Methods for producing a paired tag from a nucleic acid sequence and methods of use thereof

Abstract

Methods for producing a paired tag from a nucleic acid sequence are provided in which the paired tag comprises the 5′ end tag and 3′ end tag of the nucleic acid sequence. In one embodiment, the nucleic acid sequence comprises two restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence distally to the restriction endonuclease recognition sites. In another embodiment, the nucleic acid sequence further comprises restriction endonuclease recognition sites specific for a rare cutting restriction endonuclease. Methods of using paired tags are also provided. In one embodiment, paired tags are used to characterize a nucleic acid sequence. In a particular embodiment, the nucleic acid sequence is a genome. In one embodiment, the characterization of a nucleic acid sequence is karyotyping. Alternatively, in another embodiment, the characterization of a nucleic acid sequence is mapping of the sequence. In a further embodiment, a method is provided for identifying nucleic acid sequences that encode at least two interacting proteins.

Description

RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 516,080, filed Oct. 31, 2003. [0002] The entire teachings of the above application are incorporated herein by reference.BACKGROUND OF THE INVENTION [0003] Whole genome shotgun sequencing, assembly and finishing is typically the strategy of choice for microbial and fungal genome sequencing. The cost-advantages and simplicity of the whole genome approach relative to a BAC based or hybrid sequencing strategy argue strongly for its continued development and application in future sequencing projects. However, a major problem with the BAC-based approaches is the high cost and operational burden associated with the production of 15,000-25,000 individual BAC subclone libraries, the 15-20% waste associated with re-sequencing the vector, as well as the unavoidable E. coli contamination, the need to deal with transposon and bacteriophage insertions, and the 20-50% waste in redundant sequencing...

AI Technical Summary

Benefits of technology

[0029] In a further aspect of the invention, a method for identifying nucleic acid sequences that encode at least two interacting proteins is provided, comprising the steps of a) combining (i) a first vector comprising (1) a nucleic acid sequence that encodes a first protein that interacts with a second protein, and (2) a first restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, and (ii) a second vector comprising (1) a nucleic acid sequence that encodes the second protein; and (2) a second restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, thereby producing a combination, b) optionally maintaining the combination under conditions in which the first protein and the second protein are expressed and interact, c) joining the first vector with the second vector, thereby forming a contiguous nucleic acid sequence that comprises (i) the nucleic acid sequence that encodes a first protein that interacts with a second protein, (ii) the first restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, (iii) the nucleic acid sequence that encodes the second protein, and (iv) the second restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, d) cleaving the first restriction endonuclease recognition site and the second restriction endonuclease recognition site in the contiguous nucleic acid sequence with restriction endonucleases that cleave the contiguous nucleic acid sequence distally to the restriction endonuclease recognition sites, thereby producing a 5′ end tag and a 3′ end tag of the contiguous nucleic acid sequence, e) joining the 5′ end tag to the 3′ end tag, thereby producing a paired tag, and f) sequencing the paired tag, thereby identifying nucleic acid sequences that encode at least two interacting proteins.

[0030] In an additional embodiment of the invention, provided is a method for identifying nucleic acid sequences that encode at least two interacting proteins comprising the steps of a) combining (i) a first vector comprising (1) a nucleic acid sequence that encodes a first protein that interacts with a second protein, and (2) a first restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, and (ii) a second vector comprising (1) a nucleic acid sequence that encodes the second protein, and (2) a second restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, thereby producing a combination, b) optionally maintaining the combination under conditions in which the first protein and the second protein are expressed and interact, c) joining the first vector with the second vector, thereby forming a contiguous nucleic acid sequence that comprises (i) the nucleic acid sequence that encodes a first protein that interacts with a second protein, (ii) the first restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, (iii) the nucleic acid sequence that encodes the second protein, and (iv) the second restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, d) sequencing the contiguous nucleic acid sequence, thereby identifying nucleic acid sequences that encode at least two interacting proteins.

[0031] In another embodiment of the invention, provided is a method for identifying nucleic acid sequences that encode at least two interacting proteins comprising the steps of a) combining, (i) a first vector comprising (1) a nucleic acid sequence that encodes a first protein that interacts with a second protein, and (2) a first restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, and (ii) second vector comprising (1) a nucleic acid sequence that encodes the second protein; and (2) a second restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, thereby producing a combination, b) optionally maintaining the combination under conditions in which the first protein and the second protein are expressed and interact, c) joining the first vector with the second vector, thereby forming a contiguous nucleic acid sequence that comprises (i) the nucleic acid sequence that encodes a first protein that interacts with a second protein, (ii) the first restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, (iii) the nucleic acid sequence that encodes the second protein, and (iv) the second restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, d) cleaving the first restriction endonuclease recognition site and the second restriction endonuclease recognition site in the contiguous nucleic acid sequence with restriction endonucleases that cleave the contiguous nucleic acid sequence distally to the restriction endonuclease recognition sites, thereby producing a paired tag comprising a 5′ end tag and a 3′ end tag of the contiguous nucleic acid sequence, e) sequencing the paired tag, thereby identifying nucleic acid sequences that encode at least two interacting proteins.

[0032] In a further embodiment of the invention, provided is a method for identifying a plurality of nucleic acid sequences that encode at least two interacting proteins comprising the steps of a) combining (i) a plurality of first vectors each comprising (1) a nucleic acid sequence that encodes a first protein that interact with a second protein; and (2) a first restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, and (ii) a plurality of second vectors each comprising (1) a nucleic acid sequence that encodes the second protein; and (2) a second restriction endonuclease recognition site specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, thereby producing a combination comprising a plurality of first vectors and a plurality of second vectors, b) optionally maintaining the combination under conditions in which the plurality of first vectors encoding a first protein and the p

Problems solved by technology

However, a major problem with the BAC-based approaches is the high cost and operational burden associated with the production of 15,000-25,000 individual BAC subclone libraries, the 15-20% waste associated with re-sequencing the vector, as well as the unavoidable E. coli contamination, the need to deal with transposon and bacteriophage insertions, and the 20-50% waste in redundant sequencing of BAC overlaps.

Although these costs can be reduced by sequencing the BACs at low coverage (using a hybrid BAC / WGS strategy, for example) or by using a pooling strategy, they cannot be eliminated.

The need to generate a physical map by using restriction digest fingerprinting or by complex pooling and sequence based mapping strategies adds additional cost and operational overhead.

However, these methods suffer from the need for large numbers of transformations to be performed: one for each bait to be analyzed against one or more prey molecule.

Therefore, these methods only incrementally increase the efficiency of conventional two-hybrid systems.

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