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Method for the simultaneous amplification of a plurality of different nucleic acid target sequences

A nucleotide sequence and target sequence technology, applied in the field of DNA or RNA library, nucleic acid polymer library, can solve the problems of primer incompatibility, difference in amplification efficiency of different targets, etc.

Inactive Publication Date: 2015-09-16
UNIVSSPITAL BASEL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the main challenge of multiplex PCR is to overcome two main problems: primer incompatibility leading to non-specific amplification (e.g. primer-dimers) and variability in the amplification efficiency of different targets

Method used

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  • Method for the simultaneous amplification of a plurality of different nucleic acid target sequences
  • Method for the simultaneous amplification of a plurality of different nucleic acid target sequences
  • Method for the simultaneous amplification of a plurality of different nucleic acid target sequences

Examples

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Embodiment 1

[0154] Oligonucleotide probes were designed to target three genomic locations of the calpain-3 gene, exon 17, exons 18 and 19, and exon 22, as image 3 shown. For each target region, a first oligonucleotide probe ("reverse oligo") and a second oligonucleotide probe ("forward oligo") were synthesized. Oligonucleotide probes are shown in Table 1 below.

[0155] Reverse oligonucleotide probes (for each exon, CAPN3_Exon17_Reverse_ET1, CAPN3_Exon18-19_Reverse_ET5 and CAPN3_Exon22_ Reverse_ET1) is phosphorylated at the 5' end and contains a portion of the target sequence complementary to the original target sequence, the efficiency tag sequence (underlined), the universal reverse primer annealing sequence, and six phosphorothioates at its 3' end Nylated nucleotide analogs (indicated by an asterisk).

[0156] Forward oligonucleotide probes (CAPN3_exon17_forward_ET1, CAPN3_exon18-19_forward_ET5, and CAPN3_exon22_forward_ET1) contained their 5 The 6 phosphorothioated nucleotide ana...

Embodiment 2

[0167] result

[0168]As a model we chose the human dystrophin gene, the largest (not exon wise, but coverage wise) known human being consisting of 79 exons Gene. Since Chamberlain first reported multiplex PCR, the dystrophin gene has also been used as a multiplex PCR model by other investigators. To build our new technology, we utilized ExonPrimer to design 78 different targets covering all 79 exons. To enable rapid analysis by gel electrophoresis, we selected 12 targets of different sizes that were easily discernible when resolved on the gel ( Figure 5 ). The size of the selected targets ranged from 153bp to 725bp ( Figure 5 ).

[0169] To demonstrate the ability of etPCR to control PCR efficiency, we first generated by PCR a single template containing an efficiency tag and a common priming sequence ( Figure 5 ). The gel-purified template was subjected to quantitative PCR to analyze PCR efficiency ( Figure 6 a). We first performed standard qPCR using a universe ...

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Abstract

The present invention relates to a method for the simultaneous amplification of a plurality of different nucleic acid target sequences comprising the steps of providing a plurality of different nucleic acid polymers as templates, each template comprising a specific target sequence and a primer annealing sequence located downstream of the target sequence, and amplifying the template by a polymerase dependent amplification reaction using a primer oligonucleotide comprising a primer sequence which is at least essentially complementary to the primer annealing sequence. The method is characterized in that for the polymerase dependent amplification reaction a set of primer oligonucleotides is used, said set comprising at least two primer oligonucleotides which are able to anneal to the primer annealing sequence of the same template and which differ from each other in the efficiency for the polymerase dependent amplification reaction to take place.

Description

technical field [0001] The present invention relates to a method for the simultaneous amplification of a plurality of different nucleic acid target sequences, a kit for carrying out the method and a nucleic acid polymer library, in particular a DNA or RNA library. The invention also relates to the application of the method in gene probe detection and molecular cloning. Background technique [0002] The detection of specific nucleic acid polymers is an important tool in diagnostic medicine and molecular biology research. Gene probe detection is currently used, for example, in the identification of infectious organisms (Organisms) such as bacteria and viruses, in the detection of normal gene expression and in the identification of mutant genes such as oncogenes, in tissue typing compatibility before tissue transplantation, in forensic science It plays a role in matching tissue or blood samples, and in studying the homology of genes from different species. [0003] Ideally, g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/16C12Q1/6888C12Q1/6858C12Q1/6883C12Q2525/204C12Q2537/143C12Q2549/119
Inventor 约亨·金特迈克尔·赛恩瑞驰
Owner UNIVSSPITAL BASEL
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