Back to results
US 12,630,864 B2Granted
Methods of nucleic acid sample preparation
AssigneeLaboratory Corporation of America Holdings
InventorsJoshua Stahl, Jason Myers, Brady Culver, Brian Kudlow, Jens Eberlein
Life of this patent
Estimating the term…
Abstract
Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids, e.g., cfDNA, and nucleic acids encoding immune receptors and immunoglobulins. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.
Claims20
A method of preparing nucleic acids for analysis, the method comprising:
- (a) hybridizing a primer to a nucleic acid molecule comprising a target nucleotide sequence, wherein the nucleic acid molecule was obtained from cell-free DNA obtained from a subject having or suspected of having a cancer;
- (b) generating a double stranded nucleic acid by a process comprising conducting a polymerase extension that is primed by the hybridized primer and that uses the nucleic acid molecule as a template, wherein the conducting of the polymerase extension comprises incorporating one or more capture moiety modified nucleotides into the double stranded nucleic acid, wherein each of the capture moiety modified nucleotides comprises a capture moiety;
- (c) ligating an adapter nucleic acid to the double-stranded nucleic acid to produce a ligation product comprising the capture moiety;
- (d) isolating the ligation product by a process comprising contacting the ligation product with a binding partner of the capture moiety;
- and
- (e) amplifying the isolated ligation product by a polymerase chain reaction using (i) a first adapter primer that specifically anneals to a sequence of the adapter nucleic acid and (ii) a target-specific primer that comprises a 3′ portion that specifically anneals to the target nucleotide sequence and a 5′ tail portion that does not specifically anneal to the target nucleotide sequence, thereby providing a first amplification product.
3 more elements
A method of preparing nucleic acids for analysis, the method comprising:
- (a) hybridizing a plurality of different target-specific primers to a population of nucleic acid molecules comprising a plurality of different target nucleotide sequences, wherein the population of nucleic acid molecules was obtained from cell-free DNA obtained from a human subject having or suspected of having a cancer;
- (b) generating a plurality of double stranded nucleic acids by a process comprising conducting a polymerase extension that is primed by each of the plurality of different target-specific primers, wherein the conducting of the polymerase extension comprises incorporating one or more capture moiety modified nucleotides into the double stranded nucleic acids, and wherein each of the capture moiety modified nucleotides comprises a capture moiety;
- (c) ligating an adapter nucleic acid to each of the plurality of double-stranded nucleic acids to produce a plurality of ligation products, each comprising at least one of the capture moiety;
- (d) isolating the plurality of ligation products by a process comprising contacting the plurality of ligation products with a substrate or plurality of beads comprising a binding partner of the capture moiety;
- (e) amplifying the isolated ligation products by a polymerase chain reaction using (i) a first adapter primer that specifically anneals to a sequence of the adapter nucleic acid and (ii) a plurality of different target-specific primers, each comprising a 3′ portion that specifically anneals to one of the plurality of different target nucleotide sequences and a substantially identical 5′ tail portion that does not specifically anneal to a target nucleotide sequence, thereby providing a plurality of first amplification products;
- and
- (f) amplifying the plurality of first amplification products by a polymerase chain reaction using (i) a tail primer that comprises a 3′ portion that specifically anneals to a complementary sequence of the 5′ tail portion of the plurality of different target-specific primers and (ii) a second adapter primer that specifically anneals to a portion of the adapter nucleic acid, thereby providing a plurality of second amplification products.
4 more elements
Dependencies are inferred from the claim text.