Compositions and methods for random nucleic acid mutagenesis

Abstract

The invention relates to compositions and methods for nucleic acid PCR mutagenesis using novel error-prone DNA polymerases and a PCR enhancing factor. The invention also relates to compositions and methods for nucleic mutagenesis with two or more DNA polymerases lacking or exhibiting reduced exonuclease activity. The invention further relates to kit format of said compositions for PCR mutagenesis.

1 . A composition for PCR mutagenesis comprising an archaeal exo-DNA polymerase which substantially lacks 3′ to 5′ exonuclease activity, and a PCR enhancing factor, wherein said PCR enhancing factor comprises one or more proteins selected from the group consisting of: 
 (a) an isolated or purified naturally occurring polymerase enhancing protein from an archebacterial source;    (b) a synthetic protein comprising the amino acid sequence of Pfu P45;    (c) an analog of (b) having polymerase enhancing activity;    (d) isolated or purified Pfu P45;    (e) recombinant Pfu P45; and    (f) an isolated or purified native complex comprising Pfu P45 and Pfu P50.    
     
     
         2 . The composition of  claim 1 , wherein said archaeal exo-DNA polymerase is selected from the group consisting of: exo-Tli DNA polymerase, exo-Pfu DNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase, and exo-PGB-D DNA polymerase.  
     
     
         3 . The composition of  claim 1 , further comprising one or more DNA polymerases selected from the group consisting of: Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exo-Tli DNA polymerase, exo-Pfu DNA polymerase, exo-Tma DNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase, and exo-PGB-D DNA polymerase, wherein said one or more DNA polymerases are different from said archaeal DNA polymerase.  
     
     
         4 . The composition of  claim 1 , further comprising a PCR buffer.  
     
     
         5 . The composition of  claim 4 , wherein said PCR buffer lacks Mn 2+ .  
     
     
         6 . The composition of  claim 1 , further comprising equivalent molar amounts of dATP, dTTP, dGTP, and dCTP.  
     
     
         7 . A kit for PCR mutagenesis comprising an archaeal exo-DNA polymerase, a PCR enhancing factor comprising one or more proteins selected from the group consisting of: 
 (a) an isolated or purified naturally occurring polymerase enhancing protein from an archebacterial source;    (b) a synthetic protein comprising the amino acid sequence of Pfu P45;    (c) an analog of (b) having polymerase enhancing activity;    (d) isolated or purified Pfu P45;    (e) recombinant Pfu P45; and    (f) an isolated or purified native complex comprising Pfu P45 and Pfu P50; and packaging means therefor.    
     
     
         8 . The kit of  claim 7 , wherein said archaeal exo-DNA polymerase is selected from the group consisting of: exo-Tli DNA polymerase, exo-Pfu DNA polymerase, exo-Tma DNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase, and exo-PGB-D DNA polymerase.  
     
     
         9 . The kit of  claim 7 , further comprising one or more polymerases selected from the group consisting of: Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exo-Tli DNA polymerase, exo-Pfu DNA polymerase, exo-Tma DNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase, and exo-PGB-D DNA polymerase, wherein said one or more DNA polymerases are different from said archaeal DNA polymerase.  
     
     
         10 . The kit of  claim 7 , further comprising a PCR buffer.  
     
     
         11 . The kit of  claim 10 , wherein said PCR buffer lacks Mn 2+ .  
     
     
         12 . The kit of  claim 7 , further comprising equivalent molar amounts of dATP, dTTP, dGTP, and dCTP.  
     
     
         13 . The kit of  claim 7 , further comprising dATP, dTTP, dGTP, and dCTP, any three of which are present in equivalent molar amounts.  
     
     
         14 . The kit of  claim 7 , wherein said PCR buffer comprises Mn 2+ .  
     
     
         15 . The kit of  claim 7 , wherein said PCR buffer comprises 20 mM Tris, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 100 μg/ml BSA, and 0.1% Triton X-100 and is adjusted to a pH value in the range 7.5 to 9.2.  
     
     
         16 . The kit of  claim 15 , wherein said PCR buffer is adjusted to a pH value of 8.8 or 9.2.  
     
     
         17 . A method of PCR amplification for mutagenesis comprising incubating a reaction mixture comprising a nucleic acid template, at least two PCR primers, an archaeal exo-DNA polymerase which substantially lacks 3′ to 5′ exonuclease activity, and a PCR enhancing factor under conditions which permit amplification of said nucleic acid template by said archaeal exo-DNA polymerase to produce a mutated amplified product, wherein said PCR enhancing factor comprises one or more proteins selected from the group consisting of: 
 (a) an isolated or purified naturally occurring polymerase enhancing protein from an archebacterial source;    (b) a synthetic protein comprising the amino acid sequence of Pfu P45;    (c) an analog of (b) having polymerase enhancing activity;    (d) isolated or purified Pfu P45;    (e) recombinant Pfu P45; and    (f) an isolated or purified native complex comprising Pfu P45 and Pfu P50.    
     
     
         18 . The method of  claim 17 , wherein said archaeal exo-DNA polymerase is selected from the group consisting of: Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exo-Tli DNA polymerase, exo-Pfu DNA polymerase, exo-Tma DNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase, and exo-PGB-D DNA polymerase.  
     
     
         19 . The method of  claim 17 , wherein said incubating step further comprises incubating one or more exo-DNA polymerases selected from a group consisting of: Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, exo-Tli DNA polymerase, exo-Pfu DNA polymerase, exo-Tma DNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase, and exo-PGB-D DNA polymerase in said reaction mixture, wherein said one or more DNA polymerases are different from said archaeal DNA polymerase.  
     
     
         20 . The method of  claim 17 , wherein said incubating step is performed in a PCR reaction buffer lacking Mn 2+ .  
     
     
         21 . The method of  claim 17 , wherein said incubating step is performed in a PCR reaction buffer comprising Mn 2+ .  
     
     
         22 . The method of  claim 17 , wherein said incubating step is performed in the presence of equivalent molar amounts of dATP, dTTP, dGTP, and dCTP.  
     
     
         23 . The method of  claim 17 , wherein said incubating step is performed in the presence of 200 μM dATP, 200 μM dTTP, 200 μM dGTP, and 200 μM dCTP.  
     
     
         24 . The method of  claim 17 , wherein said incubating step is performed in the presence of equivalent molar amounts of any three of dATP, dTTP, dGTP, and dCTP.  
     
     
         25 . The method of  claim 17 , wherein said incubating step is performed in the presence of 20 mM Tris, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 100 μg/ml BSA, and 0.1% Triton X-100 at a pH value in the range of 7.5 to 9.2.  
     
     
         26 . The method of  claim 25 , wherein said method is performed at a pH value of 8.8 or 9.2.  
     
     
         27 . The method of  claim 17 , wherein said mutated amplified product comprises mutations having a transition/transversion ratio of greater than 0.5.  
     
     
         28 . The method of  claim 27 , wherein said mutated amplified product comprises mutations having a transition/transversion ratio of greater than 1.  
     
     
         29 . The method of  claim 17 , wherein said mutated amplified product comprises mutations having a GC/AT ratio of greater than 1.  
     
     
         30 . The method of  claim 17 , wherein increasing the amount of said nucleic acid template in said incubating step decreases the mutation frequency, or wherein decreasing the amount of said nucleic acid template in said incubating step increases the mutation frequency.  
     
     
         31 . The method of  claim 30 , wherein a first incubating step generates a first mutated amplified product at a first frequency using a first selected amount of said nucleic acid template, and a second incubating step generates a second mutated amplified product at a second frequency using a second selected amount of said nucleic acid template; and wherein said first incubating step and said second incubating step are performed using a single buffer composition.  
     
     
         32 . The method of  claim 31 , further comprising subsequently repeating one or more times an additional incubating step using a portion of or the total amplified product of a preceding incubating step as template.  
     
     
         33 . The method of  claim 30 , wherein said mutation frequency is proportional to the amount of said nucleic acid template.  
     
     
         34 . The method of  claim 17 , wherein said reaction mixture comprises 1 pg to 1 μg of said nucleic acid template.  
     
     
         35 . The method of  claim 30 , wherein said step of incubating produces said mutated amplified product from said nucleic acid template at a mutation frequency of at least 1,000 mutations per 10 6  base pairs.  
     
     
         36 . The method of  claim 35 , wherein said incubating produces said mutated amplified product at a mutation frequency of 1,000 to 16,000 mutations per 10 6  base pairs.  
     
     
         37 . The method of  claim 34 , wherein said reaction mixture comprises 10 ng to 100 ng of said nucleic acid template.  
     
     
         38 . The method of  claim 37 , wherein said incubating produces said mutated amplified product at a mutation frequency of 1,000 to 3,000 mutations per 10 6  base pairs.  
     
     
         39 . The method of  claim 34 , wherein said reaction mixture comprises 10 pg to 10 ng of said nucleic acid template.  
     
     
         40 . The method of  claim 39 , wherein said incubating step produces said mutated amplified product at a mutation frequency of 3,000 to 7,000 mutations per 10 6  base pairs.  
     
     
         41 . The method of  claim 39 , wherein said incubating step produces said mutated amplified product at a mutation frequency of at least 7,000 mutations per 10 6  base pairs.  
     
     
         42 . The method of  claim 41 , wherein said incubating step produces said mutated amplified product at a mutation frequency of 7,000 to 16,000 mutations per 10 6  base pairs.  
     
     
         43 . The method of  claim 41 , further comprising subsequently repeating one or more times an additional incubating step using a portion of or the total amplified product of a preceding incubating step as template.  
     
     
         44 . The method of  claim 17 , wherein the nucleic acid template is from 0.1 kb to 10 kb in length.  
     
     
         45 . The method of  claim 17 , wherein the step of incubating produces amplified product at a yield of 0.5-10 μg.  
     
     
         46 . A composition for PCR mutagenesis comprising exo-Pfu DNA polymerase and a PCR enhancing factor.  
     
     
         47 . The composition of  claim 46  further comprising a storage buffer that comprises 50 mM Tris-HCl, pH 8.2; 0.1 mM EDTA, pH 8.2, 1 mM DTT; 0.1% (v/v) Igepal CA-630; 0.1% (v/v) Tween 20; and 50% glycerol.  
     
     
         48 . The composition of  claim 46 , wherein said exo-Pfu DNA polymerase and said PCR enhancing factor are present in an activity ratio of from 40/1 to 1/400.  
     
     
         49 . The composition of  claim 48 , wherein said exo-Pfu DNA polymerase and said PCR enhancing factor are present in an activity ratio of from 2.5/1 to 1/40.  
     
     
         50 . The composition of  claim 49 , wherein said exo-Pfu DNA polymerase and said PCR enhancing factor are present in an activity ratio of 1.25/1.  
     
     
         51 . The composition of  claim 46 , wherein said exo-Pfti DNA polymerase is present at a concentration of 3.5 activity units per microliter.  
     
     
         52 . The composition of  claim 46 , wherein said PCR enhancing factor is present at a concentration of 2 activity units per microliter.  
     
     
         53 . The composition of  claim 46 , wherein said PCR enhancing factor is recombinant Pfu P45.