Methods for nucleic acid amplification
First Claim
1. A method for sequencing a target segment of nucleotides in a nucleic acid test sample, said method comprising:
- (a) amplifying said target segment of nucleotides using at least two oligonucleotide primers that flank the target segment, wherein at least one of the oligonucleotide primers comprise;
i) a complementary region with a sequence of about 15 to about 25 nucleotides that hybridizes adjacent to the target segment of nucleotides; and
ii) an extender region that includes a primer hybridization sequence wherein said extender region does not hybridize to the target segment of nucleotides and said extender region is not fully complementary to the segment of nucleotides adjacent to which the complementary region of the primer hybridizes in the target nucleic acids, with a proviso that the length of the complimentary region and the extender region excluding the primer hybridization sequence portion of the extender region together represents a contiguous segment of at least, or, about 50 nucleotides; and
(b) determining the sequence of the amplified target segment by priming with an oligonucleotide primer that is complementary to said primer hybridization sequence of the extender region.
1 Assignment
0 Petitions
Accused Products
Abstract
Disclosed are methods for amplifying and sequencing target segments of nucleotides. In the method, nucleic acids are amplified using at least two oligonucleotide primers, wherein at least one of the primers contains a complimentary region that hybridizes adjacent to the target segment of nucleotides and an extender region that includes a primer hybridization sequence. Also included are methods of amplification with an extender region includes a sequence encoding a promoter for an RNA polymerase and optionally sequences for translation. The latter amplified products may be used for transcription of a RNA including subsequent translation of the RNA to generate a protein product.
24 Citations
51 Claims
-
1. A method for sequencing a target segment of nucleotides in a nucleic acid test sample, said method comprising:
-
(a) amplifying said target segment of nucleotides using at least two oligonucleotide primers that flank the target segment, wherein at least one of the oligonucleotide primers comprise; i) a complementary region with a sequence of about 15 to about 25 nucleotides that hybridizes adjacent to the target segment of nucleotides; and ii) an extender region that includes a primer hybridization sequence wherein said extender region does not hybridize to the target segment of nucleotides and said extender region is not fully complementary to the segment of nucleotides adjacent to which the complementary region of the primer hybridizes in the target nucleic acids, with a proviso that the length of the complimentary region and the extender region excluding the primer hybridization sequence portion of the extender region together represents a contiguous segment of at least, or, about 50 nucleotides; and (b) determining the sequence of the amplified target segment by priming with an oligonucleotide primer that is complementary to said primer hybridization sequence of the extender region. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 38, 43, 44, 45)
-
-
15. A method for amplifying a target segment of nucleotides in a nucleic acid sample for nucleic acid sequence determination, said method comprising:
-
(a) contacting said nucleic acids in a test sample with at least two oligonucleotide primers that flank a target segment of nucleotides, wherein at least one of said oligonucleotide primers comprise; i) a complementary region with a sequence of about 15 to about 25 nucleotides that hybridizes adjacent to the target segment of nucleotides; and ii) an extender region that includes a primer hybridization sequence wherein said extender region does not hybridize to the target segment of nucleotides and said extender region is not fully complementary to the segment of nucleotides adjacent to which the complementary region of the primer hybridizes in the target nucleic acids, with a proviso that the length of the complimentary region and the extender region excluding the primer hybridization sequence portion of the extender region together represents a contiguous segment of at least, or, about 50 nucleotides; and (b) amplifying said target segment of nucleotides by priming with said primers, wherein said target segment of nucleotides comprises up to, or, about 150 nucleotides and said target segment is located in the nucleic acid less than 100 nucleotides from a genomic region selected from the group consisting of a repetitive element, a loop, a bulge, a stem-loop structure, triplex DNA, tertiary or quanternary nucleic acid structure, AT-rich region, GC-rich region and polymorphic region, and wherein said complementary region of the primer hybridizes to the nucleic acid between said target segment and genomic region. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 39, 46, 47, 48)
-
-
28. A method for amplifying a target segment of nucleotides in a nucleic acid sample for transcription of the amplified target segment, said method comprising:
-
(a) contacting a nucleic acid sample with at least two oligonucleotide primers that flank a target segment of nucleotides, wherein at least one of said oligonucleotide primers comprise; i) a complementary region with a sequence of about 15 to about 25 nucleotides that hybridizes adjacent to the target segment of nucleotides; and ii) an extender region that includes a sequence encoding a promoter for an RNA polymerase wherein said extender region does not hybridize to the target segment of nucleotides and said extender region is not fully complementary to the segment of nucleotides adjacent to which the complementary region of the primer hybridizes in the target nucleic acids, with a proviso that the length of the complimentary region and the extender region excluding the sequence encoding the promoter for an RNA polymerase portion of the extender region together represents a contiguous segment of at least, or, about 50 nucleotides; and (b) amplifying said target segment of nucleotides by priming with said primers, wherein said target segment of nucleotides comprises up to, or, about 150 nucleotides and said target segment is located in the nucleic acid less than 100 nucleotides from a genomic region selected from the group consisting of a repetitive element, a loop, a bulge, a stem-loop structure, triplex DNA, tertiary or quanternary nucleic acid structure, AT-rich region, GC-rich region and polymorphic region, and wherein said complementary region of the primer hybridizes to the nucleic acid between said target segment and genomic region. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 41, 42, 49, 50, 51)
-
Specification