Chimeric primers for improved nucleic acid amplification reactions

一种扩增反应、嵌合引物的技术，应用在核酸扩增领域，能够解决寡核苷酸引物不能被完全复制等问题

Active Publication Date: 2010-09-22
INFINIPLEX LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In other words, the template default oligonucleotide primers disclosed in US7,205,129 and WO 01 / 64952 cannot be fully replicated

Method used

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  • Chimeric primers for improved nucleic acid amplification reactions
  • Chimeric primers for improved nucleic acid amplification reactions
  • Chimeric primers for improved nucleic acid amplification reactions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] to 10 4 Copies of the 16s RNA gene of strain E. canis (EC) were subjected to real-time PCR using DNA primers and RNA / DNA chimeric primers.

[0090] DNA primers:

[0091] Forward primer: 5'-TCGCTATTAGATGAGCCTACGT3' (SEQ ID NO: 4)

[0092] Reverse primer: 5'-GAGTCTGGACCGTATCTCAGTT-3' (SEQ ID NO: 5)

[0093] RNA / DNA Chimeric Primers

[0094] (embedded RNA bases are in bold):

[0095] Forward primer: 5'-TCGCUATUAGATGAGCCUACGT-3' (SEQ ID NO: 6)

[0096] Reverse primer: 5'-GAGTCTGGACCGUATCTCAGTT-3' (SEQ ID NO: 7)

[0097] The results are presented in Figure 5 middle. As can be seen, when DNA primers were used, in the absence of template, obvious artifacts could be detected after 27 cycles (see non-templated control (NTC) curve), but when chimeric primers were used Artifacts remained below the detection threshold. In fact, the C(t) value of the specific template-dependent product was delayed by about 3 cycles, but the elimination of the non-specific product exten...

Embodiment 2

[0100] Real-time PCR was performed on the 16s RNA gene of the bacterial strain Ehrlichia canis (EC) with a series of dilutions, using the same DNA and chimeric primers as in Example 1. The results are presented in Figure 7A and 7B middle. An additional real-time PCR was performed on a series of dilutions of the hsp70 gene of the strain Babesia canis (BC) using the following primers and the results are presented in Figure 8A and 8B middle.

[0101] DNA primers:

[0102] Forward primer: 5'-GTCATCACTGTGCCTGCGTACT-3' (SEQ ID NO: 8)

[0103] Reverse primer: 5'-GCATGACGTTGAGACCGGCAAT-3' (SEQ ID NO: 9)

[0104] RNA / DNA chimeric primers:

[0105] (embedded RNA bases are in bold):

[0106] Forward primer: 5'-GTCATCACTGTGCCTGCGUACT-3' (SEQ ID NO: 10)

[0107] Reverse primer: 5'-GCATGACGTTGAGACCGGCAAT-3' (SEQ ID NO: 11)

[0108] This example shows that the sensitivity of the amplification reaction is increased when RNA / DNA chimeric primers are used. In both cases, the us...

Embodiment 3

[0110] In this example, serial dilutions of the canine ACTB gene were subjected to qPCR using DNA and chimeric primers, followed by Tm analysis.

[0111] DNA primers:

[0112] Forward primer: 5'-GCGCAAGTACTCTGTGTGGAT-3' (SEQ ID NO: 12)

[0113] Reverse primer: 5'-GTCGTACTCCTGCTTGCTGAT-3' (SEQ ID NO: 13)

[0114] RNA / DNA chimeric primers:

[0115] (embedded RNA bases are in bold):

[0116]Forward primer: 5'-GCGCAAGUACTCTGTGTGGAT-3' (SEQ ID NO: 14)

[0117] Reverse primer: 5'-GTCGUACTCCTGCTTGCTGAT-3' (SEQ ID NO: 15)

[0118] This example demonstrates the improved specificity obtained by using PCR chimeric primers prior to HRM analysis at low template concentrations. When using DNA primers ( Figure 9A ), in the Tm diagram, it can be seen that the non-specific product peak is in the range of 70°C to 78°C, while the Tm peak of the expected amplicon in the high concentration template is in the range of 81°C to 82°C. Figure 9B Tm plots obtained when using DNA / RNA chimeri...

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PUM

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Abstract

Methods are provided for amplification of a nucleic acid sequence. The method use RNA / DNA chimeric oligonucleotides as primers. The primers have RNA residues scattered along their length and no two ribonucleotides in the prime are adjacent to one another. The methods are useful for reducing non-specific amplification products, such as primer dimers. The invention also provides kits comprising RNA / DNA chimeric oligonucleotide primers for practicing the amplification methods.

Description

field of invention [0001] The present invention relates to the field of nucleic acid amplification. Specifically, the present invention relates to the use of ribonucleotides in DNA primers and probes to reduce non-specific products in DNA-dependent DNA polymerase amplification reactions. Background of the invention [0002] Nonspecific amplification products are generated in DNA-dependent DNA polymerase amplification reactions such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR). This artifact originates from between primers or between primers and probes (such as TaqMan TM and molecular beacon probes) resulting in inappropriate hybridization products named primer-dimers (Rychlik, 1995) and primer-probe dimers. The weak complementarity between the 3' end of the primer and the bases in the non-targeting oligonucleotide strand in the reaction mixture leads to the formation of these undesired products. This results in annealing of the primer to the no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/6853C12Q1/686C12Q2521/101C12Q2525/121C12Q2531/113
Inventor 奥菲尔·皮莱格
Owner INFINIPLEX LTD
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