Mobility cassettes

• US 20050239089A1
• Filed: 06/04/2004
• Published: 10/27/2005
• Est. Priority Date: 06/06/2003
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Johnson, Martin D., Hunkapiller, Michael W.
• Application Number:
  US10/861,314
• Publication Number:
  US 20050239089A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP

Abstract

Mobility cassettes are provided. Methods of using mobility cassettes for the detection of nucleic acids are provided.

Claims

1. A method of detecting at least one analyte polynucleotide comprising a tag sequence, the method comprising:

(a) forming a ligation reaction composition comprising the at least one analyte polynucleotide and at least one mobility cassette, wherein the at least one mobility cassette comprises;

a first nucleic acid strand comprising a mobility modifier; and

a second nucleic acid strand comprising a tag complement sequence that is complementary to the tag sequence of the analyte polynucleotide, wherein a portion of the second nucleic acid strand is hybridized to the first nucleic acid strand and wherein at least a portion of the tag complement sequence is not hybridized to the first nucleic acid strand, and

wherein the first nucleic acid strand is suitable for ligation to the analyte polynucleotide when the first nucleic acid strand and the analyte polynucleotide are hybridized adjacent to one another on the second nucleic acid strand;

(b) subjecting the ligation reaction composition to at least one cycle of ligation, wherein the first nucleic acid strand and the analyte polynucleotide are ligated to form a mobility modifier ligation product comprising the first nucleic acid strand and the analyte polynucleotide; and

(c) detecting the at least one analyte polynucleotide by analyzing the mobility modifier ligation product using a mobility-dependent analysis technique.

2. A method of detecting at least one primer extension product comprising:

(a) forming an extension reaction composition comprising a polymerase, at least one target nucleic acid sequence, and a primer for each target nucleic acid sequence, the primer comprising a target-specific portion and a tag sequence;

(b) incubating the extension reaction composition under appropriate conditions to generate a primer extension product for each target nucleic acid, the primer extension product comprising the tag sequence;

(c) forming a ligation reaction composition comprising the primer extension product for each target nucleic acid and a mobility cassette for each target nucleic acid, wherein the mobility cassette comprises;

a first nucleic acid strand comprising a mobility modifier; and

a second nucleic acid strand comprising a complementary tag sequence that is complementary to the tag sequence of the primer extension product, wherein a portion of the second nucleic acid strand is hybridized to the first nucleic acid strand and wherein at least a portion of the complementary tag sequence is not hybridized to the first nucleic acid strand, and

wherein the first nucleic acid strand is suitable for ligation to the primer extension product when the first nucleic acid strand and the primer extension product are hybridized adjacent to one another on the second nucleic acid strand;

(d) subjecting the ligation reaction composition to at least one cycle of ligation, wherein the first nucleic acid strand and the primer extension product for each target nucleic acid that are adjacently hybridized on the second nucleic acid strand for each target nucleic acid are ligated to form a mobility modifier ligation product comprising the first nucleic acid strand and the primer extension product; and

(e) detecting the primer extension product for each target nucleic acid sequence by analyzing the mobility modifier ligation product or a portion of the mobility modifier ligation product for each primer extension product using a mobility-dependent analysis technique.

3. A method of detecting at least one target nucleic acid sequence in a sample comprising:

(a) forming a first ligation reaction composition comprising the sample and a probe set for each target nucleic acid sequence, the probe set comprising (i) at least one first probe, comprising a first target-specific portion, and (ii) at least one second probe, comprising a second target-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence, and wherein at least one probe in each probe set further comprises at least one tag sequence;

(b) subjecting the first ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to form a first ligation product comprising the first and second target-specific portions and the tag sequence;

(c) forming a second ligation reaction composition comprising the first ligation product and a mobility cassette, wherein the mobility cassette comprises;

a first nucleic acid strand comprising a mobility modifier; and

a second nucleic acid strand comprising a complementary tag sequence that is complementary to the tag sequence of the first ligation product, wherein a portion of the second nucleic acid strand is hybridized to the first nucleic acid strand and wherein at least a portion of the complementary tag sequence is not hybridized to the first nucleic acid strand, and

wherein the first nucleic acid strand is suitable for ligation to the first ligation product when the first nucleic acid strand and the first ligation product are hybridized adjacent to one another on the second nucleic acid strand;

(d) subjecting the second ligation reaction composition to at least one cycle of ligation, wherein the first nucleic acid strand and the first ligation product that are adjacently hybridized on the second nucleic acid strand are ligated to form a mobility modifier ligation product comprising the first nucleic acid strand and the first ligation product; and

(e) detecting the at least one target nucleic acid sequence by analyzing the mobility modifier ligation product or a portion of the mobility modifier ligation product using a mobility-dependent analysis technique.

4. A method for detecting at least one target nucleic acid sequence in a sample comprising:

(a) forming a first ligation reaction composition comprising;

the sample and a probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a first target-specific portion, a tag sequence, and a 5′

primer-specific portion, wherein the 5′

primer-specific portion comprises a sequence, and wherein the tag sequence is located between the 5′

primer-specific portion and the first target-specific portion and (b) at least one second probe, comprising a second target-specific portion and a 3′

primer-specific portion, wherein the 3′

primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence;

(b) subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to form a first ligation product comprising the first and second target-specific portions, the tag sequence, and the 3′ and

5′

primer-specific portions;

(c) forming a first amplification reaction comprising a DNA polymerase, the first ligation product, and at least one primer set comprising at least one first primer comprising the sequence of the 5′

primer-specific portion, and at least one second primer comprising a sequence complementary to the sequence of the 3′

primer-specific portion;

(d) subjecting the first amplification reaction composition to at least one cycle of amplification to generate an amplification product;

(e) forming a second ligation reaction composition comprising the amplification product and a mobility cassette, wherein the mobility cassette comprises;

a first nucleic acid strand comprising a mobility modifier; and

a second nucleic acid strand comprising a complementary tag sequence that is complementary to the 5′

primer-specific portion and the tag sequence of the amplification product, wherein a portion of the second nucleic acid strand is hybridized to the first nucleic acid strand and wherein at least a portion of the complementary tag sequence is not hybridized to the first nucleic acid strand, and

wherein the first nucleic acid strand is suitable for ligation to the amplification product when the first nucleic acid strand and the amplification product are hybridized adjacent to one another on the second nucleic acid strand;

(f) subjecting the second ligation reaction composition to at least one cycle of ligation, wherein the first nucleic acid strand and the amplification product that are adjacently hybridized on the second nucleic acid strand are ligated to form a mobility modifier ligation product comprising the first nucleic acid strand and the amplification product; and

(g) detecting the at least one target nucleic acid sequence by analyzing the mobility modifier ligation product or a portion of the mobility modifier ligation product using a mobility-dependent analysis technique.

5. A method for detecting at least one target nucleic acid sequence in a sample comprising:

(a) forming a first ligation reaction composition comprising;

the sample and a probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a first target-specific portion and a 5′

primer-specific portion, wherein the 5′

primer-specific portion comprises a sequence, and (b) at least one second probe, comprising a second target-specific portion, a tag sequence, and a 3′

primer-specific portion, wherein the 3′

primer-specific portion comprises a sequence, and wherein the tag sequence is located between the 3′

primer-specific portion and the second target-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence;

(b) subjecting the first ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to form a first ligation product comprising the first and second target-specific portions, the tag sequence, and the 3′ and

5′

primer-specific portions;

(c) forming a first amplification reaction composition comprising a DNA polymerase, the first ligation product, and at least one primer set comprising at least one first primer comprising the sequence of the 5′

primer specific portion, and at least one second primer comprising a sequence complementary to the sequence of the 3′

primer-specific portion;

(d) subjecting the first amplification reaction composition to at least one cycle of amplification to generate an amplification product;

(e) forming a second ligation reaction composition comprising the amplification product and a mobility cassette, wherein the mobility cassette comprises;

a first nucleic acid strand comprising a mobility modifier; and

a second nucleic acid strand comprising a complementary tag sequence that is complementary to the 3′

primer-specific portion and the tag sequence of the amplification product, wherein a portion of the second nucleic acid strand is hybridized to the first nucleic acid strand and wherein at least a portion of the complementary tag sequence is not hybridized to the first nucleic acid strand, and

wherein the first nucleic acid strand is suitable for ligation to the amplification product when the first nucleic acid strand and the amplification product are hybridized adjacent to one another on the second nucleic acid strand;

(f) subjecting the second ligation reaction composition to at least one cycle of ligation, wherein the first nucleic acid strand and the amplification product that are adjacently hybridized on the second nucleic acid strand are ligated to form a mobility modifier ligation product comprising the first nucleic acid strand and the amplification product; and

(g) detecting the at least one target nucleic acid sequence by analyzing the mobility modifier ligation product or a portion of the mobility modifier ligation product using a mobility-dependent analysis technique.

6. A mobility cassette comprising:

a first nucleic acid strand comprising a mobility modifier; and

a second nucleic acid strand comprising a complementary tag sequence that is complementary to a tag sequence of a target product, wherein a portion of the second nucleic acid strand is hybridized to the first nucleic acid strand and wherein at least a portion of the complementary tag sequence is not hybridized to the first nucleic acid strand, and

wherein the first nucleic acid strand is suitable for ligation to the target product when the first nucleic acid strand and the target product are hybridized adjacent to one another on the second nucleic acid strand.

7. The mobility cassette of claim 6, wherein the mobility modifier comprises a polyethylene oxide molecule.

8. The mobility cassette of claim 6, wherein the mobility modifier is a polynucleotide.

9. A kit for detecting at least one target nucleic acid sequence comprising:

(a) at least one of (i) a probe comprising a tag sequence and a target-specific portion that can hybridize to a target nucleic acid sequence; and

(ii) a primer comprising a tag sequence and a target-specific portion that can hybridize to a target nucleic acid sequence;

(b) a mobility cassette comprising;

a first nucleic acid strand comprising a mobility modifier; and

a second nucleic acid strand comprising a complementary tag sequence that is complementary to the tag sequence, wherein a portion of the second nucleic acid strand is hybridized to the first nucleic acid strand and wherein at least a portion of the complementary tag sequence is not hybridized to the first nucleic acid strand, and

wherein the first nucleic acid strand is suitable for ligation to the tag sequence when the first nucleic acid strand and the tag sequence are hybridized adjacent to one another on the second nucleic acid strand.