[미국특허]
Methods and compositions for size-controlled homopolymer tailing of substrate polynucleotides by a nucleic acid polymerase
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12N-009/12
C12P-019/34
출원번호
US-0384113
(2013-03-13)
등록번호
US-9896709
(2018-02-20)
국제출원번호
PCT/US2013/031104
(2013-03-13)
국제공개번호
WO2013/138536
(2013-09-19)
발명자
/ 주소
Makarov, Vladimir
Kurihara, Laurie
출원인 / 주소
SWIFT BIOSCIENCES, INC.
대리인 / 주소
Gibson, Matthew S.
인용정보
피인용 횟수 :
0인용 특허 :
103
초록▼
The present invention is directed to methods and compositions for adding tails of specific lengths to a substrate polynucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support. The disclosure contemplates that the attenuator molec
The present invention is directed to methods and compositions for adding tails of specific lengths to a substrate polynucleotide. The invention also contemplates methods and compositions for immobilization of tailed substrates to a solid support. The disclosure contemplates that the attenuator molecule is any biomolecule that associates with a tail sequence added to a substrate polynucleotide and controls the addition of a tail sequence to the 3′ end of the substrate polynucleotide. The sequence that is added to the substrate polynucleotide is referred to herein as a tail sequence, or simply a tail, and the process of adding a nucleotide to a substrate polynucleotide is referred to herein as tailing.
대표청구항▼
1. A kit comprising: a template-independent nucleic acid polymerase;an attenuator polynucleotide comprising an attenuator sequence and a sequence W positioned adjacent to the attenuator sequence, wherein the attenuator sequence is from about 10 nucleotides to about 100 nucleotides in length, and whe
1. A kit comprising: a template-independent nucleic acid polymerase;an attenuator polynucleotide comprising an attenuator sequence and a sequence W positioned adjacent to the attenuator sequence, wherein the attenuator sequence is from about 10 nucleotides to about 100 nucleotides in length, and wherein the attenuator polynucleotide comprises a 3′ blocking group selected from the group consisting of at least one ribonucleotide, at least one deoxynucleotide, a C3 spacer, a phosphate, a dideoxynucleotide, an amino group, and an inverted deoxythymidine;an adaptor polynucleotide, wherein the adaptor polynucleotide comprises a sequence X which is complementary to sequence W of the attenuator polynucleotide;nucleotides complementary to the attenuator sequence; anda ligase. 2. The kit of claim 1, wherein the sequence W is positioned 5′ adjacent to the attenuator sequence. 3. The kit of claim 1, wherein the template-independent nucleic acid polymerase is terminal deoxynucleotidyl transferase (TdT). 4. The kit of claim 1, wherein the template-independent nucleic acid polymerase is an RNA-specific nucleotidyl transferase selected from the group consisting of poly(A) polymerase and poly(U) polymerase. 5. The kit of claim 1, wherein the adaptor polynucleotide further comprises a 5′ phosphate. 6. The kit of claim 1 further comprising a primer, a polymerase, and a second adaptor polynucleotide comprising a sequence Y and a sequence V, wherein sequence V is complementary to sequence Y when sequence V is the same length as sequence Y, or wherein sequence V is complementary to a portion of sequence Y when sequence V is less than the length of sequence Y, the second adaptor polynucleotide being a separate molecule from the attenuator polynucleotide. 7. The kit of claim 1, wherein the attenuator polynucleotide comprises an affinity label for immobilization. 8. The kit of claim 1 wherein the attenuator sequence of the attenuator polynucleotide comprises a homopolymeric sequence selected from the group consisting of poly (dA), poly (dT), poly (dC), poly (dG), and poly (dU). 9. The kit of claim 1, wherein the attenuator sequence of the attenuator polynucleotide comprises a homopolymeric sequence selected from the group consisting of poly (rA), poly (U), poly (rC), and poly (rG). 10. The kit of claim 1, wherein the attenuator sequence of the attenuator polynucleotide comprises a heteropolymeric sequence selected from the group consisting of (i) dA and rA bases, (ii) dT, dU and U bases, (iii) dC and rC bases, and (iv) dG and rG bases. 11. The kit of claim 1, wherein the attenuator sequence of the attenuator polynucleotide comprises a dinucleotide sequence comprising a plurality of random sequences comprised of the following dinucleotide combinations: (i) dG and dC; (ii) dA and dT; (iii) dG and dT; (iv) dG and dA; (v) dA and dC; or (vi) dC and dT. 12. The kit of claim 1, wherein sequence X is a sequence complementary to any one of SEQ ID NOS: 44-51. 13. The kit of claim 1, wherein the attenuator sequence is from about 10 nucleotides to about 20 nucleotides in length.
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