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Annealing control primer and the use of the same annealing control primer

A technology of annealing and primer pairs, applied in the field of application

Inactive Publication Date: 2005-02-09
视基因公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0026] In an effort to solve the problems of such conventional primers and various methods for nucleic acid amplification, the present inventors have developed a new annealing control primer that can be used in all technical fields based on nucleic acid amplification with more Highly specific nucleic acid amplification and unlimited applications

Method used

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  • Annealing control primer and the use of the same annealing control primer
  • Annealing control primer and the use of the same annealing control primer
  • Annealing control primer and the use of the same annealing control primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0407] Example 1: Estimating the role of universal bases in ACP

[0408] The role of universal base residues located between the 3'- and 5'-terminal parts of ACP can be assessed by RT-PCR using mouse embryonic tissue.

[0409] Total RNA was obtained from intact embryos of murine ICR at gestational days 4.5, 11.5, and 18.5 by using Tri-reagent (Sigma) or the LiCl / urea method (Hogan et al., 1994) were extracted. Two independent cDNA amplification experiments using ACP were performed to detect the following effects of universal bases located between the 3'- and 5'-terminal parts of ACP, in particular, deoxyinosine residues: A.ACP 3' Effect of deoxyinosine residues between - and 5'-terminal parts compared with ACP and conventional primers without deoxyinosine groups; B. Deoxyinosine residues between 3'- and 5'-terminal parts of ACP The effect of xanthosine residues as a function of the number of deoxyinosines.

[0410] These experiments were conducted based on the following a...

Embodiment 2

[0439] Example 2: Method for Amplifying a Target Nucleic Acid Sequence Using ACP

[0440] The ACP of the subject invention was used to amplify the target nucleotide sequence of the mouse placenta-specific homebox gene Esx1 cDNA. The procedure and results of using ACPs to amplify the target nucleotide sequence of Esx1 cDNA are described here. Total RNA (3 μg) extracted from placentas of 18.5-day-old mice was used as starting material. In addition to using oligo-dT 15 First-strand cDNAs were prepared under the same conditions as the cDNA synthesis in Example 1 as primers for first-strand cDNA synthesis.

[0441] oligo-dT 15 5'-TTTTTTTTTTTTTTTT-3' (SEQ ID NO: 54)

[0442] The resulting first-strand cDNAs were used as templates to amplify the target cDNA fragment of Esx1 using ACPs. These experiments performed a two-stage PCR amplification which is one of the unique features of the present invention.

[0443] The conventional primers of Esx1 used in this example are:

[...

Embodiment 3

[0492] Example 3: Differentially Expressed mRNAs During Mouse Embryo Development Using ACP Identification and Characterization

[0493] The ACP of the subject invention has been used to detect differentially expressed mRNAs in embryonic development. In particular, three different protocols and results are described here using total RNAs from embryonic tissues at different developmental stages as starting material. The primers used in the subject invention are shown in Table 1.

[0494] A1. Process 1

[0495] step 1): Synthesis of first-strand cDNA

[0496] Use dT 10 -ACP1 or JYC5-T 15 -ACP as a primer for cDNA synthesis First-strand cDNAs were prepared under the same conditions as in Example 1 for cDNA synthesis. The resulting cDNAs were purified by spin column (PCR purification kit QIAGEN) to remove primers, dNTPs and the above reagents. A purification step must be performed before measuring the concentration of cDNAs absorbing at 260 nm using a UV spectrometer...

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Abstract

The present invention relates to an annealing control primer for improving annealing specificity in nucleic acid amplification and its applications to all fields of nucleic acid amplification-involved technology. The present primer comprises (a) a 3'-end portion having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith; (b) a 5'-end portion having a pre-selected arbitrary nucleotide sequence; and (c) a regulator portion positioned between said 3'-end portion and said 5'-end portion comprising at least one universal base ornon-discriminatory base analog, whereby said regulator portion is capable of regulating an annealing portion of said primer in association with annealing temperature.

Description

technical field [0001] The invention relates to an annealing control primer and its application. More specifically, the present invention relates to an annealing control primer for improving annealing specificity in nucleic acid amplification and its use in all fields involving nucleic acid amplification. Background technique [0002] Nucleic acid amplification is a key process for various methods in the field of molecular biology, so various amplification methods have been proposed. For example, Miller, H.I. et al. (WO 89 / 06700) disclose nucleic acid sequence amplification based on hybridization of a promoter / primer sequence to 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). [0003] The protocol for amplifying a di-o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C07H21/04C12Q1/68
CPCC12Q1/686C12Q1/6809C12Q2539/113C12Q2525/161C12Q2525/101C12Q2525/155C12N15/11C12Q1/6869
Inventor 千锺润
Owner 视基因公司
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