Methods and Compositions for DNA Manipulation

Abstract

Methods and compositions are provided for generating a single-stranded extension on a polynucleotide molecule, the single-stranded extension having a desired length and sequence composition. Methods for forming single-stranded extensions include: the use of a cassette containing at least one nicking site and at least one restriction site at a predetermined distance from each other and in a predetermined orientation; or primer-dependent amplification which introduces into a polynucleotide molecule, a modified nucleotide which is excised to create a nick using a nicking agent. The methods and compositions provided can be used to manipulate a DNA sequence including introducing site specific mutations into a polynucleotide molecule and for cloning any polynucleotide molecule or set of joined polynucleotide molecules in a recipient molecule such as a vector of choice.

1 . A method of generating a single-stranded extension on a polynucleotide molecule, the single-stranded extension having a desired length and sequence composition, comprising:
 (a) introducing a cassette into a polynucleotide molecule at a predetermined location;   (b) cleaving the polynucleotide molecule with a nicking endonuclease specific for a nicking site in the cassette and with a restriction endonuclease specific for a restriction site in the cassette; and   (c) dissociating the cleaved polynucleotide molecule between the nicking site and the restriction site to generate the single-strand extension with the desired length and sequence composition.   
     
     
         2 . A method according to  claim 1 , wherein the cassette contains one nicking site and one restriction site for generating a 3′ or 5′, left side or right side single-stranded extension. 
     
     
         3 . A method according to  claim 1 , wherein the cassette contains one restriction site flanked on each side by a nicking site for generating a left side and a right side 3′ or 5′ single-strand extension. 
     
     
         4 . A method according to  claim 1 , wherein the cassette contains two restriction sites positioned between two nicking sites for generating two single-stranded extensions. 
     
     
         5 . A method according to  claim 4 , wherein a spacer sequence is located between the two restriction sites. 
     
     
         6 . A method according to  claim 5 , wherein the spacer sequence encodes a marker. 
     
     
         7 . A method according to  claim 6 , wherein the marker is selected from: a toxin, a drug-resistant factor, an enzyme, an antigen, a fluorescent molecule, and an siRNA. 
     
     
         8 . A method according to  claim 1 , wherein the composition of the cassette determines the composition of the single-strand extension. 
     
     
         9 . A method according to  claim 1 , wherein the cassette has a length greater than about 5 nucleotides. 
     
     
         10 . A method according to  claim 2 , wherein the nicking site is positioned upstream from the restriction site in the cassette in an orientation suitable for nicking a first of two strands in the polynucleotide molecule, by the nicking endonuclease. 
     
     
         11 . A method according to  claim 2 , wherein the nicking site is positioned upstream in the cassette in an orientation suitable for nicking a second of two strands in the polynucleotide molecule, by the nicking endonuclease. 
     
     
         12 . A method according to  claim 2 , wherein the nicking site is positioned downstream from the restriction site in the cassette in an orientation suitable for nicking a first of two strands in the polynucleotide molecule, by the nicking endonuclease. 
     
     
         13 . A method according to  claim 2 , wherein the nicking site is positioned downstream from the restriction site in the cassette in an orientation suitable for nicking a second of two strands in the polynucleotide molecule, by the nicking endonuclease. 
     
     
         14 . A method according to  claim 3  or  4 , wherein the two nicking sites are each inversely oriented with respect to each other. 
     
     
         15 . A method according to  claim 1 , wherein the single-stranded extension in (c) has a length of no more than about 20 nucleotides. 
     
     
         16 . A method according to  claim 8 , wherein the predetermined sequence between the one or more nicking sites and the one or more restriction sites in the cassette is selected according to the desired composition of the single-stranded extension. 
     
     
         17 . A method according to  claim 1 , wherein the double-stranded polynucleotide molecule is a double-stranded DNA. 
     
     
         18 . A method according to  claim 17 , wherein the double-stranded polynucleotide molecule is a recipient molecule capable of being replicated in a host cell. 
     
     
         19 . A method according to  claim 18 , wherein the recipient molecule is a vector. 
     
     
         20 . A method according to  claim 19 , wherein the vector is selected from: pNEB205A, pNEB200A, pNEB210A, and pUC-TT. 
     
     
         21 . A cassette, comprising: a double-stranded DNA having a nicking site located less than about 50 nucleotides from a restriction site, the DNA being capable of insertion into a polynucleotide molecule, wherein the restriction site in the cassette does not occur in the polynucleotide molecule. 
     
     
         22 . A cassette according to  claim 21 , wherein the double-stranded DNA has a single nicking site and a single restriction site for generating a left side or right side, 3′ or 5′ single-stranded extension. 
     
     
         23 . A cassette according to  claim 21 , wherein the double-stranded DNA has two nicking sites and one restriction site for generating a left side and a right side, 3′ or 5′ single-stranded extension. 
     
     
         24 . A cassette according to  claim 21 , wherein the double-stranded DNA has two restriction sites positioned between two nicking sites for generating two 3′ or 5′ single-stranded extensions. 
     
     
         25 . A cassette according to  claim 24 , wherein the double-stranded DNA has a spacer sequence located between the two restriction sites. 
     
     
         26 . A cassette according to  claim 25 , wherein the spacer sequence encodes a marker. 
     
     
         27 . A cassette according to  claim 26 , wherein the marker is selected from: a toxin, a drug-resistant factor, an enzyme, an antigen, a fluorescent molecule, and an siRNA. 
     
     
         28 . A cassette according to  claim 21 , wherein the double-stranded DNA has a length greater than about 5 nucleotides. 
     
     
         29 . A cassette according to  claim 22 , wherein the double-stranded DNA has the nicking site positioned upstream from the restriction site in an orientation for nicking a first strand of the double-stranded DNA by a nicking endonuclease. 
     
     
         30 . A cassette according to  claim 22 , wherein the double-stranded DNA has the nicking site positioned upstream from the restriction site in an orientation for nicking a second strand of the double-stranded DNA by a nicking endonuclease. 
     
     
         31 . A cassette according to  claim 22 , wherein the double-stranded DNA has the nicking site positioned downstream from the restriction site in an orientation for nicking a first strand of the double-stranded DNA by a nicking endonuclease. 
     
     
         32 . A cassette according to  claim 22 , wherein the double-stranded DNA has the nicking site positioned downstream from the restriction site in an orientation for nicking a second strand of the double-stranded DNA by a nicking endonuclease. 
     
     
         33 . A cassette according to  claims 23  or  24 , wherein the two nicking sites are each inversely oriented with respect to each other in the double-stranded DNA. 
     
     
         34 . A cassette according to  claim 28 , wherein the sequence between the one or more nicking sites and the one or more restriction sites in the double-stranded DNA is predetermined. 
     
     
         35 . A polynucleotide molecule, comprising: any of the cassettes according to  claims 21 ,  22 ,  23 ,  24  and  25  and being capable of replication in a host cell. 
     
     
         36 . A polynucleotide molecule, comprising a vector selected from: pNEB205A, pNEB200A, pNEB210A, and pUC-TT. 
     
     
         37 - 55 . (canceled) 
     
     
         56 . A method of generating a single-strand extension on a polynucleotide molecule, the single-stranded extension having a desired length and composition, comprising:
 (a) introducing a modified nucleotide into the polynucleotide molecule at a specific location;   (b) cleaving the polynucleotide molecule at the modified nucleotide with a nicking agent to create a terminal sequence flanked by a nick; and   (c) dissociating the terminal sequence to generate the single-strand extension with the desired length and sequence composition.   
     
     
         57 . A method according to  claim 56 , wherein the polynucleotide molecule is a product of primer pair dependent DNA amplification of a target molecule. 
     
     
         58 . A method according to  claim 57 , wherein each primer in the primer pair contains the modified nucleotide. 
     
     
         59 . A method according to  claim 57 , wherein one of the primers in the primer pair contains the modified nucleotide. 
     
     
         60 . A method according to  claim 56 , wherein the single-stranded extension on the polynucleotide molecule is complementary to a single-stranded extension on a second polynucleotide molecule. 
     
     
         61 . A method of creating a site-specific mutation in a target molecule, comprising:
 (a) selecting two pairs of primers for amplifying the target molecule, wherein a first pair of primers produces a first amplification product and a second pair of primers produces a second amplification product, and wherein one primer from each of the first and second primer pair contains a modified nucleotide, the sequence of such primers complementing each other at the 5′ end, and optionally one or both such primers containing a mutation in the complementary or in a non-complementary 5′ sequence with respect to the target molecule;   (b) amplifying the target molecule using the first and second primer pairs of (a) to form a first and second polynucleotide molecule;   (c) nicking the first and second polynucleotide molecules at the modified nucleotide with a nicking agent;   (d) dissociating the first and second polynucleotide molecules between the nick and the 5′ end to produce a first 3′ single-strand extension on the first polynucleotide molecule which is complementary to a second single-stranded extension produced on the second polynucleotide molecule; and   (e) allowing the first and second polynucleotide molecules to re-associate through their complementary single-strand extensions to form a target molecule having a site-specific mutation.   
     
     
         62 . The method according to  claim 61 , wherein the primer sequence at the 5′ end, adjacent to the modified nucleotide, is not complementary to the target molecule. 
     
     
         63 . The method according to  claim 61 , wherein the modified nucleotide is positioned between a priming sequence and a 5′ terminal region, wherein the priming sequence is complementary to the target molecule and wherein the 5′ terminal regions of such primers adjacent to the modified nucleotide are complementary to each other. 
     
     
         64 . The method according to  claim 63 , wherein the modified nucleotide on at least one primer is positioned at a junction between the priming sequence and the 5′ terminal region. 
     
     
         65 . The method according to  claim 63 , wherein the modified nucleotide on at least one primer is positioned between the 5′ sequence and an insertion sequence wherein the insertion sequence is adjacent to the priming sequence. 
     
     
         66 . The method according to  claim 63 , wherein the priming sequence on each of the primers complements sequences on the target molecule that are separated by an intervening sequence. 
     
     
         67 . The method of  claim 63 , wherein the site-specific mutation is an alteration in one or more nucleotides. 
     
     
         68 . The method of  claim 63 , wherein the site-specific mutation is an inserted nucleotide sequence. 
     
     
         69 . An oligonucleotide suitable for priming a target molecule, comprising a 5′ sequence selected from GGAGACAU, GGGAAAGU, ACGAGACU, ACCAGACU and GGGGG(8-oxo-G) adjacent to a sequence identical to the 5′ end of the target molecule. 
     
     
         70 . A method for joining a plurality of linear polynucleotide molecules to form a single molecule, comprising:
 (a) forming a single-stranded extension on one or both ends of each of the plurality of polynucleotide molecules using the method of  claim 1 , such that at least one single-stranded extension on one polynucleotide molecule is complementary to a single-stranded extension on another polynucleotide molecule; and   (b) allowing the plurality of polynucleotide molecules to associate to form the single molecule.   
     
     
         71 . A method for joining a plurality of linear polynucleotide molecules to form a single molecule, comprising;
 (a) forming a single-strand extension on one or both ends of each the plurality of polynucleotide molecules using the method of claim  53 , such that at least one single-strand extension on one polynucleotide molecule is complementary to a single-strand extension on another polynucleotide molecule; and   (b) allowing the plurality of polynucleotide molecules to associate to form the single molecule.   
     
     
         72 . A method for inserting a target molecule into a recipient molecule, comprising:
 (a) forming a first and a second single-stranded extension on a first and a second end of a recipient molecule using the method of  claim 1 , wherein the first and second single-stranded extension may be the same or different from each other;   (b) forming single-stranded extensions on the ends of a target molecule using the method of claim  53 , wherein the single-stranded extensions are complementary to the first and the second single-stranded extension on the recipient molecule; and   (c) allowing the recipient molecule and the target molecule to associate to form a single molecule.   
     
     
         73 . A method according to  claim 72 , wherein the target molecule is a product of joining a plurality of polynucleotide molecules according to  claim 71 . 
     
     
         74 . A method according to  claim 73 , wherein the plurality of polynucleotides molecules comprise DNA domains. 
     
     
         75 . A method according to  claim 73 , wherein the DNA domains are exons. 
     
     
         76 - 79 . (canceled) 
     
     
         80 . A host cell containing a recipient molecule into which a target molecule has been inserted according to  claim 72 .