Methods of preparing nucleic acids for sequencing
Inventors
Iafrate, Anthony John • Le, Long Phi • Zheng, Zongli • Myers, Jason • Stahl, Joshua
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10450597-B2
Publication Date
2019-10-22
Expiration Date
Abstract
Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.
Core Innovation
The invention provides a method of preparing nucleic acids for analysis in which an extension product is generated that contains both a sequence characteristic of a target-specific primer and a sequence characteristic of a different primer pool. A first nucleic acid template comprising a sequence of a first strand of a double-stranded target nucleic acid is contacted with a complementary target-specific primer that comprises a target-specific hybridization sequence to promote template-specific hybridization and extension.
A second nucleic acid template comprising a sequence of a second strand complementary to the first strand is contacted with a plurality of different primers that share a common sequence that is 5′ to different hybridization sequences to promote template-specific hybridization and extension of at least one different primer. The different hybridization sequences have different 3′ ends, and each primer of the plurality does not anneal to the same sequence of the double-stranded target nucleic acid as any other primer.
Following the two contacting and extension steps, the resulting extension product contains both target-specific primer sequence information and sequence information derived from at least one of the plurality of different primers. The extension product is subjected to amplification comprising successive rounds of polymerase extension using a tail primer and a primer that anneals to the target-specific hybridization sequence, and embodiments include sequencing-compatible amplification design for analyzing contiguous sequence adjacent to a known target using nested tail primers and nested target-specific primers.
Claims Coverage
The independent claim coverage includes 2 inventive features.
Sequential extension from target-specific primer and tailed primer plurality
Contacting a first nucleic acid template with a complementary target-specific primer comprising a target-specific hybridization sequence under conditions to promote template-specific hybridization and extension; contacting a second complementary strand template with a plurality of different primers that share a common 5′ sequence to different hybridization sequences, wherein the different hybridization sequences have different 3′ ends and each primer does not anneal to the same sequence as any other primer; generating an extension product containing both a sequence characteristic of the target-specific primer and a sequence characteristic of at least one of the plurality of different primers.
Amplification with tail primer and target-specific primer
Subjecting the extension product to an amplification reaction comprising successive rounds of polymerase extension using a tail primer that specifically anneals to the complement of the common sequence and comprises a 5′ tail sequence, together with a primer that specifically anneals to the complement of the target-specific hybridization sequence.
The claim coverage centers on generating an extension product containing both a target-specific primer component and a sequence derived from a plurality of different primers sharing a common 5′ sequence with different 3′ ends, followed by amplification using a tail primer complementary to the common sequence and a primer complementary to the target-specific hybridization sequence.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?