Genetic sequence verification compositions, methods and kits
AssigneeLife Technologies Corporation
InventorsEdgar Schreiber, Kamini Varma, Mark Andersen
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Abstract
Methods, compositions and kits are described for resequencing, confirming or verifying Next Generation Sequencing (NGS) results with Sanger Sequencing. These methods are particularly useful for samples having very limited quantities such as formalin-fixed, paraffin-embedded (FFPE), Laser Capture Microdissection (LCM), fine needle biopsies or aspirates.
Claims99
A method for sequencing at least one amplicon, comprising the steps of:
- a) providing at least one amplicon, wherein the at least one amplicon comprises a sequence of interest and a preceding sequence 5′ to the sequence of interest incorporated from a first priming sequence;
- b) amplifying the at least one amplicon in a first reaction mixture comprising a plurality of nuclease-sensitive amplification primers to form an amplified DNA product;
- c) contacting the first reaction mixture containing the amplified DNA product with a second reaction mixture comprising a nuclease and at least one chemically-enhanced primer whereby the plurality of nuclease sensitive amplification primers are degraded by the nuclease;
- d) inactivating the nuclease;
- e) priming the amplified DNA product with the at least one chemically-enhanced primer in a sequencing reaction;
- and
- f) producing extension products of the at least one chemically enhanced primer.
The method of any one of the preceding claims, wherein the first priming sequence was used to produce the amplicon.
The method of any one of the preceding claims, wherein the first priming sequence comprises at least one cleavable moiety.
The method of any one of the preceding claims, wherein the steps of contacting the first reaction mixture with the second reaction mixture, inactivating the nuclease, and producing the extension products of the chemically enhanced primer are performed in the same reaction vessel.
The method of any one of the preceding claims, wherein the steps of amplifying the at least one amplicon, contacting the first reaction mixture with the second reaction mixture, inactivating the nuclease, and producing the extension products of the chemically enhanced primer are performed without intermediate purification steps.
The method of any one of the preceding claims, wherein the at least one amplicon further comprises a succeeding sequence 3′ to the sequence of interest wherein the succeeding sequence is complementary to a second priming sequence used to produce the at least one amplicon.
The method of any one of the preceding claims, wherein the at least one amplicon has a length of about 100 nucleotides to about 400 nucleotides.
The method of any one of the preceding claims, wherein the sequence of interest has a length of about 100 nucleotides to about 300 nucleotides.
The method of any one of the preceding claims, wherein the at least one amplicon is a plurality of amplicons.
The method of any one of the preceding claims, further comprising:
- a) obtaining sequencing results based on the sequencing reaction;
- and
- b) determining a nucleotide base sequence of at least the sequence of interest based on the results.
The method of any one of the preceding claims, wherein the second reaction mixture further comprises a polymerase, deoxynucleotide triphosphates, and dye-labelled dideoxynucleotide triphosphates.
The method of any one of the preceding claims, wherein amplifying DNA comprises polymerase chain reaction amplification.
The method of any one of the preceding claims, wherein the first reaction mixture further comprises a polymerase.
The method of any one of the preceding claims, wherein the polymerase of the second reaction mixture is a thermostable polymerase.
The method of any one of the preceding claims, wherein the nuclease is selected from exonuclease I, Exo III, Pfu and DNA pol I.
The method of any one of the preceding claims, wherein the chemically-enhanced primer comprises an oligonucleotide sequence, a NCM and none or at least one nuclease-resistant linkage.
The method of any one of the preceding claims, wherein the chemically-enhanced primer comprises one nuclease-resistant linkage at a terminal 3′ end.
The method of any one of the preceding claims, wherein the sequencing reaction comprises cycle sequencing.
The method of any one of the preceding claims, wherein the chemically-enhanced primer comprises a plurality of NCMs either at a terminal 5′ end or within a oligonucleotide sequence of the chemically-enhanced primer.
The method of any one of the preceding claims, wherein the NCM is a (Cn) spacer wherein n is any integer from 1 to 9.
The method of any one of the preceding claims, wherein the chemically-enhanced primer has a structure of the formula:
- (Cn)x-OLIGO, wherein (Cn)x has a structure of the following formula:
- wherein each instance of n is independently an integer of 1 to 9;
- and x is an integer of 1 to about 30;
- OLIGO has a structure of the following formula:
- wherein B is a nucleobase;
- K is S or O;
- m is 0 or 1;
- z is an integer of 3 to about 100;
- W is OH, F, OMe, or H;
- and
- Nt is a moiety having a formula:
The method of any one of the preceding claims, wherein each of the plurality of nuclease-sensitive amplification primers is configured to prime a sequence of interest of a specific disease state.
The method of any one of the preceding claims, wherein the plurality of nuclease-sensitive amplification primers prime a set of sequences connected to a specific disease state.
A method for confirming a DNA sequence, comprising the steps of:
- a) amplifying a sample comprising nucleic acid using at least a first priming sequence to provide a plurality of amplicons, wherein each of the plurality of amplicons comprises a sequence of interest and a preceding sequence 5′ to the sequence of interest incorporated from a first priming sequence;
- b) amplifying a first aliquot of the plurality of amplicons in a first reaction mixture comprising a plurality of nuclease-sensitive amplification primers to form an amplified DNA product;
- c) contacting the first reaction mixture containing the amplified DNA product with a second reaction mixture comprising a nuclease and at least one chemically-enhanced primer whereby the nuclease sensitive amplification primers are degraded by the nuclease;
- d) inactivating the nuclease;
- e) priming the amplified DNA product with the at least one chemically-enhanced primer in a sequencing reaction;
- and
- f) producing extension products of the chemically enhanced primer.
The method of any one of claims 35-55, wherein the polymerase of the second reaction mixture is a thermostable polymerase.
A method of preparing DNA for sequencing, comprising the steps of:
- a) amplifying a sample comprising nucleic acid using at least a first priming sequence to provide a plurality of amplicons, wherein each of the plurality of amplicons comprises a sequence of interest and a preceding sequence 5′ to the sequence of interest incorporated from a first priming sequence;
- b) amplifying an aliquot of the plurality of amplicons in a first reaction mixture comprising nuclease-sensitive amplification primers to form an amplified DNA product;
- c) contacting the first reaction mixture containing the amplified DNA product with a second reaction mixture comprising a nuclease and a chemically-enhanced primer whereby the nuclease sensitive amplification primers are degraded by the nuclease;
- d) inactivating the nuclease;
- e) priming the amplified DNA product with the chemically-enhanced primer in a sequencing reaction;
- and
- f) producing extension products of the chemically enhanced primer.
A method of sequencing and verifying a variant nucleic acid sequence of interest, comprising the steps:
- a) amplifying a sample comprising nucleic acid using at least a first priming sequence to provide a plurality of amplicons, wherein each of the plurality of amplicons comprises a sequence of interest and a preceding sequence 5′ to the sequence of interest incorporated from a first priming sequence;
- b) splitting the plurality of amplicons into a first aliquot and a second aliquot;
- c) amplifying the first aliquot of the plurality of amplicons in a first reaction mixture comprising nuclease-sensitive amplification primers to form a first amplified DNA product;
- d) contacting the first reaction mixture containing the first amplified DNA product with a second reaction mixture comprising a nuclease and a chemically-enhanced primer whereby the nuclease sensitive amplification primers are degraded by the nuclease;
- e) inactivating the nuclease;
- f) priming the first amplified DNA product with the chemically-enhanced primer in a sequencing reaction;
- g) producing extension products of the chemically enhanced primer;
- h) obtaining sequencing results of at least the sequence of interest of the extended chemically enhanced primer using a mobility dependent separation;
- and
- i) determining a nucleotide base sequence of at least the sequence of interest of the extended chemically enhanced primer;
- j) amplifying the second aliquot of the amplicons to form a second DNA product;
- k) obtaining sequencing results of at least the sequence of interest of the second DNA product using a NGS sequencing method;
- and
- l) verifying a nucleotide sequence of the second DNA product by comparing it to the nucleotide base sequence of at least the sequence of interest of the extended chemically enhanced primer.
A composition for sequencing nucleic acid comprising:
- a PCR amplification reaction product that comprises:
- a) a DNA product amplified from at least one amplicon, wherein the amplicon comprises a sequence of interest and a preceding sequence 5′ to the sequence of interest incorporated from a first priming sequence;
- and
- b) non-nuclease-resistant amplification primer(s);
- and
- c) a chemically enhanced primer wherein the chemically enhanced primer comprises an oligonucleotide sequence, a NCM and none or at least one nuclease-resistant linkage.
The composition of any one of claims 70-73, wherein the NCM comprises a plurality of (Cn) spacers.
A chemically enhanced primer, comprising:
- an oligonucleotide sequence, at least one NCM and none or at least one nuclease-resistant linkage, and wherein at least the 10 of the nucleotides at a 3′ terminus of the chemically enhanced primer are complementary to at least 10 of the nucleotides at the 5′ terminus of an amplicon, wherein the amplicon comprises a sequence of interest and a preceding sequence 5′ to the sequence of interest incorporated from a first priming sequence.
The chemically enhanced primer of any one of claims 86-90, wherein the NCM comprises a plurality of (Cn) spacers.
The chemically enhanced primer of any one of claims 86-91, wherein the chemically-enhanced primer has a structure of the formula:
- (Cn)x-OLIGO, wherein (Cn)x has a structure of the following formula:
- wherein each instance of n is independently an integer of 1 to 9;
- and x is an integer of 1 to about 30;
- OLIGO has a structure of the following formula:
- wherein B is a nucleobase;
- K is S or O;
- m is 0 or 1;
- z is an integer of 3 to about 100;
- W is OH, F, OMe, or H;
- and
- Nt is a moiety having a formula:
A kit, comprising:
- a polymerase, a nuclease, at least one deoxynucleotide triphosphate, and dideoxynucleotide triphosphates.
Dependencies are inferred from the claim text.