Method for making full-length coding sequence cDNA libraries
First Claim
1. A method for making a full-length coding sequence cDNA library, comprising:
- (a) forming RNA-DNA hybrids by reverse transcription starting from primers using mRNAs as templates;
(b) binding a tag molecule to a diol structure present in the 5′
cap site of a mRNA forming a RNA-DNA hybrid; and
(c) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length mRNA from the RNA-DNA hybrids formed above by binding the tag molecule, wherein said DNA corresponding to a full-length mRNA are first cDNA strands;
wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs.
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Abstract
The present invention relates to a method for making cDNA libraries wherein the cDNA inserts comprise the full-length of the coding sequences but having lengths less than the full-length of the mRNA. The method comprises binding a tag molecule to a diol structure present in the 5′ Cap sites of mRNAs, forming RNA-DNA hybrids by reverse transcription to synthesize the first cDNA strand, separating RNA-DNA hybrids carrying a DNA corresponding to a full-length of mRNAs from RNA-DNA hybrids formed above by using a function of the tag molecule, and synthesizing the second cDNA strand by self-priming the first cDNA strand. The resulting cDNA libraries do not contain the full-length of the mRNAs but do contain the full-length of the coding sequences of the mRNAs.
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Citations
33 Claims
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1. A method for making a full-length coding sequence cDNA library, comprising:
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(a) forming RNA-DNA hybrids by reverse transcription starting from primers using mRNAs as templates;
(b) binding a tag molecule to a diol structure present in the 5′
cap site of a mRNA forming a RNA-DNA hybrid; and
(c) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length mRNA from the RNA-DNA hybrids formed above by binding the tag molecule, wherein said DNA corresponding to a full-length mRNA are first cDNA strands;
wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method for constructing a full-length coding sequence cDNA library, comprising:
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(a) binding a tag molecule to a diol structure present in 5′
cap sites of mRNAs by oxidizing the 5′
cap site diol to form a dialdehyde and reacting the resulting dialdehyde with a tag molecule having a group reactive with the dialdehyde;
(b) forming RNA-DNA hybrids by reverse transcription using primers and the mRNAs binding the tag molecule as templates; and
(c) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length of mRNA from the RNA-DNA hybrids formed above by using a function of the tag molecule, wherein said DNA corresponding to a full-length mRNA are first cDNA strands;
wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A method for making a full-length cDNA library, comprising:
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(a) binding a biotin molecule to a diol structure present in 5′
cap sites of mRNAs by oxidizing the 5′
cap site diol to form a dialdehyde and reacting the resulting dialdehyde with a biotin molecule having a group reactive with the dialdehyde;
(b) forming RNA-DNA hybrids by reverse transcription using primers and the mRNAs bound to biotin molecules as templates;
(c) digesting the RNA-DNA hybrids with an RNase capable of cleaving single strand RNA to cleave the single strand RNA parts of the hybrids carrying a DNA not corresponding to a full-length mRNA to remove biotin molecules from the hybrids, wherein said DNA not corresponding to a full-length mRNA is a first cDNA strand; and
(d) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length mRNA and binding the biotin molecules by (1) allowing them to react with avidin fixed on a solid support or (2) affinity chromatography to a solid support;
wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs. - View Dependent Claims (25, 26, 27, 28)
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29. A method for making a fall-length cDNA library, comprising:
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(a) binding an avidin molecule to a diol structure present in 5′
cap sites of mRNAs by oxidizing the 5′
cap site diol to form a dialdehyde and reacting the resulting dialdehyde with an avidin molecule having a group reactive with the dialdehyde;
(b) forming RNA-DNA hybrids by reverse transcription using primers and the mRNAs bound to avidin molecules as templates;
(c) digesting the RNA-DNA hybrids with an RNase capable of cleaving single strand RNA to cleave the single strand RNA parts of the hybrids carrying a DNA not corresponding to full-length mRNAs to remove avidin molecules from the hybrids, wherein said DNA not corresponding to a full-length mRNA is a first cDNA strand; and
(d) separating RNA-DNA hybrids carrying a DNA corresponding to a full-length mRNA and binding avidin molecules by (1) allowing them to react with biotin fixed on a solid support or (2) affinity chromatography to a solid support;
wherein said full-length coding sequence cDNA library is a library of cDNAs comprising the full-length of the coding sequences and having lengths less than the full-length of mRNAs. - View Dependent Claims (30, 31, 32, 33)
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Specification