NEW METHOD FOR QUALITATIVE AND QUANTITATIVE DETECTION OF SHORT NUCLEIC ACID SEQUENCES OF ABOUT 8-50 NUCLEOTIDES IN LENGTH
First Claim
1. A method of qualitative and quantitative detecting a short nucleic acid sequence of interest, preferably a DNA oligonucleotide or a modified DNA oligonucleotide in a sample, the method comprising (a) contacting the sample with a capture probe;
- wherein the capture probe comprises a portion complementary to part of the sequence of interest and so capable of hybridising thereto, and a portion non-complementary to the sequence of interest;
(b) causing extension of the sequence of interest with a nucleic acid polymerase, using the capture probe as a template;
causing extension of the capture probe with a nucleic acid polymerase, using the sequence of interest as a template; and
(c) qualitative and quantitative detecting directly or indirectly the extended sequence of interest and the extended capture probe using a nucleic acid amplification reaction, so as to indicate the presence and amount of the sequence of interest;
characterised in that the primers used for nucleic acid amplification comprising a portion complementary to the extension of the sequence of interest and of the extension of the capture probe, thereby preventing nucleic acid amplification in the absence of the sequence of interest.
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Abstract
The present invention concerns a new analytical method for qualitative and quantitative detection of short nucleic acid sequences, preferably a DNA oligonucleotide or a modified DNA oligonucleotide such as antisense oligonucleotides or fragmented nucleic acid sequences of about 8-50 nucleotides in length. The invention relates to the introduction of modified nucleic acids into an oligonucleotide probe that hybridizes to the target sequence such that amplification and quantitation of the short nucleic acid sequence is enabled and sensitivity and specificity of the reaction is increased. The invention also embraces test kits for performing nucleic acid amplification to detect and quantitate short nucleic acid sequences and processes for preparing such and the use of new analytical methods.
3 Citations
30 Claims
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1. A method of qualitative and quantitative detecting a short nucleic acid sequence of interest, preferably a DNA oligonucleotide or a modified DNA oligonucleotide in a sample, the method comprising (a) contacting the sample with a capture probe;
- wherein the capture probe comprises a portion complementary to part of the sequence of interest and so capable of hybridising thereto, and a portion non-complementary to the sequence of interest;
(b) causing extension of the sequence of interest with a nucleic acid polymerase, using the capture probe as a template;
causing extension of the capture probe with a nucleic acid polymerase, using the sequence of interest as a template; and
(c) qualitative and quantitative detecting directly or indirectly the extended sequence of interest and the extended capture probe using a nucleic acid amplification reaction, so as to indicate the presence and amount of the sequence of interest;
characterised in that the primers used for nucleic acid amplification comprising a portion complementary to the extension of the sequence of interest and of the extension of the capture probe, thereby preventing nucleic acid amplification in the absence of the sequence of interest. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22, 24, 26, 28, 29, 30)
- wherein the capture probe comprises a portion complementary to part of the sequence of interest and so capable of hybridising thereto, and a portion non-complementary to the sequence of interest;
- 19. A capture probe for use in a method of qualitative and quantitative detecting a short nucleic acid sequence of interest, preferably a DNA oligonucleotide or a modified DNA oligonucleotide, comprising a portion complementary to part of the sequence of interest and so capable of hybridizing thereto, and a portion non-complementary to the sequence of interest.
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21. A forward (upstream-, 5′
- -) primer for use in a method of qualitative and quantitative detecting a short nucleic acid sequence of interest, preferably a DNA oligonucleotide or a modified DNA oligonucleotide, comprising a portion complementary to the extension of the capture probe and so capable of hybridizing thereto.
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23. A reverse (downstream-, 3′
- -) primer for use in a method of qualitative and quantitative detecting a short nucleic acid sequence of interest, preferably a DNA oligonucleotide or a modified DNA oligonucleotide, comprising a portion complementary to the extension of the sequence of interest and so capable of hybridizing thereto.
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25. A DNA probe for use in a method of qualitative and quantitative detecting a short nucleic acid sequence of interest, preferably a DNA oligonucleotide or a modified DNA oligonucleotide, comprising a portion complementary to part of the sequence of interest and a portion complementary to the extension of the sequence of interest and so capable of hybridizing thereto.
Specification