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-9310304-B2
Publication Date
2016-04-12
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 methods detect single nucleotide polymorphisms in a nucleic-acid sample using fluorescently distinguishable hybridization products or primer-extension products labeled with at least eight different fluorescent dyes. Laser-induced fluorescence detection employs a spectrograph and a galvanometer operating in step-and-stare mode. These approaches contact the sample with unlabeled oligonucleotides to generate labeled extension products or with differently labeled oligonucleotides to form hybridization products.
The technology encompasses multicolor multiplex amplification and detection of short tandem repeat loci alongside single nucleotide polymorphism analysis for genetic-marker profiling. Increased dye counts permit the detection of more loci, higher multiplex density, and smaller amplicons that facilitate the analysis of degraded nucleic acids. Primer modifications further optimize multiplex assays by reducing incomplete nontemplate addition.
Claims Coverage
The claims cover two inventive features across two independent claims for detecting single nucleotide polymorphisms in a nucleic-acid sample using multicolor fluorescence detection.
Detection using fluorescent primer-extension products
The claim covers contacting a nucleic-acid sample with at least eight unlabeled oligonucleotides, generating primer-extension products labeled with at least eight different fluorescent dyes using fluorescently labeled ddNTPs, and detecting the products by laser-induced fluorescence using a galvanometer in step-and-stare mode and a spectrograph.
Detection using labeled oligonucleotide hybridization
The claim covers contacting a nucleic-acid sample with at least eight oligonucleotides, each labeled with a different fluorescent dye, under conditions permitting hybridization, and detecting the resulting hybridization products by laser-induced fluorescence using a galvanometer in step-and-stare mode and a spectrograph.
The independent claims cover two single nucleotide polymorphism detection methods based on labeled primer-extension products and labeled oligonucleotide hybridization products, both specifying laser-induced fluorescence detection using a galvanometer and spectrograph.
Stated Advantages
Increasing the number of detectable dyes allows more single nucleotide polymorphisms to be analyzed in a lane, supports higher multiplex density, and enables the use of shorter products or smaller amplicons.
Smaller amplicons improve profiling of degraded DNA.
Primer modifications reduce incomplete nontemplate addition in multiplex assays.
Documented Applications
Iris-color analysis.
Combined single nucleotide polymorphism and short tandem repeat testing.
Human identification.
Forensic and kinship analysis.
Database searching.
Multicolor multiplex DNA-marker analysis and genetic-marker profiling.
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