Conversion of Target Specific Amplification to Universal Sequencing
First Claim
1. A method of performing a re-sequencing reaction comprising;
- performing a polymerase chain reaction (PCR) with a forward primer and a reverse primer to form a PCR amplicon,wherein the forward primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises a type IIs restriction enzyme recognition site, and,wherein the reverse primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises a type IIs restriction enzyme recognition site;
digesting the PCR amplicon with a type IIs restriction enzyme to form a digested amplicon with a first end and a second end;
ligating an adapter to the first end of the digested amplicon and ligating an adapter to the second end of the digested amplicon to form an adapter-ligated amplicon with a first end and a second end; and
,sequencing the adapter-ligated amplicon.
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Abstract
The present teachings provide methods, compositions, and kits for amplifying nucleic acids. The amplified nucleic acids can then be sequenced. The sequencing reaction for such amplified nucleic acids can provide additional sequence information, and less redundancy, as compared to conventional approaches. In some embodiments, a target polynucleotide is amplified with a primer to form an amplicon comprising a type IIs restriction enzyme recognition site. Following digestion with a type IIs restriction enzyme, and ligation of an adapter, a sequencing primer can be hybridized to the adapter, and a sequencing reaction performed. The sequencing reaction results in the omission of unwanted sequence information that was present in the amplification primer.
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Citations
57 Claims
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1. A method of performing a re-sequencing reaction comprising;
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performing a polymerase chain reaction (PCR) with a forward primer and a reverse primer to form a PCR amplicon, wherein the forward primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises a type IIs restriction enzyme recognition site, and,wherein the reverse primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises a type IIs restriction enzyme recognition site;digesting the PCR amplicon with a type IIs restriction enzyme to form a digested amplicon with a first end and a second end; ligating an adapter to the first end of the digested amplicon and ligating an adapter to the second end of the digested amplicon to form an adapter-ligated amplicon with a first end and a second end; and
,sequencing the adapter-ligated amplicon. - View Dependent Claims (2, 3, 4, 5, 13, 22)
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6. A method of forming an adapter-ligated amplicon comprising a first end that differs in sequence from a second end, said method comprising;
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amplifying a short nucleic acid in a polymerase chain reaction (PCR) comprising a forward primer and a reverse primer to form a PCR amplicon, wherein the forward primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises a type IIs restriction enzyme recognition site, and,wherein the reverse primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises a type IIs restriction enzyme recognition site;digesting the PCR amplicon with a type IIs restriction enzyme to form a digested amplicon with a first end and a second end; and
,ligating a first adapter to the first end of the digested amplicon and ligating a second adapter to the second end of the digested amplicon to form an adapter-ligated amplicon with a first end that differs in sequence from a second end. - View Dependent Claims (7, 8)
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9. A method of increasing the percent of novel sequence information in a sequencing reaction of a digested PCR amplicon less than 100 bases in length comprising;
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performing a polymerase chain reaction (PCR) with a forward primer and a reverse primer to form a PCR amplicon, wherein the forward primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises a type IIs restriction enzyme recognition site, and,wherein the reverse primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises a type IIs restriction enzyme recognition site;digesting the PCR amplicon with a type IIs restriction enzyme to form a digested PCR amplicon with a first end and a second end, wherein the digested PCR amplicon is less than one hundred bases in length; ligating an adapter to the first end of the digested PCR amplicon and ligating an adapter to the second end of the digested PCR amplicon to form an adapter-ligated PCR amplicon with a first end and a second end; sequencing the adapter-ligated amplicon; and
,increasing the percent of novel sequence information in a sequencing reaction of the PCR amplicon as compared to sequencing a PCR amplicon that was amplified in a PCR not comprising primers with a type IIs restriction enzyme site. - View Dependent Claims (10, 11, 12)
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14. A method of sequencing a target polynucleotide comprising;
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amplifying the target polynucleotide with at least one primer to form an amplicon comprising a type IIs restriction enzyme recognition site on at least one end; digesting the PCR amplicon with a type IIs restriction enzyme to form a digested amplicon with at least one ligatable end; ligating an adapter to the ligatable end of the digested amplicon to form an adapter-ligated amplicon with a known end; hybridizing a sequencing primer to the known end; and
,sequencing the adapter-ligated amplicon, wherein the first sequenced base is target polynucleotide information. - View Dependent Claims (15, 16, 17)
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18. A method of performing a sequencing reaction comprising;
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performing a polymerase chain reaction (PCR) with a forward primer and a reverse primer to form a PCR amplicon, wherein the forward primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises an exonuclease-resistant moiety, and,wherein the reverse primer comprises a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises an exonuclease-resistant moiety;digesting the PCR amplicon with an exonuclease to form a digested PCR amplicon with a first end and a second end; ligating an adapter to the first end of the digested PCR amplicon and to the second end of the digested PCR amplicon to form an adapter-ligated amplicon with a first end and a second end; and
,sequencing the adapter-ligated amplicon. - View Dependent Claims (19, 20, 21)
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23. A method of performing a sequencing reaction, wherein the first sequenced base is target polynucleotide information, said method comprising;
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performing a primer-mediated amplification reaction to form an amplicon, wherein the primer comprises a target-specific portion and cleavage-related moiety; digesting the amplicon with a cleavage agent directed to the cleavage-related moiety to form a digested amplicon with a cleaved end; ligating an adapter to the cleaved end of the digested amplicon to form an adapter-ligated amplicon; and
,sequencing the adapter-ligated amplicon, wherein the first sequenced base is target polynucleotide information. - View Dependent Claims (24, 25, 26, 27, 28, 29)
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30. A kit comprising;
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a forward primer comprising a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises a type IIs restriction enzyme recognition site;a reverse primer comprising a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises a type IIs restriction enzyme recognition site;a first adapter comprising a first universal primer portion; a second adapter comprising a second universal primer portion; a ligase; a polymerase; and
,dNTPs. - View Dependent Claims (31, 32, 33, 34)
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35. A composition comprising;
a digested PCR amplicon less than 50 nucleotides in length, the digested PCR amplicon comprising a first single-stranded end resulting from cleavage of a first type IIs restriction enzyme recognition site by a type IIs restriction enzyme that was introduced by a forward primer, and, a second single-stranded end resulting from cleavage of a type IIs restriction enzyme site by a type IIs restriction enzyme that was introduced by a reverse primer. - View Dependent Claims (36, 37)
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38. A kit comprising;
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a forward primer comprising a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises a exonuclease-resistant moiety;a reverse primer comprising a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises an exonuclease-resistant moiety;a first adapter comprising a first universal primer portion; a second adapter comprising a second universal primer portion; a ligase; a polymerase; and
,dNTPs. - View Dependent Claims (39, 40, 41, 42)
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43. A composition comprising;
a digested PCR amplicon less than 50 nucleotides in length, the digested PCR amplicon comprising a first single-stranded end resulting from cleavage with a first exonuclease of a region upstream from a first exonuclease-resistant moiety that was introduced by a forward primer, and, a second single-stranded end resulting from cleavage with a second exonuclease of a region upstream from a second exonuclease-resistant moiety that was introduced by a reverse primer. - View Dependent Claims (44, 45)
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46. A kit comprising;
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a forward primer comprising a target-specific portion and a 5′
tail, wherein the 5′
tail of the forward primer comprises cleavage-related moiety;a reverse primer comprising a target-specific portion and a 5′
tail, wherein the 5′
tail of the reverse primer comprises a cleavage-related moiety;a first adapter comprising a first universal primer portion; a second adapter comprising a second universal primer portion; a ligase; a polymerase; and
,dNTPs. - View Dependent Claims (47, 48, 49, 50, 51, 52)
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53. A composition comprising;
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a digested amplicon ligated to an adapter, and, a sequencing primer hybridized to the adapter, wherein the digested amplicon is less than 50 nucleotides in length and comprises a single-stranded end hybridized to the adapter, wherein the single-stranded end results from cleavage of a cleavage-related moiety introduced by an amplification primer. - View Dependent Claims (54, 55, 56, 57)
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Specification