Primers, probes and methods for nucleic acid amplification

A nucleic acid and probe technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc.

Active Publication Date: 2007-11-21
BRANDEIS UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Primers, probes and methods for nucleic acid amplification
  • Primers, probes and methods for nucleic acid amplification
  • Primers, probes and methods for nucleic acid amplification

Examples

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example 11

[0112] Example 11 and Figures 18-19 illustrate strategies for LATE-PCR amplification of more than one product from the same DNA template in the same reaction. Thus, these reactions contain two pairs of primers (each pair consisting of an excess primer and a limiting primer) that amplify two separate sequences within a contiguous template. The two pairs of primers can be configured such that both the excess primer and both the limiting primer hybridize to the same strand of the template, or to opposite strands of the template. As will be appreciated by those skilled in the art, two excess primers may extend "in" or "out" on their respective template strand when similar primers hybridize to opposite strands of the template. Figure 19 also shows the sequences of both excess primer strands that can be obtained from the same reaction mixture via the "dilution and removal" method.

[0113] Example 12 and Figure 20 demonstrate that the amounts of ssDNA and dsDNA produced by LATE-PCR...

example 14

[0115] Example 14 and Figure 23 demonstrate that LATE-PCR together with at least one single mismatch-tolerant probe can be used to generate endpoint melting curves which in turn can be used to quantify the mixing of two or more closely related but distinct sequences. Relative amounts of LATE-PCR amplicons. Quantitative end-point dissolution analysis (QE) of LATE-PCR for mixtures of related amplicons is made possible by the fact that LATE-PCR produces single-stranded products. Thus, when one or more labeled mismatch-tolerant probes are present in a reaction, the probes hybridize first to the most complementary target sequence, and then if the temperature is sufficiently lowered, to all related target sequences. Each probe / target hybrid in the set thus has its own melting temperature, and the magnitude of the melting peak derived from each probe / target hybrid accurately reflects the amount of each accumulated target sequence. Quantitative measurements of the amplitude or two-di...

example 1

[0118] Example 1. Conjugated Dye vs. Conjugated Dye Plus Labeled Primer

[0119] To compare the performance of the incorporated dye with that of the dye used in conjunction with the primer comprising the interacting fluorophore, an extension assay was performed. The dye utilized was SYBR Green I at a dilution of 1:40,000.

[0120] Contains three nucleotide chains. DNA template, extendable DNA primer (5' labeled with Cy5, complementary to the template, and having a T of 60°C m ), and a non-extensible DNA oligonucleotide (blocked with a phosphate group at the 3′ end), which is also complementary to the target, complementary to the primer at position 3′, also labeled with a Cy5 fluorophore, and has a relatively high temperature of 79°C High T m . The spacing between the primer and the non-extendable nucleotide is chosen such that the products of primer extension determined by the non-extendable oligonucleotide will all have a T below 79°C m .

[0121] The reaction mixture f...

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Abstract

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit ''single-tube'' processing.

Description

technical field [0001] The present invention relates to nucleic acid amplification reactions, including amplification utilizing the polymerase chain reaction, and assays utilizing such reactions in combination with sequencing and hybridization probe detection methods. Background technique [0002] Nucleic acid amplification techniques and assays are well known. Certain amplification reactions are isothermal, such as nucleic acid sequence based amplification (NASBA). Other amplification reactions use thermal cycling, such as the polymerase chain reaction (PCR). Preferred amplification assays using fluorescent detection of amplified products are homogeneous, i.e., they do not require physical separation of reagents to allow detection (e.g., to separate bound probes from unbound probes) and can be performed on a single Execute in airtight container. Such assays can be end-point, wherein the product is detected after amplification, or such assays can be real-time, wherein the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6876C12Q1/6869C12Q1/6851C12Q1/6844C12Q2600/156C12Q2600/16C12Q2545/114C12Q2531/107C12Q2527/107C12Q2561/113C12N15/11
Inventor 劳伦斯·J·王约翰·赖斯阿基莱斯·J·桑切斯肯尼思·皮尔斯杰西·索尔克阿瑟·瑞斯
Owner BRANDEIS UNIV
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