US 2003/0165911 A1 · Gene expression analysis using nicking agents

A method for determining the presence or absence of a target cDNA molecule in a cDNA population or for determining the presence or absence of a target mRNA molecule in a biological sample, comprising:

(A) forming a mixture comprising:
(i) the cDNA molecules of the cDNA population or the RNA molecules of the biological sample,
(ii) a template nucleic acid molecule that
(a) comprises one strand of a nicking agent recognition sequence, and
(b) is at least substantially complementary to the target cDNA if the target cDNA is single-stranded,
is at least substantially complementary to one strand of the target cDNA if the target cDNA is double-stranded, or
is at least substantially complementary to the target mRNA,
(iii) a nicking agent that recognizes the recognition sequence,
(iv) a DNA polymerase, and
(v) one or more deoxynucleoside triphosphate(s);
(B) maintaining the mixture at conditions that amplify a single-stranded nucleic acid molecule using a portion of the target cDNA, a portion of the target mRNA, or a portion of the template nucleic acid molecule as a template, if the target cDNA is present in the cDNA population or if the target mRNA is present in the biological sample;
and
(C) detecting the presence or absence of the single-stranded nucleic acid molecule to determine the presence or absence of the target cDNA molecule in the cDNA population, or to determine the presence or absence of the target mRNA in the biological sample.

10 more elements

A method for determining the presence or absence of a target cDNA molecule in a cDNA population, or for determining the presence or absence of a target mRNA in a biological sample, comprising

(A) forming a mixture comprising:
(i) the cDNA molecules in the cDNA population, or the RNA molecules of the biological sample;
(ii) a partially double-stranded nucleic acid probe that comprise:
(a) a sequence of a sense strand of a nicking agent recognition sequence, a sequence of an antisense strand of the nicking agent recognition sequence, or both;
and
(b) a 5′ overhang in the strand that either the strand itself or an extension product thereof contains a nicking site nickable by a nicking agent that recognizes the nicking agent recognition sequence, or
a 3′ overhang in the strand that either the strand nor an extension product thereof contains the nicking site,
wherein each overhang comprises a nucleic acid sequence at least substantially complementary to the target cDNA if the target cDNA is single-stranded, to one strand of the target cDNA if the target cDNA is double-stranded, or to the target mRNA;
(B) separating the probe molecules that have hybridized to the cDNA or mRNA molecules from those that have not;
(C) performing an amplification reaction in the presence of the hybridized probe molecules and a nicking agent that recognizes the nicking agent recognition sequence to amplify a single-stranded nucleic acid fragment using one strand of the partially double-stranded nucleic acid probe as a template, if the target cDNA is present in the cDNA population or if the target mRNA is present in the biological sample;
and
(D) detecting the presence or absence of the single-stranded nucleic acid fragment of step (C) to determine the presence or absence of the target cDNA in the cDNA population, or to determine the presence or absence of the target mRNA in the biological sample.

9 more elements

A method for determining the presence or absence of a target cDNA molecule in a cDNA population, comprising

(A) forming a mixture of a first oligonucleotide primer (ODNP), a second ODNP, and the cDNA molecules in the cDNA population, wherein
(i) if the target cDNA is a double-stranded nucleic acid having a first strand and a second strand,
the first ODNP comprises a nucleotide sequence of a sense strand of a nicking endonuclease recognition sequence and a nucleotide sequence at least substantially complementary to a first portion of the first strand of the target nucleic acid, and
the second ODNP comprises a nucleotide sequence at least substantially complementary to a second portion of the second strand of the target nucleic acid and comprises a sequence of one strand of a restriction endonuclease recognition sequence, the second portion being located 3′ to the complement of the first portion in the second strand of the target nucleic acid,
or
(ii) if the target nucleic acid is a single-stranded nucleic acid,
the first ODNP comprises a nucleotide sequence of a sense strand of a nicking endonuclease recognition sequence and a nucleotide sequence at least substantially identical to a first portion of the target nucleic acid, and
the second ODNP comprises a nucleotide sequence at least substantially complementary to a second portion of the target nucleic acid and comprises a sequence of one strand of a restriction endonuclease recognition sequence, the second portion being located 5′ to the first portion in the target nucleic acid;
(B) subjecting the mixture to conditions that, if the target cDNA is present in the cDNA population,
(i) extend the first and the second ODNPs to produce an extension product comprising the first ODNP and the second ODNP;
(ii) optionally digesting the extension product of step (i) with a restriction endonuclease that recognizes the restriction endoculease recognition sequence to provide a digestion product;
(iii) amplify a single-stranded nucleic acid fragment using one strand of the extension product of step (B)(i) or the digestion product of step (B)(ii) as a template in the presence of a nicking endonuclease that recognizes the nicking endonuclease recognition sequence;
and
(C) detecting the presence or absence of the single-stranded nucleic acid fragment of step (B)(ii) to determine the presence or absence of the target cDNA in the cDNA population.

11 more elements

A method for determining the presence or absence of a target cDNA in a cDNA population, comprising

(A) forming a mixture of a first oligonucleotide primer (ODNP), a second ODNP, and the cDNA molecules of the cDNA population, wherein
(i) if the target cDNA is a double-stranded nucleic acid having a first strand and a second strand,
the first ODNP comprises a nucleotide sequence of a sense strand of a first nicking endonuclease recognition sequence (NERS) and a nucleotide sequence at least substantially complementary to a first portion of the first strand of the target cDNA, and
the second ODNP comprises a nucleotide sequence at least substantially complementary to a second portion of the second strand of the target nucleic acid and comprises a sequence of the sense strand of a second NERS, the second portion being located 3′ to the complement of the first portion in the second strand of the target cDNA,
or
(ii) if the target cDNA is a single-stranded nucleic acid,
the first ODNP comprises a nucleotide sequence of a sense strand of a first NERS and a nucleotide sequence at least substantially identical to a first portion of the target cDNA, and
the second ODNP comprises a nucleotide sequence at least substantially complementary to a second portion of the target nucleic acid and comprises a sequence of the sense strand of a second NERS, the second portion being located 5′ to the first portion in the target cDNA;
(B) subjecting the mixture to conditions that, if the target cDNA is present in the cDNA population,
(i) extend the first and the second ODNPs to produce an extension product comprising both the first and the second NERSs;
(ii) amplify a single-stranded nucleic acid fragment using one strand of the extension product of step (B)(i) as a template in the presence of one or more nicking endonucleases (NEs) that recognizes the first and the second NERSs;
and
(C) detecting the presence or absence of the single-stranded nucleic acid fragment of step (B)(ii) to determine the presence or absence of the target nucleic acid in the sample.

10 more elements

A method for determining the presence or absence of a target cDNA in a cDNA population, comprising

(A) forming a mixture of a first oligonucleotide primer (ODNP), a second ODNP, and the cDNA molecules of the cDNA population, wherein
(i) if the target cDNA is a double-stranded nucleic acid having a first strand and a second strand,
the first ODNP comprises a nucleotide sequence of a sense strand of a restriction endonuclease recognition sequence (RERS) and a nucleotide sequence at least substantially complementary to a first portion of the first strand of the target cDNA, and
the second ODNP comprises a nucleotide sequence at least substantially complementary to a second portion of the second strand of the target nucleic acid and comprises a sequence of the sense strand of a second RERS, the second portion being located 3′ to the complement of the first portion in the second strand of the target cDNA,
or
(ii) if the target cDNA is a single-stranded nucleic acid,
the first ODNP comprises a nucleotide sequence of a sense strand of a first RERS and a nucleotide sequence at least substantially identical to a first portion of the target cDNA, and
the second ODNP comprises a nucleotide sequence at least substantially complementary to a second portion of the target nucleic acid and comprises a sequence of the sense strand of a second RERS, the second portion being located 5′ to the first portion in the target cDNA;
(B) subjecting the mixture to conditions that, if the target cDNA is present in the cDNA population,
(i) extend the first and the second ODNPs to produce an extension product comprising both the first and the second RERSs;
(ii) amplify a single-stranded nucleic acid fragment using one strand of the extension product of step (B)(i) as a template in the presence of one more restriction endonucleases (REs) that recognizes the first and the second RERSs;
and
(C) detecting the presence or absence of the single-stranded nucleic acid fragment of step (B)(ii) to determine the presence or absence of the target cDNA in the cDNA population.

10 more elements

A method for determining the presence or absence of a target cDNA molecule in a cDNA population, or for determining the presence or absence of a target mRNA molecule in a biological sample, comprising:

(A) forming a mixture comprising:
(i) the cDNA molecules of the cDNA population, or the RNA molecule of the biological sample,
(ii) a first single-stranded template nucleic acid molecule (T1 ) that
(a) comprises one strand of a first nicking agent recognition sequence, and
(b) is at least substantially complementary to the target cDNA if the target cDNA is single-stranded,
is at least substantially complementary to one strand of the target cDNA if the target cDNA is double-stranded, or
is at least substantially complementary to the target mRNA,
(iii) a first nicking agent that recognizes the first nicking agent recognition sequence,
(iv) a DNA polymerase, and
(v) one or more deoxynucleoside triphosphate(s);
(B) maintaining the mixture at conditions that amplify a first single-stranded nucleic acid molecule (A1 ) using a portion of the target cDNA, a portion of the target mRNA, or a portion of the template nucleic acid molecule as a template, if the target cDNA is present in the cDNA population or if the target mRNA is present in the biological sample;
(C) providing a second single-stranded template nucleic acid molecule (T2 ) that is at least substantially complementary to A1 and comprises one strand of a second nicking agent recognition sequence;
(D) performing an amplification reaction in the presence of a second nicking agent that recognizes the second nicking agent recognition sequence to amplify a second single-stranded nucleic acid molecule (A2 ) using either A1 or T2 as a template;
and
(E) detecting the presence or absence of A2 to determine the presence or absence of the target cDNA molecule in the cDNA population or the presence or absence of the target mRNA in the biological sample.

12 more elements

A method for determining the presence or absence of a target cDNA molecule in a cDNA population, comprising:

(A) forming a mixture comprising:
(i) the cDNA molecules of the cDNA population,
(ii) a first single-stranded template nucleic acid molecule (T1 ) that
(a) comprises a sequence of the antisense strand of a first nicking agent recognition sequence, and
(b) is at least substantially complementary to the target cDNA if the target cDNA is single-stranded, or
is at least substantially complementary to one strand of the target cDNA if the target cDNA is double-stranded,
(iii) a second single-stranded template nucleic acid molecule (T2 ) that comprises, from 3′ to 55′:
(a) a sequence that is at least substantially identical to the sequence of the T1 located 5′ to the sequence of the antisense strand of the first nicking agent recognition sequence, and
(b) a sequence of the antisense strand of a second nicking agent recognition sequence,
(iv) a first nicking agent that recognizes the first nicking agent recognition sequence,
(v) a second nicking agent that recognizes the second nicking agent recognition sequence,
(vi) a DNA polymerase, and
(vii) one or more deoxynucleoside triphosphate(s);
(B) maintaining the mixture at conditions that amplify a first single-stranded nucleic acid molecule (A2 ) using the T2 as a template, if the target cDNA is present in the cDNA population;
and
(C) detecting the presence or absence of A2 to determine the presence or absence of the target cDNA molecule in the cDNA population.

13 more elements

A method for determining the presence or absence of a target cDNA molecule in a cDNA population, or for determining the presence or absence of a target mRNA molecule in a biological sample, comprising:

(A) forming a mixture comprising:
(i) the cDNA molecules of the cDNA population, or the RNA molecule of the biological sample,
(ii) a first template nucleic acid molecule (T1 ) that comprises, from 3′ to 55′:
(a) a first sequence that is at least substantially complementary to the 3′ portion of the target cDNA if the target cDNA is single-stranded, or
a first sequence that is at least substantially complementary to the 3′ portion of one strand of the target cDNA if the target cDNA is double-stranded, or
a first sequence that is at least substantially complementary to the 3′ portion of the target mRNA,
(b) a sequence of the antisense strand of a first nicking agent recognition sequence, and
(c) a second sequence,
(iii) a second template nucleic acid molecule (T2 ) comprising, from 3′ to 55′:
(a) a first sequence that is at least substantially identical to the second sequence of T1 ,
(b) a sequence of the antisense strand of a second nicking agent recognition sequence, and
(c) a second sequence,
(iv) a first nicking agent that recognizes the first nicking agent recognition sequence,
(v) a second nicking agent that recognizes the second nicking agent recognition sequence,
(vi) a DNA polymerase, and
(vii) one or more deoxynucleoside triphosphate(s);
(B) maintaining the mixture at conditions that amplify a single-stranded nucleic acid molecule (A2 ) using the second sequence of T2 as a template, if the target cDNA is present in the cDNA population;
and
(C) detecting the presence or absence of A2 to determine the presence or absence of the target cDNA molecule in the cDNA population or the presence or absence of the target mRNA in the biological sample.

16 more elements

A method for determining the presence or absence of a target cDNA molecule in a cDNA population, comprising:

(A) forming a mixture comprising:
(i) the cDNA molecules of the cDNA population;
(ii) an oligonucleotide primer that
(a) comprises a sequence of the sense strand of a double-stranded nicking agent recognition sequence recognizable by a nicking agent that nicks outside the recognition sequence, and
(b) is at least substantially complementary to a first region of the single-stranded target nucleic acid or of one strand of the double-stranded target nucleic acid;
and
(iii) a partially double-stranded nucleic acid that
(a) comprises a double-stranded type IIs restriction endonucelase recognition sequence, and
(b) a 3′ overhang that is at least substantially complementary to a second region of the single-stranded target cDNA or of the one strand of the double-stranded target cDNA located 5′ to the first region the single-stranded target cDNA or of the one strand of the double-stranded target cDNA;
under conditions that allow for hybridization between the oligonucleotide primer and the first region of the single-stranded target cDNA or of the one strand of the double-stranded nucleic acid and between the 3′ overhang of the partially double-stranded nucleic acid and the second region of the single-stranded target cDNA or of the one strand of the double-stranded nucleic acid;
(B) digesting the single-stranded target cDNA or the one strand of the double-stranded target cDNA that have hybridized to the oligonucleotide primer and to the partially double-stranded nucleic acid in the second region.
(C) performing an amplification reaction that amplify a single-stranded nucleic acid molecule using a portion of the single-stranded target cDNA or of the one strand of the double-stranded target cDNA digested in step (B) as a template in the presence of the nicking agent, and
(D) detecting the presence or absence of the single-stranded nucleic acid molecule of step (C) to determine the presence or absence of the target cDNA molecule in the cDNA population.

10 more elements

Gene expression analysis using nicking agents

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Abstract

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Gene expression analysis using nicking agents

Life of this patent

Abstract

Claims181