Solid-phase reactions
View Patent ↗The present invention relates to systems, compositions, and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for attaching nucleic acids to solid supports and modifying nucleic acids. For example, in some embodiments, the 5′ nuclease activity of a cleavage agent is used to cleave a cleavage structure formed on the solid support, the occurrence of the cleavage event indicating the presence of specific nucleic acid sequences.
1. A method for detecting polymorphisms in unamplified DNA, comprising:
a. providing:
i. unamplified genomic DNA, wherein said unamplified genomic DNA comprises 50 micrograms or less of genomic DNA;
ii. a microsphere;
iii. oligonucleotides and enzymes for conducting an invasive cleavage reaction, wherein said oligonucleotides comprise a probe oligonucleotide and an upstream oligonucleotide each attached to said microsphere via a spacer molecule, wherein said spacer molecule has a length of 10 hexaethylene glycol molecules and, wherein said upstream oligonucleotide is configured to hybridize to a target sequence in a manner that overlaps with said probe oligonucleotide; and
b. treating said unamplified genomic DNA with said oligonucleotides and enzymes under conditions such that said upstream oligonucleotide and said probe oligonucleotide hybridize to said unamplified genomic DNA such that said upstream oligonucleotide overlaps said probe oligonucleotide, and an invasive cleavage reaction occurs on said microsphere such that the presence or absence of a polymorphism in said unamplified genomic DNA is identified.
2. The method of claim 1 , wherein said oligonucleotides and enzymes are configured to detect a plurality of different polymorphisms in said unamplified genomic DNA.
3. The method of claim 2 , wherein said plurality of different polymorphisms comprises at least 100 different polymorphisms.
4. The method of claim 2 , wherein said plurality of different polymorphisms comprises at least 1000 different polymorphisms.
5. The method of claim 2 , wherein said plurality of different polymorphisms comprises at least 1,000,000 different polymorphisms.
6. The method of claim 1 , wherein said treating step is carried out in a single reaction vessel.
7. The method of claim 1 , wherein said oligonucleotides and enzymes are present in approximately equal concentrations.
8. The method of claim 1 , wherein said treating comprises use of flow cytometry.
9. A method for detecting polymorphisms in unamplified DNA, comprising:
a. providing:
i. unamplifled genomic DNA, wherein said unamplified genomic DNA comprises 50 micrograms or less of genomic DNA;
ii. a solid surface;
iii. oligonucleotides and enzymes for conducting an invasive cleavage reaction, wherein said oligonucleotides comprise a probe oligonucleotide and an upstream oligonucleotide each attached to said solid surface via a spacer molecule, wherein said spacer molecule has a length of 10 hexaethylene glycol molecules, and wherein said upstream oligonucleotide is configured to hybridize to a target sequence in a manner that overlaps with said probe oligonucleotide;
iv. a flow cytometer;
b. treating said unamplified genomic DNA with said oligonucleotides and enzymes under conditions such that said probe oligonucleotide and said upstream oligonucleotide hybridize to said unamplified genomic DNA such that said upstream oligonucleotide overlaps said probe oligonucleotide, and an invasive cleavage reaction, occurs on said solid surface such that a signal is generated, said signal indicating the presence or absence of said polymorphism; and
c. detecting said signal with said flow cytometer.