Signal amplification with lollipop probes
First Claim
1. A method of amplifying nucleic acid sequences, the method comprising(a) mixing one or more different lollipop oligomers with one or more target samples each comprising one or more target sequences, and incubating under conditions that promote hybridization between the oligomers and the target sequences, wherein the lollipop oligomers each comprise a branched oligomer comprising a tail portion, wherein the tail portion comprises a rolling circle replication primer, wherein the rolling circle replication primer comprises a complementary portion that is complementary to a primer complement portion of an amplification target circle, (b) prior to, simultaneous with, or following step (a), mixing one or more amplification target circles with the oligomers, and incubating under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer portions of the oligomers, and (c) mixing DNA polymerase with the oligomers and amplification target circles, and incubating under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA.
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Accused Products
Abstract
Disclosed is a method and compositions for the sensitive detection of the amount and location of specific nucleic acid sequences. The method makes use of a branched oligomer, referred to as a lollipop oligomer, that has a tail portion, a right arm portion, and a left arm portion. These three components are joined at a common junction making a three-tailed structure. The two arms each end with sequences complementary to adjacent sequences in a target sequence. This allows the right and left arms to be ligated together when the oligomer is hybridized to the target sequence, thus topologically linking the oligomer to the target sequence. The tail portion can then be detected at the location of the target sequence.
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Citations
30 Claims
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1. A method of amplifying nucleic acid sequences, the method comprising
(a) mixing one or more different lollipop oligomers with one or more target samples each comprising one or more target sequences, and incubating under conditions that promote hybridization between the oligomers and the target sequences, wherein the lollipop oligomers each comprise a branched oligomer comprising a tail portion, wherein the tail portion comprises a rolling circle replication primer, wherein the rolling circle replication primer comprises a complementary portion that is complementary to a primer complement portion of an amplification target circle, (b) prior to, simultaneous with, or following step (a), mixing one or more amplification target circles with the oligomers, and incubating under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer portions of the oligomers, and (c) mixing DNA polymerase with the oligomers and amplification target circles, and incubating under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA.
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27. A method of detecting target nucleic acid sequences, the method comprising
(a) mixing one or more different lollipop oligomers with one or more target samples each comprising one or more target sequences, and incubating under conditions that promote hybridization between the oligomers and the target sequences, wherein the lollipop oligomers each comprise a branched oligomer comprising a tail portion, a right arm portion, and a left arm portion, wherein the right arm portion and the left arm portion are coupled together, wherein the tail portion is coupled to the oligomer at the junction of the right arm portion and the left arm portion, wherein the tail portion comprises an address tag, wherein the right arm portion of at least one of the oligomers comprises a right target probe portion and a right backbone portion, wherein the left arm portion comprises a left target probe portion and a left backbone portion, wherein the right target probe portion is at the end of the right arm portion, wherein the left target probe portion is at the end of the left arm portion, wherein the target probe portions are complementary to at least one of the target sequences, wherein the target sequence comprises a 5′ - region and a 3′
region, wherein the left target probe portion and the right target probe portion of the oligomer are each complementary to the 3′
region and the 5′
region, respectively, of the target sequence,(b) detecting the address tags. - View Dependent Claims (28)
- region and a 3′
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29. A method of amplifying nucleic acid sequences, the method comprising
replicating one or more amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein the amplification target circles are mixed with one or more different lollipop oligomers, wherein the lollipop oligomers each comprise a branched oligomer comprising a tail portion, wherein the tail portion comprises a rolling circle replication primer, wherein the rolling circle replication primer comprises a complementary portion that is complementary to a primer complement portion of at least one of the amplification target circles, wherein the rolling circle replication primer primes replication of the amplification target circle, wherein at least one of the lollipop oligomers is hybridized to a target sequence in a target sample.
Specification