3 rd nucleotide to 5 th nucleotide at the 3′-end of the UBP and the remainder is located in a region ranging from 4 th nucleotide at the 5′-end of the UBP to 6 th nucleotide at the 3′-end of the UBP.
  3. 3 . The method of claim 1 , wherein both of primers in the at least one primer pair are UBPs.
  4. 4 . The method of claim 1 , wherein either or both of primers in each primer pair are UBPs.
  5. 5 . The method of claim 1 , wherein at least one of the UBPs does not comprise a degenerate base.
  6. 6 . The method of claim 1 , wherein at least five primer pairs are used for amplifying at least five target nucleic acid molecules.
  7. 7 . The method of claim 1 , wherein the primer annealing in the multiplex real-time amplification reaction is performed at 50° C. or higher.
  8. 8 . The method of claim 1 , wherein the method exhibits a target amplification efficiency of at least 3% higher than that of using primer(s) containing naturally occurring nucleotides (A, C, G or T (U)) complementary to the target nucleic acid sequence instead of the at least one UBPs.
  9. 9 . The method of claim 1 , wherein a Ct value obtained from a reaction using a primer pair containing the UBP in the absence of a target nucleic acid molecule is increased by 1 or more, as compared with that using a primer pair consisting of conventional primers in the absence of a target nucleic acid molecule.
  10. 10 . The method of claim 1 , wherein the at least three probes do not have a universal base nucleotide.
  11. 11 . The method of claim 1 , wherein the universal base nucleotide is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 2-aza-2′-deoxyinosine, 2′-OMe inosine, 2′-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2′-OMe 3-nitropyrrole, 2′-F 3-nitropyrrole, 1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole, 5-nitroindole, 2′-OMe 5-nitroindole, 2′-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2′-F nebularine, 2′-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2′-O-methoxyethyl inosine, 2′-O-methoxyethyl nebularine, 2′-O-methoxyethyl 5-nitroindole, 2′-O-methoxyethyl 4-nitrobenzimidazole, 2′-O-methoxyethyl 3-nitropyrrole, and combinations thereof.
  12. 12 . A method for reducing primer dimer formation in a multiplex real-time amplification reaction for at least three target nucleic acid molecules, comprising the steps of: (a) determining at least three target nucleic acid molecules to be amplified; (b) determining nucleotide sequences of at least three primer pairs and at least three probes, each primer pair comprising a forward primer and a reverse primer; wherein each primer comprises a hybridizing nucleotide sequence to a corresponding target nucleic acid molecule; wherein each of the at least three probes comprises a hybridizing nucleotide sequence to the target nucleic acid molecule to be amplified and is located between the forward primer and the reverse primer; (c) replacing 1-3 nucleotides in at least 50% of primers in said at least three primer pairs with universal base nucleotides to prepare universal base primers (UBPs); wherein the primers in said at least three primer pairs are different from each other; wherein the replacement with the universal base nucleotides allows the inhibition of primer dimer formation; wherein the UBP has 1-3 universal base nucleotides; wherein one or two of the universal base nucleotides are located in a core region ranging from 3 rd nucleotide to 6 th nucleotide at the 3′-end of the UBP, and the remainder is located in a region ranging from 4 th nucleotide at the 5′-end of the UBP to 7 th nucleotide at the 3′-end of the UBP; wherein the nucleotide sequence in the 5′ direction from the core region comprises a hybridizing nucleotide sequence to the corresponding target nucleic acid molecule; wherein when the UBP has 2 or 3 universal base nucleotides the universal base nucleotides are nonconsecutive in the UBP; and wherein each of the UBPs is at least 12 nucleotides in length; and (d) performing a multiplex real-time amplification reaction comprising at least two cycles of primer annealing, primer extension and denaturation by using the at least three primer pairs and the at least three probes; wherein the multiplex real-time amplification reaction generates amplification products of the target nucleic acid molecules with reduced primer dimer formation; and (c) determining the presence of the at least three target nucleic acid molecules by detecting the amplification products.
  13. 13 . The method of claim 12 , wherein the core region ranges from 3 rd nucleotide to 5 th nucleotide at the 3′-end of the UBP.
  14. 14 . The method of claim 12 , wherein both of primers in the at least one primer pair are UBPs.
  15. 15 . The method of claim 12 , wherein either or both of primers in each primer pair are UBPs.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national phase application of PCT Application No. PCT/KR2017/015386, filed on Dec. 22, 2017, which claims the benefit and priority to Korean Patent Application No. 10-2016-0182955, filed Dec. 29, 2016. The entire disclosures of the applications identified in this paragraph are incorporated herein by references. FIELD OF THE INVENTION The present invention relates to a method for reducing primer dimer formation and increasing amplification efficiency in multiplex amplification reactions. BACKGROUND OF THE INVENTION Nucleic acid amplification is a pivotal process for a wide variety of methods in molecular biology, such that various amplification methods have been proposed. For example, Miller, H. I. et al. (WO 89/06700) discloses a method for amplifying nucleic acids, comprising the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. Other known nucleic acid amplification procedures include transcription-based amplification systems (Kwoh, D. et al., Proc. Natl. Acad. Sci. U.S.A., 86:1173 (1989); and Gingeras T. R. et al., WO 88/10315). The most predominant process for a nucleic acid amplification known as polymerase chain reaction (hereinafter referred to as “PCR”) is based on repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354). PCR-based techniques have been widely used not only for amplification of a target DNA sequence, but also for scientific applications or methods in the fields of biological and medical research, such as reverse transcriptase PCR (RT-PCR), differential display PCR (DD-PCR), cloning of known or unknown genes by PCR, rapid amplification of cDNA ends (RACE), arbitrary priming PCR (AP-PCR), multiplex PCR, SNP genome typing, and PCR-based genomic analysis (McPherson and Moller, (2000) PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY). Since the PCR-based techniques involve amplification of the target nucleic acid molecule, it is required to amplify only the target sequence accurately and efficiently. For this purpose, it is necessary to select primers of appropriate sequence and length and perform an amplification reaction by appropriately adjusting the type and content of the components used (e.g., DNA polymerase, dNTPs, Mg ions and buffers) and the reaction temperature/time. However, in some cases, it is difficult to select optimum reaction conditions, and satisfactory amplification efficiency may not be achieved even under the selected reaction conditions. Various methods have been developed for more efficient amplification. Meanwhile, reduced amplification efficiency may be caused by the formation of primer dimers. In this regard, Lebedev et at discloses a method for improving the specificity and the amplification efficiency by using a primer in which 4-oxo-1-pentyl (OXP) phosphotriester (PTE) group is introduced at the 3′ terminal phosphodiester linkage or at the penultimate phosphodiester linkage of the primer, to prevent the non-specific hybridization of the primers occurring under low temperature condition at an initial reaction (See Lebedev et al., Nucleic Acids Res 2008; 36: e131). Primers have a significant effect on the specificity and efficiency of the amplification reaction, because they specifically hybridize to a target nucleic acid molecule and initiate its amplification. Particularly, the importance of primers is further emphasized in multiplex amplification reactions using multiple primers. Typically, a method of altering the site where the primer is hybridized or adjusting the sequence or length of the primer has been used to minimize primer dimer formation; however, it has limitations in applying to the design of primers used in multiplex amplification reactions. Thus, there remains a need for a novel method for effectively inhibiting the primer dimer formation, and such method would improve the amplification efficiency in a more economical and convenient manner alone or in combination with known methods as described above. Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entirety are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains. SUMMARY OF THE INVENTION The present inventors have endeavored to develop a method capable of effectively reducing the formation of the primer dimer in a multiplex amplification reaction, particularly for at least three target nucleic acid molecules. As a result, the present inventors have found that