US 5,629,179 AGranted

Method and kit for making CDNA library

AssigneeNovagen, Inc.

InventorsRobert C. Mierendorf, Barbara B. Morris

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Abstract

A method for forming directionally clonable randomly primed cDNA molecules includes the step of priming first strand synthesis using a set of first strand cDNA primers having the sequence 5'-XXNNNNNN-3' where XX is a constant dinucleotide pair across the set and NNNNNN is a random hexanucleotide, the set including primers representing all random hexanucleotides.

Claims6

Dependent claims

A method for making a cDNA library, the method comprising the steps of:

annealing a set of primers to a mixed population of template RNA strands under suitable annealing conditions, the set of primers having a sequence of 5'-XXNNNNNN-3', each X end each N being a deoxyribonucleotide selected from the group consisting of A, G, C and T, every primer comprising an identical 5' terminal dinucleotide portion XX selected so as to define a 3' portion of a first endonuclease recognition sequence cleavable by a first endonuclease, each primer further comprising a single random hexanucleotide portion NNNNNN, the set of primers comprising every random hexanucleotide;
reverse transcribing the primed template RNA strands to form a mixed population of cDNA first strands hybridized to the template RNA strands, each cDNA first strand having at its 5' terminus the 5'-XXNNNNNN-3' sequence of a single primer;
adding a DNA dependent DNA polymerase and synthesizing a cDNA second strand complementary to the cDNA first strands, displacing the template RNA strands, and creating a mixed population of fully double-stranded cDNA molecules, each cDNA second strand having at its 3'-terminus a dinucleotide sequence complementary to the selected 5' terminal dinucleotide portion XX of the cDNA first strands;
ligating together under suitable ligation conditions the mixed population of cDNA molecules and a double-stranded palindromic linker that comprises an internal second endonuclease recognition sequence, different from the first recognition sequence and cleavable by a second endonuclease, and a 5' portion of the first endonuclease recognition sequence, the 5' portion comprising at least two complementary nucleotide pairs at each 3' terminus of the palindromic linker, whereupon the second recognition sequence is formed;
cleaving the products of the preceding ligation step using the first endonuclease and the second endonuclease to form a mixed population of directional cDNA fragments each having two ends, the two ends of each fragment being distinct and incompatible with each other;
ligating the population of directional cDNA fragments in a desired orientation into a vector capable of propagating a cDNA library, the vector having been prepared with termini that are ligation-compatible with the ends of the directional cDNA fragments;
introducing the vector-ligated fragments into a bacterial host cell that supports transcription of introduced sequences.

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Dependencies are inferred from the claim text.

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Abstract

A method for making a cDNA library, the method comprising the steps of:

annealing a set of primers to a mixed population of template RNA strands under suitable annealing conditions, the set of primers having a sequence of 5'-XXNNNNNN-3', each X end each N being a deoxyribonucleotide selected from the group consisting of A, G, C and T, every primer comprising an identical 5' terminal dinucleotide portion XX selected so as to define a 3' portion of a first endonuclease recognition sequence cleavable by a first endonuclease, each primer further comprising a single random hexanucleotide portion NNNNNN, the set of primers comprising every random hexanucleotide;
reverse transcribing the primed template RNA strands to form a mixed population of cDNA first strands hybridized to the template RNA strands, each cDNA first strand having at its 5' terminus the 5'-XXNNNNNN-3' sequence of a single primer;
adding a DNA dependent DNA polymerase and synthesizing a cDNA second strand complementary to the cDNA first strands, displacing the template RNA strands, and creating a mixed population of fully double-stranded cDNA molecules, each cDNA second strand having at its 3'-terminus a dinucleotide sequence complementary to the selected 5' terminal dinucleotide portion XX of the cDNA first strands;
ligating together under suitable ligation conditions the mixed population of cDNA molecules and a double-stranded palindromic linker that comprises an internal second endonuclease recognition sequence, different from the first recognition sequence and cleavable by a second endonuclease, and a 5' portion of the first endonuclease recognition sequence, the 5' portion comprising at least two complementary nucleotide pairs at each 3' terminus of the palindromic linker, whereupon the second recognition sequence is formed;
cleaving the products of the preceding ligation step using the first endonuclease and the second endonuclease to form a mixed population of directional cDNA fragments each having two ends, the two ends of each fragment being distinct and incompatible with each other;
ligating the population of directional cDNA fragments in a desired orientation into a vector capable of propagating a cDNA library, the vector having been prepared with termini that are ligation-compatible with the ends of the directional cDNA fragments;
introducing the vector-ligated fragments into a bacterial host cell that supports transcription of introduced sequences.

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