Back to results
US 2009/0023151 A1Published application
Method For The Labeling And Detection Of Small Polynucleotides
OwnerElliott P. Dawson, Kristie E. Wombleinventors
Life of this patent
Estimating the term…
Abstract
Methods and kits for use in isolating, labeling or detecting a small polynucleotide of interest from a sample. The method entails hybridizing the small polynucleotide to a capture probe, lengthening the small polynucleotide by primer extension and/or ligation, and degrading the capture probe to provide a single stranded extension product. The kits include the capture probe and additional reagents for the extension, ligation and/or degradation reactions.
Claims32
A method of labeling and/or detecting a small polynucleotide of interest, comprising:
- (a) providing one or more than one capture probe, wherein the capture probe is a polynucleotide comprising;
- (1) a small polynucleotide binding segment having a small polynucleotide binding segment sequence, the small polynucleotide binding segment having a 3′ end and a 5′ end;
- and
- (2) a template segment having an template segment sequence, the template segment having a 3′ end and a 5′ end;
- where the small polynucleotide binding segment is substantially complementary to, and capable of hybridizing to, one or more than one small polynucleotide of interest by Watson-Crick base pairing, where the small polynucleotide of interest is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide and a combination thereof;
- and
- where the 3′ end of template segment is connected to the 5′ end of the small polynucleotide binding segment;
- (b) providing a sample comprising a small polynucleotide of interest, where the small polynucleotide of interest is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide and a combination thereof;
- (c) combining the capture probe and the sample;
- (d) allowing the small polynucleotide of interest to hybridize with the small polynucleotide binding segment of the capture probe to form a small polynucleotide/capture probe complex;
- (e) combining the small polynucleotide/capture probe complex with a polynucleotide polymerase and a set of nucleoside triphosphates;
- (f) extending the hybridized small polynucleotide of interest to form an extension product, the extension product comprising the small polynucleotide of interest connected at the 3′ end to an extended segment, the extended segment sequence comprising a sequence complementary to the template segment sequence of the capture probe, where the extension product is hybridized to the capture probe to form an extension product/capture probe complex;
- and
- (g) degrading the capture probe to obtain a single stranded extension product.
11 more elements
A method for labeling and/or detecting a small polynucleotide of interest in a sample comprising:
- (a) providing one or more than one capture probe, the capture probe comprising
- (1) a spacer segment having a spacer segment sequence, the spacer segment having a 3′ end and a 5′ end.
- (2) a small polynucleotide binding segment having a small polynucleotide binding segment sequence, the small polynucleotide binding segment having a 3′ end and a 5′ end;
- and
- (3) a template segment having an template segment sequence, the template segment having a 3′ end and a 5′ end;
- where the 5′ end of the spacer segment is connected to the 3′ end of the small polynucleotide binding segment;
- where the small polynucleotide binding segment is substantially complementary to, and capable of hybridizing to, one or more than one small polynucleotide of interest by Watson-Crick base pairing, where the small polynucleotide of interest is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide and a combination thereof;
- and
- where the 3′ end of template segment is connected to the 5′ end of the small polynucleotide binding segment.
- (b) providing a sample comprising a small polynucleotide of interest, where the small polynucleotide of interest is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide and a combination thereof;
- (c) combining the capture probe and the sample;
- (d) allowing the small polynucleotide of interest to hybridize with the small polynucleotide binding segment of the capture probe to form a small polynucleotide/capture probe complex;
- (e) combining the small polynucleotide/capture probe complex with a polynucleotide polymerase and a set of nucleoside triphosphates;
- (f) extending the hybridized small polynucleotide of interest to form an extension product, the extension product comprising the small polynucleotide of interest connected at the 3′ end to an extended segment, the extended sequence comprising a sequence complementary to the template segment of the capture probe, where the extension product is hybridized to the capture probe to form a extension product/capture probe complex;
- and
- (g) providing a ligase and a linker segment, the linker segment comprising a polynucleotide having 3′ end and a 5′ end, the linker segment having a linker segment sequence, wherein the linker segment sequence is substantially complementary to, and capable of hybridizing to, the spacer segment sequence by Watson-Crick base pairing;
- (h) allowing the linker segment to hybridize to the spacer segment;
- (i) ligating the 3′ end of the linker segment to the 5′ end of the small polynucleotide of interest to form a ligated extension product substantially complementary to, and capable of hybridizing to, the capture probe sequence;
- and
- (j) degrading the capture probe by nuclease treatment to obtain a single stranded ligated extension product.
17 more elements
A kit for the isolation, labeling and/or detection of small polynucleotides, the kit comprising:
- (a) one or more capture probes comprising
- (1) a small polynucleotide binding segment having a small polynucleotide binding segment sequence, the small polynucleotide binding segment having a 3′ end and a 5′ end;
- and
- (2) a template segment having an template segment sequence, the template segment having a 3′ end and a 5′ end;
- where the small polynucleotide binding segment is substantially complementary to, and capable of hybridizing to, one or more than one small polynucleotide of interest by Watson-Crick base pairing, where the small polynucleotide of interest is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide and a combination thereof;
- and
- where the 3′ end of template segment is connected to the 5′ end of the small polynucleotide binding segment;
- (3) an optional spacer segment having a spacer segment sequence, the spacer segment having a 3′ end and a 5′ end, where the 5′ end of the spacer segment is connected to the 3′ end of the small polynucleotide binding segment;
- and
- (4) an optional solid phase binding segment containing biotin;
- and
- (b) one or more than one substance selected from the group consisting of:
- (1) a deoxynucleoside triphosphate mix comprising the alpha thio triphosphate forms of dATP, dCTP, and dGTP and a labeled form of dUTP;
- (2) a polymerase capable of extending the 3′ end of a polynucleotide of interest hybridized to the capture probe by using the extension template segment of the capture probe as a template for an extension reaction;
- (3) an oligonucleotide linker that is substantially complementary to and capable of hybridizing to the spacer segment of the capture probe;
- (4) a ligase enzyme;
- (5) DNase I;
- (6) alkaline phosphatase;
- (7) suitable buffers compatible with one or more of the polymerase, ligase, DNase I and alkaline phosphatase reactions;
- (8) spin columns for separation of unincorporated nucleoside triphosphates from labeled extension products;
- and
- (9) streptavidin coated paramagnetic beads.
19 more elements
Dependencies are inferred from the claim text.