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Method for genetic detection using interspersed genetic elements: multiplexed DNA analysis system

A technology for analysis and markers, which is applied in the field of human identification and biological ancestry testing, and can solve the problems of RE unavailability and size difference

Active Publication Date: 2015-08-05
素德赫·辛哈
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, until now, the use of REs has not been available in a practical sense due to the inherent size differences associated with INNUL
Although REs constitute more than 40% of the human genome (Lander, E.S. et al., Initial sequencing and analysis of the human genome, Nature, 409(6822):860-921 (2001)), and there are a large number of REs for human identity testing. potential targets, but for use in forensic human identity testing, these INNULS (i.e., insertion and null alleles rather than INDELs, since one form of these alleles is not the result of a deletion) have received limited attention (Zangenberg et al., Multiplex PCR: Optimization Guidelines, in PCR Applications: Protocols for Functional Genomics, Academic Press, San Diego, CA, 1999, pp. 73-94)

Method used

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  • Method for genetic detection using interspersed genetic elements: multiplexed DNA analysis system
  • Method for genetic detection using interspersed genetic elements: multiplexed DNA analysis system
  • Method for genetic detection using interspersed genetic elements: multiplexed DNA analysis system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Four dye multiplex system for forensic applications.

[0082] A few markers are selected for multiplexing that can be used in forensic kits. Using three fluorophores, 6-carboxyfluorescein (6-FAM TM ), 4,5-dichloro-dimethoxy-fluorescein (JOE TM ) or carboxytetramethylrhodamine (TAMRA TM ) (using 5-carboxy-X-rhodamine (ROX TM ) and a fifth fluorophore at an orange wavelength as a size standard) to label the forward primer for each marker. The amplicon sizes of the selected markers ranged from about 46-124 bp, and the amplicon sizes of the individual INNUL alleles varied by 3-8 bp. The sex marker amelogenin was also added to the multiplex. Multiplex optimization experiments to address primer concentration and peak height issues were performed.

[0083] Markers were selected from http: / / dbRIP.org, existing literature, and by BLAST sequence analysis (A.F.A. Smit et al.; Batzer, M.A. et al. (2002); Batzer, M.A. et al. (1994); Feng, Q. et al.; Houck , C.M. et...

Embodiment 2

[0086] Example 2: Primer Design

[0087] Primers were designed using Primer3 (input version 0.4.0, http: / / frodo.wi.mit.edu / primer3 / ). Sets of three primers were designed for each marker: one forward primer and two reverse primers, one for the insertion allele and one for the null allele. All designed primers had Tm values ​​in the range of 58-61°C. The program "Reverse Complement" from the Harvard Medical Technology Group and the Lipper Center for Computational Genomics (arep.med.harvard.edu / ) was used. Primers were then screened against GenBank's non-redundant database (National Center for Biotechnology Information, U.S. National Library of Medicine, National Institutes of Health) to determine if they were unique DNA sequences). Table 1 provides available markers and Table 2 provides primer sequences for selected markers.

[0088] Table 1. RE markers available for selection.

[0089]

[0090]

[0091]

[0092] Table 2. Primer sequences used for each INNUL marker ...

Embodiment 3

[0097] Example 3: Primer Preparation

[0098] Fluorescently labeled or unlabeled oligonucleotide primers were synthesized by Eurofins MWG Operon (Huntsville, AL, USA) or Integrated DNA Technologies (Skokie, IL). All lyophilized primers (labeled or unlabeled) were dissolved in 10 mM TE (tris(hydroxymethyl)aminomethane (“Tris”) and ethylenediaminetetraacetic acid (“EDTA”)) buffer (pH 8.0 ) to 100 μM stock solution concentration (10×). Store the stock primers at 4°C until use. After reconstitution, each primer was diluted to a final concentration of 10 μM (1×) using TE buffer. Each primer mix consisted of three primers: a labeled forward primer and two corresponding unlabeled reverse primers. The combined volume of the two reverse primers is equal to the volume of the forward primer. Store all labeled primers in opaque polypropylene tubes to avoid quenching of fluorescent labels.

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Abstract

By utilizing a Mini-Primer strategy targeting the target site duplication (TSD) sequence of retrotransposons, INNUL markers, which include SINEs, LINEs, and SVAs, can be effectively used as markers for human identification and bio-ancestry studies regardless of the size of the inserted element. The size of the amplicons for INNULs and the difference between allelic states can be reduced substantially such that these markers have utility for analyzing high and low quality human DNA samples. A 15 RE marker and Amelogenin (for sex determination) multiplex for a single tube amplification of DNA, in four color detection was successfully designed. The multiplex provided power of discrimination suitable for forensic and paternity analysis. In addition, sensitivity of detection can enable human identity and bio-ancestry studies on forensic and anthropological samples. Depending on the distribution of the alleles in global populations, INNULs can be selected for human identity testing or for bio-ancestry studies.

Description

[0001] claim to priority [0002] This application, pursuant to 35 U.S.C. §119, references, incorporates, and claims an earlier application, METHOD FOR GENETIC DETECTION USING INTERSPERSED GENETIC, filed with the U.S. Patent and Trademark Office on October 15, 2012 and assigned Serial No. 61 / 714,088 at that time. All rights in ELEMENTS: A MULTIPLEXED DNA ANALYSIS SYSTEM (Methods for genetic testing using discrete genetic elements: a multiplexed DNA analysis system). technical field [0003] The present invention relates generally to human identification and biological ancestry testing, and more particularly to improvements to increase detection sensitivity during analysis of human DNA samples for human identity testing or for biological ancestry studies. Background of the invention [0004] Short tandem repeat (STR) loci are the main genetic markers used in human identity testing. These markers are highly polymorphic and provide high detection sensitivity, allowing analysis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34C07H21/04
CPCC12Q2600/16C12Q2600/156C12Q1/6876A61B18/245A61B2018/00345A61B2018/00577A61B2018/2211A61B2018/2238
Inventor 素德赫·辛哈
Owner 素德赫·辛哈
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