Constant length signatures for parallel sequencing of polynucleotides
OwnerAchim Fischer, Holger Hiemisch +5inventors
InventorsAchim Fischer, Holger Hiemisch, Steven Williams, Sydney Brenner, Roger Walker, Eric Vermaas, Rongdian Fu
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Abstract
Methods are disclosed for producing solid phase cloned libraries of oligonucleotide tag-DNA signature sequence constructs, in which the DNA signature components are all of the same length. Such libraries are especially useful for large-scale parallel sequencing of DNA signature sequences prepared from a source population, such as mRNA or genomic DNA.
Claims20
A method of preparing a library of same-length signature sequences from a source nucleic acid population, the method comprising:
- (a) attaching to an end of each of a population of polynucleotides, a first adapter containing a recognition site for a first restriction endonuclease, such that the cleavage site for the endonuclease is within the polynucleotide,
- wherein the end to which the adapter is attached is the same for each polynucleotide of the population, and is selected from:
- (i) the 5′ end of a full length cDNA transcript, (ii) the 3′ end of a cDNA transcript from which the polyA/polyT tract has been removed, (iii) the 5′ end of a cDNA fragment produced by cleavage of cDNA with a restriction endonuclease, and (iv) the 3′ end of a cDNA fragment produced by cleavage of cDNA with a restriction endonuclease;
- (b) cleaving the polynucleotides with the first restriction endonuclease, to produce a population of adapter-signature conjugates, each containing a same-length signature sequence of source nucleic acid, at least six basepairs in length, having a newly cleaved end;
- and
- (c) ligating to the newly cleaved ends of said signatures, a second adapter containing a recognition and cleavage site for a second restriction endonuclease, to produce a library of adapter-signature-adapter constructs.
A kit for use in parallel sequencing of signature sequences from a sample nucleic acid population, comprising:
- an oligonucleotide tag vector library, each tag vector in the library comprising:
- a left primer binding site, a left restriction cleavage site, an oligonucleotide tag selected from a minimally cross-hybridizing set of oligonucleotides, a cloning site for insertion of a signature fragment, a right restriction cleavage site, and a right primer binding site;
- a corresponding set of oligonucleotide tag complements, each bound to a spatially distinct solid phase support;
- and
- a population of same-length signature fragments having right and left cloning sites, each containing a same-length signature sequence from the sample nucleic acid population.
A kit for use in preparing same-length signature sequences from a sample nucleic acid population, comprising:
- a first restriction endonuclease which is a type IIs restriction endonuclease having a cleavage site at least 10 nucleotides from its recognition site;
- a first double stranded adapter containing a recognition site for the second restriction endonuclease;
- a second adapter having a single stranded overhang effective to hybridize with an overhang produced by cleavage of a double stranded cDNA or cDNA fragment by said second restriction endonuclease, and containing a recognition and cleavage site for a second restriction endonuclease;
- and
- the second restriction endonuclease.
Dependencies are inferred from the claim text.