Methods and compositions for rapid multiplex amplification of STR loci
Inventors
Schumm, James W. • Selden, Richard F. • Tan, Eugene
Assignees
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Publication Number
US-9797841-B2
Publication Date
2017-10-24
Expiration Date
Abstract
Provided are methods for multiplex polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci that can be used to rapidly generate a highly specific STR profile from target nucleic acids. The resulting STR profiles are useful for human identification purposes in law enforcement, homeland security, military, intelligence, and paternity testing applications.
Core Innovation
The invention describes a method for generating a short tandem repeat (STR) profile from a sample containing nucleic acids using multiplex amplification. Primer pairs labeled with different fluorescent dyes amplify multiple selected STR loci in a reaction chamber, followed by laser-induced fluorescence detection of the amplified nucleic acid products. The disclosed designs provide expanded locus coverage with substantially non-overlapping profiles, including a balanced 27-locus, six-color assay, amelogenin, and arrangements for 13-locus and 24-locus multiplexes.
The content identifies amplification artifacts such as stutter and incomplete nontemplate addition, demonstrating reduced incomplete nontemplated adenylation through primer redesign, dye-label changes, and 5′ terminal primer modifications. Because the background section is not included, the problem being solved as stated in that section is not explicitly described. The designs additionally support rapid profile generation, analysis of degraded DNA, and broader single nucleotide polymorphism (SNP), combined-marker, and sequencing applications.
Claims Coverage
The provided claims identify one independent claim directed to generating an STR profile, comprising three inventive features.
Multiplex amplification of STR loci
The claim covers amplification of at least seven selected STR loci using corresponding primer pairs in one reaction chamber to generate amplified nucleic acid products.
Differential fluorescent labeling
The claim specifies primer pairs labeled with different fluorescent dyes, using at least seven different fluorescent dyes to enable distinct detection.
Laser-induced fluorescence detection
The claim covers detecting the amplified nucleic acid products by laser-induced fluorescence to generate the STR profile.
The independent claim combines multiplex amplification of selected STR loci with differential fluorescent labeling and laser-induced fluorescence detection, while dependent claims add an STR locus for sex identification and progressively shorter limits for generating the STR profile.
Stated Advantages
The described assay changes and 5′ terminal primer modifications reduce incomplete nontemplate addition and adenylation from about 80–150% to under 10%.
ATTO488 labeling improves the SE33 signal.
The described assay provides a balanced 27-locus, six-color profile and is demonstrated with a 19.5-minute amplification.
The disclosed profiles provide expanded locus coverage and are described as dense and substantially non-overlapping.
The approach enables rapid STR profile generation.
The disclosed approach supports analysis of degraded DNA.
Documented Applications
Generation of multiplex STR profiles from samples containing nucleic acids, including use of an STR locus for sex identification.
SNP analysis and combined STR/SNP marker analysis.
DNA sequencing.
Forensic human identification.
Kinship analysis.
Cross-database compatibility for STR profiles.
Analysis of degraded DNA.
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