Message abundance and allele copy number determination using IVT with single-stranded primer-promoter-selector constructs
View Patent ↗Disclosed is a single stranded primer-promoter-selector construct comprising (in 3′ to 5′ orientation) a primer subsequence annealing to the target, a T7 or other promoter subsequence (the template strand), and a selector subsequence. The primer can be extended by template mediated elongation, including reverse transcription, or ligation to another oligonucleotide. The promoter sequence is oriented to direct the in-vitro transcription (IVT) opposite to that of primer extension, where the selector subsequence serves as a template for IVT. The selector is associated with the target subsequence of interest and it, and the amplified product are unique subsequences, dissimilar to other sequence present in the sample. The construct's is useful for determination of the presence and relative abundance of designated subsequences in the sample, multiplex gene expression analysis, multiplex allele counting, determination of polymorphic/mutation site, and loss of heterozygosity.
1. A primer-promoter-selector nucleic acid construct for detecting the presence or the relative quantity of a target subsequence in a sample, comprising three subsequences including:
a target specific primer subsequence complementary to said target subsequence, having its 5′ end adjacent to the 3′ end of a promoter subsequence, said promoter subsequence oriented to direct transcription of a selector subsequence, said selector sequence having its 3′ end adjacent to the 5′ end of the promoter subsequence, wherein the selector subsequence and its complementary sequence do not anneal any subsequences present in the sample and wherein said selector subsequence serves as a template for single stranded in vitro transcription.
2. The construct of claim 1 wherein the promoter is a T7 or T3 or SP6 promoter subsequence.
3. The construct of claim 1 including an identifiable label.
4. A kit comprising a plurality of different types of primer-promoter-selector nucleic acid constructs each for detecting the presence or relative quantity of a different target subsequence in a sample, each comprising three subsequences including:
a target specific primer subsequence complementary to said target subsequence, having its 5′ end adjacent to the 3′ end of a promoter subsequence, said promoter subsequence oriented to direct transcription of a selector subsequence, said selector sequence having its 3′ end adjacent to the 5′ end of the promoter subsequence, wherein the selector subsequence and its complementary sequence do not anneal to any subsequences present in the sample and wherein said selector subsequence serves as a template for single stranded in vitro transcription; and
a set of oligonucleotides, wherein oligonucleotides with different sequences are displayed on differently-encoded microparticles, and wherein the oligonucleotides are capable of annealing to the transcription product of the selector subsequence.
5. The kit of claim 4 wherein the different types of constructs differ in their respective primer subsequence and selector subsequences.
6. The kit of claim 5 wherein each type of construct has different primer subsequence and the same selector subsequences.
7. The kit of claim 4 further including labeled NTPs capable of being incorporated into the transcription product.
8. The kit of claim 7 wherein the NTPs are optically labeled.
9. The kit of claim 4 wherein the promoter is a T7 or T3 or SP6 promoter subsequence template strand.
10. The kit of claim 9 further including a nontemplate T7, T3 or SP6 promoter subsequence capable of forming a duplex with said T7, T3 or SP6 promoter subsequence.
11. The kit of claim 4 further including a set of differently-encoded microparticles adapted for attaching different types of oligonucleotides thereto which are complementary to the transcription products.
12. The kit of claim 4 further including a set of labeled ddNTPs or dNTPs designed for incorporation into an extension product when the primer subsequence is extended.
13. The kit of claim 4 further including hapten labeled ddNTPs or dNTPs.
14. The kit of claim 13 further including magnetic particles.
15. The kit of claim 4 further including an oligonucleotide capable of annealing to a subsequence in the sample which is located to the 3′ side of a primer's targeted subsequence or to the primer.
16. The kit of claim 15 wherein the oligonucleotide is labeled.
17. The kit of claim 15 wherein the oligonucleotide includes the promoter subsequence.