Multiplex nucleic acid assay methods capable of detecting closely related alleles, and reagents therefor

An assay method and multiplexing technology, which are applied in the fields of nucleic acid amplification and detection and determination, and can solve the problems of multiple assays without proof of multiplex assays.

Active Publication Date: 2019-02-05
RUTGERS THE STATE UNIV
View PDF9 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, we did not demonstrate a multiplex assay or describe a multiplex assay for closely related rare mutant target sequences with SuperSelective primer designs and methods capable of detecting as few as ten mutant target sequences

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Multiplex nucleic acid assay methods capable of detecting closely related alleles, and reagents therefor
  • Multiplex nucleic acid assay methods capable of detecting closely related alleles, and reagents therefor
  • Multiplex nucleic acid assay methods capable of detecting closely related alleles, and reagents therefor

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0090] Example 1 presents a singleplex experiment using restriction enzyme digested plasmid DNA to study the effect of varying foot sequence length. We investigated the effect of shortening the foot length of the SuperSelective primer directed against the EGFR L858R mutant sequence to overcome the possibility that the foot will form a hybrid with the G-C rich sequence present in the EGFR wild-type target sequence. We performed three sets of symmetric PCR assays, each using SuperSelective primers with foot sequences 6, 7, or 8 nucleotides in length. In all other respects, the design of the primers was identical: (i) the interrogation nucleotide was located at the 3' penultimate position of each foot; (ii) the anchor sequence was 24 nucleotides long; and (iii) Both the bridge sequence and the intervening sequence are 14 nucleotides long. Each set of PCR assays used different amounts of mutant templates (10 6 、10 5 、10 4 、10 3 、10 2 and 10 1 copies) at 10 6 Primed in the ...

example 2

[0091] Example 2 presents a singleplex experiment using restriction enzyme digested plasmid DNA to study the effect of varying the perimeter of the vesicle (the length of the bridge sequence plus the length of the intervening sequence plus 4). A single-stranded vesicle functionally separates the anchor hybrid from the foot hybrid (see figure 1 ). We performed three sets of symmetric PCR assays identical to the experiment described in Example 1, except that three different SuperSelective primers were used, each of which when hybridized to its template Form symmetrical bubbles. Each of these three primers has the same 7-nucleotide long foot sequence, where the interrogation nucleotide is located at the 3' penultimate position, and the length of the anchor sequence in each primer is maintained at 24 nucleotides. However, the length of the bridge sequence in each primer was chosen to be 10, 14 or 18 nucleotides, and the identity of the anchor sequence in each primer was chosen ...

example 3

[0092] Example 3 presents a singleplex experiment using restriction enzyme digested plasmid DNA to study the effect of altering the position of a single interrogation nucleotide within the foot sequence, including the ability of the primers to discriminate between mutant and wild-type templates. We performed a series of symmetric PCR assays using six different SuperSelective primers, each with an interrogating nucleotide at a different position within the 7 nucleotide long foot sequence. The lengths of the anchor, bridge and intervening sequences were maintained at 24, 14 and 14 nucleotides, respectively. These are 6:1:0, 5:1:1, 4:1:2, 3:1:3, 2:1:4 and 1:1:5. Two reactions were performed with each primer, one with 10 6 copies of the mutant template were primed, and one was primed with 10 6 copies of the wild-type template are primed. The observed threshold cycles are listed in Table 4 of Example 3. The results show that the threshold cycle (C T ) and wild-type threshold c...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

This invention discloses multi-part primers for primer-dependent nucleic acid amplification methods. Also disclosed are multiplex assay methods, related reagent kits, and oligonucleotides for such methods.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 62 / 319,332, filed April 7, 2016. The content of this application is incorporated herein by reference in its entirety. technical field [0003] The present invention relates to nucleic acid amplification and detection assays, such as PCR amplification and detection methods, and to primers, reaction mixtures and kits for use in such methods. Background of the invention [0004] A long-sought medical goal is the ability to detect extremely rare mutations at an extremely early stage, the presence of which in clinical samples can be used to diagnose cancer, determine prognosis and indicate the choice of effective treatment. Detection and quantitative assessment of relevant somatic mutations have multiple uses including: (i) detecting cancer at a treatable stage in patients who inherit genes that make cancer more likely to develop; (ii) detecting mutations i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/68C12Q1/6858C12Q2525/161C12Q2527/125C12Q2561/113C12Q2563/107C12Q2565/1015C12Q2537/143C12P19/30C12Q1/6853C12Q1/686C12Q1/6876C12Q2600/16
Inventor 塞尔瓦托·A·E·马拉斯D·瓦尔加斯-戈尔德S·泰尔吉F·R·克雷默
Owner RUTGERS THE STATE UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap