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Materials and methods for isothermal nucleic acid amplification

A technology for isothermal amplification and target nucleic acid, which is applied in the field of thermal amplification of target RNA, and can solve problems such as lack of proofreading ability

Inactive Publication Date: 2012-11-21
QIAGEN GAITHERSBURG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Unfortunately, the enzyme reverse transcriptase can be highly error-prone because they usually do not possess the ability to proofread

Method used

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  • Materials and methods for isothermal nucleic acid amplification
  • Materials and methods for isothermal nucleic acid amplification
  • Materials and methods for isothermal nucleic acid amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Use of PYROPHAGE 3173 as a replacement for RT enzyme in RT-HAD

[0060] PYROPHAGE 3173 DNA polymerase has several advantages over Transcriptor and Thermoscript reverse transcriptases. For example, Thermoscript and Transcriptor are known to have limited activity in HAD buffer at 65°C. PYROPHAGE 3173 DNA polymerase was tested to determine if it could replace these reverse transcriptases in a one-step isothermal RT-HAD amplification.

[0061] Briefly, a 25 μL reaction mixture was created containing: (1) 0, 10, or 100 copies of an in vitro transcribed, synthetic RNA comprising the Chlamydia trachomatis cryptic plasmid RNA (ct-RNA) ( GenBank accession number X06707) (SEQ ID NO:1); (2) forward primer 5'-ATC GCA TGC AAG ATA TCG AGT ATG CGT-3'(SEQ ID NO:2) and reverse primer 5'-CTC ATA ATT AGC AAG CTG CCT CAG AAT-3' ("ct-orf primer") (SEQ ID NO:3); (3) 2.5 U of Thermoscript, Thermo-X, Transcriptor, or Pyrophage 3173; (4) 2 U of Bst polymerase; and (5) 1 U of uvrD ...

Embodiment 2

[0065] Example 2: Use of PYROPHAGE 3173 as a replacement for both RT and DNA-dependent DNA polymerases

[0066] PYROPHAGE 3173 DNA polymerase was tested to determine if it could replace both reverse transcriptase and DNA-dependent DNA polymerase in a one-step isothermal RT-HAD amplification. Briefly, a 25 μL reaction mixture was created containing: (1) 0, 25, or 100 copies of ct-RNA; (2) ct-orf primers; (3) 2.5 U of Pyrophage 3173; (4) 0 U or 2 U Bst polymerase; and (5) 1 UuvrD helicase. The reaction mixture contained the reagents at the final concentrations listed in Table 1. Amplification was performed at 62°C or 65°C for 75 minutes. exist figure 2 show result. As can be seen, PYROPHAGE 3173 is able to replace both reverse transcriptase and DNA-dependent DNA polymerase in a one-step isothermal RT-HAD.

[0067] PYROPHAGE 3173 DNA polymerase was also compared to other enzymes with reverse transcriptase activity in its ability to perform RT-HAD in the absence of different...

Embodiment 3

[0068] Example 3: DNA Amplification Using PYROPHAGE 3173

[0069] target amplification . Use of HPV16 DNA as target DNA in the HAD assay. Double-stranded DNA targets were denatured in 5 μl of 0.1 M NaOH at 65°C for 10 min. Then, an equal volume of 0.2M Hepes was added to neutralize the denatured target. 15 μl premix and 25 μl amplification mix were added to the targets and incubated at 65°C for 1.5 hours. Table 2 lists the master mix and amplification mix components.

[0070] Amplicon Detection . The HAD product (5 μL) was transferred to a U-bottom hybridization plate and then diluted in 5 μl of 1X Denaturing Reagent (Digene HC2DNR, Qiagen Gaithersburg, Inc., Gaithersburg, MD). The plate was then sealed and shaken at 1100 rpm for 30 seconds in a Digene shaker and incubated at room temperature for 15 minutes. Hybridization diluent (5 μl 1X hc2 Probe Diluent, Qiagen Gaithersburg, Inc., Gaithersburg, MD) was added and the plate was resealed and shaken at 1100 rpm for 30 s...

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Abstract

A method for isothermal amplification of a target nucleic acid sequence is disclosed. The target nucleic acid is amplified by an enzyme with helicase activity and an enzyme with reverse transcriptase activity and DNA-dependant DNA polymerase activity. Also disclosed is a kit for isothermal amplification of a target nucleic acid sequence, including HPV nucleic acids. The kit comprises a first enzyme with helicase activity and a second enzyme having both reverse transcriptase activity and DNA-dependant DNA polymerase activity.

Description

[0001] References to related applications [0002] This application claims priority to US Provisional Patent Application No. 61 / 293,372, filed January 8, 2010, which is hereby incorporated by reference in its entirety. Background of the invention [0003] Nucleic acid amplification is widely used in research, forensics, medicine, and agriculture. Polymerase chain reaction (PCR) is the most widely used method for in vitro DNA amplification. PCR reactions typically utilize two oligonucleotide primers that hybridize to the 5' and 3' boundaries of the target sequence and DNA-dependent DNA polymerization that extends the annealed primers by polymerizing deoxyribonucleotide triphosphates (dNTPs) to generate double-stranded products enzyme. By raising and lowering the temperature of the reaction mixture (called thermocycling), the two strands of the DNA product are separated and can serve as templates for the next round of annealing and extension, and the process repeats. [0004]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N9/12C12N9/90
CPCC12Q1/6844C12Q1/686C12Q2527/101C12Q2521/513C12Q2521/107C12Q2525/179C12Q2521/519
Inventor B.洛维A.福尔布莱特
Owner QIAGEN GAITHERSBURG
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