Methods for generating a continuous nucleotide sequence from noncontiguous nucleotide sequences
First Claim
1. A method for producing a nucleic acid molecule that comprises a continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest, the nucleic acid molecule derived from noncontiguous nucleotide sequences, comprising:
- (a) amplifying at least two nucleotide sequences from a single nucleic acid molecule template using primer pairs to produce double-stranded amplified products, wherein the amplified nucleotide sequences reside noncontiguously in the nucleic acid molecule template, wherein each primer of a primer pair comprises an uninterrupted recognition sequence for a class IIS restriction endonucicase which is located near the 5′
-end of the primer and a portion of an amino acid coding sequence, such that cleavage of the amplified products with the class IIS restriction endonuclease yields at least two nucleic acid molecule fragments with cohesive ends that, when ligated to each other, produce a continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest, (b) cleaving amplified products with the class IIS restriction endonuclease to produce nucleic acid molecule fragments, and (c) ligating cleaved nucleic acid molecule fragments to produce a nucleic acid molecule comprising the continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest.
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Abstract
The increased use of nucleotide sequence data mining techniques has amplified the demand for efficient methods of producing recombinant proteins in host cells. New genes identified from genomic sequences may lack an identified source for obtaining an mRNA template to produce cDNA. A strategy is provided for generating nucleic acid molecules comprising a continuous amino acid-encoding sequence using a genomic template that contains non-continuous amino acid-encoding sequences.
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Citations
17 Claims
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1. A method for producing a nucleic acid molecule that comprises a continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest, the nucleic acid molecule derived from noncontiguous nucleotide sequences, comprising:
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(a) amplifying at least two nucleotide sequences from a single nucleic acid molecule template using primer pairs to produce double-stranded amplified products, wherein the amplified nucleotide sequences reside noncontiguously in the nucleic acid molecule template, wherein each primer of a primer pair comprises an uninterrupted recognition sequence for a class IIS restriction endonucicase which is located near the 5′
-end of the primer and a portion of an amino acid coding sequence, such that cleavage of the amplified products with the class IIS restriction endonuclease yields at least two nucleic acid molecule fragments with cohesive ends that, when ligated to each other, produce a continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest,(b) cleaving amplified products with the class IIS restriction endonuclease to produce nucleic acid molecule fragments, and (c) ligating cleaved nucleic acid molecule fragments to produce a nucleic acid molecule comprising the continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 17)
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2. A method for producing a nucleic acid molecule that comprises a continuous nucleofide sequence capable of being transcribed and translated to produce a protein of interest, the nucleic acid molecule derived from nonconziguous nucleolide sequences, comprising:
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(a) amplifying at least two nucleotide sequences from at least two nucleic acid molecule templates using primer pairs to produce double-stranded amplified products, wherein at least one primer of a primer pair comprises an uninterrupted recognition sequence for a class IIS restriction endonuclease which is located near the 5′
-end of the primer and a portion of an amino acid coding sequence, such that cleavage of the amplified products with the class IIS restriction endonuclease yields at least two nucleic acid molecule fragments with cohesive ends that, when ligated to each other, produce a continuous nucleotide sequence capable of being transcribed and translated to produce a protein of interest,(b) cleaving amplified products with the class IIS restriction endonuclease to produce nucleic acid molecule fragments, and (c) ligating cleaved nucleic acid molecule fragments to produce a nucleic acid molecule comprising the continuous nucleotide sequence capable of being transcribed and translatcd to produce a protein of interest. - View Dependent Claims (10)
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16. The method of claim 18, wherein at least one mutation resides in an amino acid encoding sequence.
Specification