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Method for amplification of nucleic acid sequences

A nucleic acid sequence and target nucleic acid sequence technology, applied in the field of amplification and identification of target nucleic acid sequences, can solve problems that are unfavorable for low copy number and forensic applications, unrealistic, unsuitable for decoding, etc.

Pending Publication Date: 2019-10-18
NUCLEOTRACE PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This fact raises a number of issues: (1) each taggant must contain a unique set or sets of distinguishing primer pairs for recovery and amplification, (2) each taggant in the library must use substantially different sequence to encode the same symbol, (3) if the taggant present in the product is not known in advance (identification, by definition), one must target the library W n To screen samples for all possible primer pairs in a sample, (4) large-scale screening (e.g., >300 PCR reactions) is impractical, expensive, and detrimental to low fragment copy number recovery, and (5) these constraints Limit current technology to w n Practical taggant pool size limit of <3000 and stratification limit of 20 taggants (US 8,735,327)
However, existing nucleotide taggant systems remain cumbersome, impractical, and expensive for identification purposes, and are not adapted to be effective in a manner that allows identification of subsets of unknown taggants (and taggant stratification) to decode
The large number of samples required to identify a product is also detrimental to low copy number and forensic applications

Method used

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  • Method for amplification of nucleic acid sequences
  • Method for amplification of nucleic acid sequences
  • Method for amplification of nucleic acid sequences

Examples

Experimental program
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Effect test

Embodiment 1

[0281] Example 1 - Molecular taggant design and preparation for Series 1 experiments (9mm pistol).

[0282]For the series 1 experiments described here, single-stranded DNA oligonucleotides (ssDNA) were synthesized by Sigma-Aldrich and purified using high performance liquid chromatography (HPLC). Production is spread across several different locations over several weeks to ensure that no cross-contamination of the manufacturing facility occurs. ssDNA oligonucleotides are designed with 5' and 3' end capping regions (3bp), universal forward and reverse primer sites (20bp), and variable lengths (14bp, 20bp, 24bp, 28bp , 32bp) code word area.

[0283] A total of 40 complementary ssDNA oligonucleotides were sequenced and subsequently annealed to form 20 dsDNA tagger duplexes (OligoTag_1_Ser1 to OligoTag_20_Ser1 in Table 1). The 20 taggants included four of each length: 80bp, 76bp, 70bp, 66bp, and 60bp, so that they could be identified by fragment length separation (UniKey-Tag 2). ...

Embodiment 2

[0298] Example 2 - Molecular Taggant Design and Preparation for Series 2 Experiments (0.22 Caliber Rifle, 0.207 Caliber Rifle and Drug Labeling).

[0299] In Series 2 experiments (0.22 and 0.207 caliber firearms ammunition tracking and drug labeling), the taggant was similarly designed according to UniKey-Tag Embodiment 1 . Specifically, the taggant was designed with no (ie, 0-3 bp) end capping region, and a universal forward and reverse primer site (22 bp) flanking a variable coding region of 46 bp in length. Still consistent with UniKey-Tag Embodiment 1, but one difference compared to Series 1 experiments is that the variable region is assembled from six Hamming (l,d,p) coding blocks (abbreviated as: Hamming (l,d,p)).

[0300] Specifically, the variable region of the taggant in series 2 experiments is composed of six crumbs (equivalent to binary bytes) of symbol length l=8 encoded by Hamming (l,d,p), including d=4 data and p = 4 odd-even nucleotides, ie, Hamming (8,4,4) co...

Embodiment 3

[0312] Example 3 - Design of base pair encoded taggant (UniKey-Tag 1)

[0313] For the UniKey-Tag 1 system, each symbol (L) in the symbol set (S) is encoded by a nucleic acid sequence, and the codeword encoding is decoded by ATD PCR and sequencing.

[0314] exist Figure 8 In the UniKey-Tag 1 system shown in (a), the nucleotide sequence in the variable region v is used to encode string n, which is decoded by sequencing. For the template strand, the fragment length k=100bp consists of capping region and 5' end and 3' end (Cp, 0-3bp), universal forward primer site (UPF, 10-30bp), complementary universal reverse primer site point (UPR c , 10-30bp) and variable region (V_x, 20-160bp). The letters in a codeword are constructed from the set {A,T,C,G,U} of nucleic acids, but it is more common to use the set {A,C,G,T} (ie, U is not present in DNA). The length of each symbol in the codeword is l=1 to vbp. exist Figure 8 In the embodiment shown in (a), v=27bp and l=3bp, allowing ...

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Abstract

The present invention relates generally to a method for the amplification of nucleic acid sequences, more specifically, to a method for the amplification and identification of target nucleic acid sequences.

Description

technical field [0001] The present invention relates generally to methods of amplifying nucleic acid sequences, and more particularly to methods of amplifying and identifying target nucleic acid sequences. Background technique [0002] Over the past two decades, counterfeiting and piracy have increased significantly, with counterfeit and pirated products found in almost all countries and almost all economic sectors around the world. Estimates of the level of counterfeiting and the value of such products vary. However, the global trade in counterfeit and pirated goods was estimated at $461 billion in 2013 (OECD and EUIPO, 2016, “Trade in Counterfeit and Pirated Goods: Mapping the Economic Impact”). Many manufacturers employ anti-counterfeiting measures to minimize the impact of counterfeiting. These include secure printing using special watermarks, inks and dyes, holograms, tamper-evident labels, laser surface authentication, and magnetic and radio-frequency identification ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6848C12N15/11
CPCC12Q1/6848C12Q2525/113C12Q2527/101C12Q2549/119C12Q2563/185
Inventor 尼古拉斯·亚历山大·欧文
Owner NUCLEOTRACE PTY LTD