Optimized real time nucleic acid detection processes
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12Q-001/6818
C12Q-001/6844
C12Q-001/6876
C12Q-001/70
C12Q-001/6853
출원번호
US-0084983
(2016-03-30)
등록번호
US-10144957
(2018-12-04)
발명자
/ 주소
Rabbani, Elazar
Donegan, James J.
Liu, Dakai
출원인 / 주소
Enzo Life Sciences, Inc.
대리인 / 주소
Diamond, Esq., Paul
인용정보
피인용 횟수 :
0인용 특허 :
114
초록▼
This invention provides for compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time n
This invention provides for compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time nucleic acid detection allows for the qualitative or quantitative detection or determination of single-stranded or double-stranded nucleic acids of interest in a sample. Other processes are provided by this invention including processes for removing a portion of a homopolymeric sequence, e.g., poly A sequence or tail, from an analyte or library of analytes. Compositions useful in carrying out such removal processes are also described and provided. Paneling and multiplex analyses of more than one nucleic acid analyte using one sample are also provided.
대표청구항▼
1. A process for detecting qualitatively or quantitatively the presence of more than one single-stranded or double-stranded nucleic acid analyte in a sample, said process comprising the steps of: (a) providing (i) the sample for analysis;(ii) for each analyte, a first nucleic acid primer having a 5′
1. A process for detecting qualitatively or quantitatively the presence of more than one single-stranded or double-stranded nucleic acid analyte in a sample, said process comprising the steps of: (a) providing (i) the sample for analysis;(ii) for each analyte, a first nucleic acid primer having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of one strand of the nucleic acid analyte;(iii) for each analyte, a second nucleic acid primer having a 5′ end and a 3′ end and comprising a nucleic acid sequence identical to at least a portion of said one strand for the analyte;(iv) labeled nucleotides comprising a first energy transfer element; and(v) reagents for carrying out nucleic acid strand extension,wherein the first nucleic acid primer, the second nucleic acid primer, or both the first nucleic acid primer and the second nucleic acid primer comprise a second energy transfer element, andwherein said first energy transfer element is an energy transfer donor and said second energy transfer element is an energy transfer acceptor, or said first energy transfer element is an energy transfer acceptor and said second energy transfer element is an energy transfer donor;(b) for each analyte, forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;(c) for each analyte, contacting under hybridization conditions said first nucleic acid primer with one strand of said nucleic acid analyte and contacting under hybridization conditions said second nucleic acid primer with the complementary strand of said nucleic acid analyte if present;(d) for each analyte, extending said first nucleic acid primer by more than one nucleotide to form a first primer-extended nucleic acid sequence and, if said complementary strand is present, extending said second nucleic acid primer by more than one nucleotide to form a second primer-extended nucleic acid sequence, thereby incorporating more than one labeled nucleotide into (i) the first primer-extended nucleic acid sequence and (ii) the second primer-extended nucleic acid sequence if said complementary strand is present;(e) for each analyte, separating said first primer-extended nucleic acid sequence from said nucleic acid analyte and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid analyte if present;(f) for each analyte, contacting under hybridization conditions said first nucleic acid primer with said nucleic acid analyte or said second primer-extended nucleic acid sequence from step (e), and contacting under hybridization conditions said second nucleic acid primer with said first primer-extended nucleic acid sequence from step (e); and(g) for each analyte, detecting the presence or quantity of said nucleic acid analyte by detecting energy transfer between a second energy transfer element of said first nucleic acid primer, said second nucleic acid primer, or both, and a first energy element of incorporated labeled nucleotides,wherein the same sample is used for the detection of each analyte, andwherein the sample comprises nucleic acid purified from a tissue preparation or a serum preparation. 2. The process of claim 1, wherein a separate aliquot of the sample is used for detection of each analyte. 3. The process of claim 1, wherein the same aliquot of the sample is used for detection of each analyte. 4. The process of claim 1, wherein the sample comprises nucleic acid purified from a tissue preparation. 5. The process of claim 1, wherein the sample comprises nucleic acid purified from a serum preparation. 6. The process of claim 1, wherein the sample comprises RNA purified from a tissue or serum preparation. 7. The process of claim 1, wherein the sample comprises DNA purified from a tissue or serum preparation. 8. The process of claim 1, wherein for at least one analyte, at least one of the first nucleic acid primer and the second nucleic acid primer comprises said second energy transfer element near the 3′ end of the primer. 9. The process of claim 1, consisting of the steps: (a) providing (i) the sample for analysis;(ii) for each analyte, a first nucleic acid primer having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of one strand of the nucleic acid analyte;(iii) for each analyte, a second nucleic acid primer having a 5′ end and a 3′ end and comprising a nucleic acid sequence identical to at least a portion of said one strand for the analyte;(iv) labeled nucleotides comprising a first energy transfer element; and(v) reagents for carrying out nucleic acid strand extension,wherein the first nucleic acid primer, the second nucleic acid primer, or both the first nucleic acid primer and the second nucleic acid primer comprise a second energy transfer element, andwherein said first energy transfer element is an energy transfer donor and said second energy transfer element is an energy transfer acceptor, or said first energy transfer element is an energy transfer acceptor and said second energy transfer element is an energy transfer donor;(b) for each analyte, forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;(c) for each analyte, contacting under hybridization conditions said first nucleic acid primer with one strand of said nucleic acid analyte and contacting under hybridization conditions said second nucleic acid primer with the complementary strand of said nucleic acid analyte if present;(d) for each analyte, extending said first nucleic acid primer by more than one nucleotide to form a first primer-extended nucleic acid sequence and, if said complementary strand is present, extending said second nucleic acid primer by more than one nucleotide to form a second primer-extended nucleic acid sequence, thereby incorporating more than one labeled nucleotide into (i) the first primer-extended nucleic acid sequence and (ii) the second primer-extended nucleic acid sequence if said complementary strand is present;(e) for each analyte, separating said first primer-extended nucleic acid sequence from said nucleic acid analyte and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid analyte if present;(f) for each analyte, contacting under hybridization conditions said first nucleic acid primer with said nucleic acid analyte or said second primer-extended nucleic acid sequence from step (e), and contacting under hybridization conditions said second nucleic acid primer with said first primer-extended nucleic acid sequence from step (e); and(g) for each analyte, detecting the presence or quantity of said nucleic acid analyte by detecting energy transfer between a second energy transfer element of said first nucleic acid primer, said second nucleic acid primer, or both, and a first energy element of incorporated labeled nucleotides,wherein the same sample is used for the detection of each analyte. 10. The process of claim 9, wherein a separate aliquot of the sample is used for detection of each analyte. 11. The process of claim 9, wherein the same aliquot of the sample is used for detection of each analyte. 12. The process of claim 9, wherein for at least one analyte, at least one of the first nucleic acid primer and the second nucleic acid primer comprises said second energy transfer element near the 3′ end of the primer. 13. The process of claim 12, wherein a separate aliquot of the sample is used for detection of each analyte. 14. The process of claim 12, wherein the same aliquot of the sample is used for detection of each analyte.
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