Methods and compositions for rapid multiplex application of STR loci
Inventors
Schumm, James W. • Selden, Richard F. • Tan, Eugene
Assignees
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Publication Number
US-11022555-B2
Publication Date
2021-06-01
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
A nucleic-acid sample is multiplex amplified with a high number of short tandem repeat (STR) loci by contacting the sample in one solution with at least 18, at least 27, or at least 37 different primer pairs, where at least one primer of each primer pair is labeled with a fluorescent dye. At least six different fluorescent dye-labels are used, and the primer pairs include autosomal STR loci primer pairs and at least one Y-STR locus primer pair. The resultant STR multiplex has a Multiplex Density equal to or greater than 2.0.
The nucleic-acid products are simultaneously amplified by polymerase chain reaction (PCR) in one reaction chamber using the at least 18, at least 27, or at least 37 different primer pairs to produce amplified nucleic acid products. The approach integrates high-plex amplification and detection as a single workflow in which all STR loci in the defined set are produced together in one chamber.
The amplified nucleic acid products are detected by laser induced fluorescence. The strategy uses multi-dye labeled primers in combination with laser induced fluorescence detection to support substantially non-overlapping allele sizing, and to improve performance with degraded DNA while reducing adventitious matches. The described multiplex density metric and the stated locus set coverage support compatibility across multiple locus sets including CODIS and European/international locus sets.
In addition to STR-only multiplex amplification, the described dye-multiplex concept extends to other marker types such as SNP assays and combined STR+SNP analyses. The document further discusses multiplex performance considerations and artifact mitigation concepts including iNTA, stutter, and primer interaction artifacts through primer modifications or label swapping, while retaining the laser induced fluorescence detection framework.
Claims Coverage
The document presents three independent claims covering multiplex amplification and laser induced fluorescence detection of high-plex STR sets (at least 18, at least 27, or at least 37 loci) using fluorescently labeled primer pairs with at least six dye labels in one reaction chamber, with a stated Multiplex Density threshold of at least 2.0.
High-plex STR multiplex with minimum multiplex density
Using a nucleic-acid sample contacted in one solution with at least 37 different primer pairs for at least 37 STR loci, where at least one primer of each primer pair is labeled with a fluorescent dye, where at least six different fluorescent dye-labels are used, where at least 26 primer pairs are for autosomal STR loci and at least 11 primer pairs are for Y-STR loci, and wherein the resultant STR multiplex has a Multiplex Density equal to or greater than 2.0.
Simultaneous PCR in a single reaction chamber
Simultaneously amplifying by polymerase chain reaction (PCR) in one reaction chamber using the at least 37 different primer pairs to produce amplified nucleic acid products.
Laser induced fluorescence detection
Detecting the nucleic acid products by laser induced fluorescence.
High-plex STR multiplex with minimum multiplex density
Using a nucleic-acid sample contacted in one solution with at least 27 different primer pairs for at least 27 STR loci, where at least one primer of each primer pair is labeled with a fluorescent dye, where at least six different fluorescent dye-labels are used, where at least 26 primer pairs are for autosomal STR loci and at least 1 primer pair is for Y-STR locus, and wherein the resultant STR multiplex has a Multiplex Density equal to or greater than 2.0.
Simultaneous PCR in a single reaction chamber
Simultaneously amplifying by polymerase chain reaction (PCR) in one reaction chamber using the at least 27 different primer pairs to produce amplified nucleic acid products.
Laser induced fluorescence detection
Detecting the nucleic acid products by laser induced fluorescence.
High-plex STR multiplex with minimum multiplex density
Using a nucleic-acid sample contacted in one solution with at least 18 different primer pairs for at least 18 STR loci, where at least one primer of each primer pair is labeled with a fluorescent dye, where at least six different fluorescent dye-labels are used, where at least 17 primer pairs are for autosomal STR loci and where at least 1 primer pair is for Y-STR locus, and wherein the resultant STR multiplex has a Multiplex Density equal to or greater than 2.0.
Simultaneous PCR in a single reaction chamber
Simultaneously amplifying by polymerase chain reaction (PCR) in one reaction chamber using the at least 18 different primer pairs to produce amplified nucleic acid products.
Laser induced fluorescence detection
Detecting the nucleic acid products by laser induced fluorescence.
Across the independent claims, the core claim coverage is the combination of contacting a nucleic-acid sample with high numbers of fluorescently labeled primer pairs using at least six dye labels to produce a resultant STR multiplex having Multiplex Density equal to or greater than 2.0, performing simultaneous PCR in one reaction chamber, and detecting the amplified products by laser induced fluorescence, with specific numeric locus counts and autosomal/Y-STR primer-pair distributions for the 18-, 27-, and 37-locus formats.
Stated Advantages
Improved performance with degraded DNA.
Reduced adventitious matches.
Substantially non-overlapping allele sizing.
International database compatibility across CODIS/European/Austrian/German/China/Interpol locus sets.
Documented Applications
Compatibility with CODIS and European/international locus sets, supporting analyses using those loci.
Use in combined STR+SNP analyses.
Familial searching/kinship analysis.
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