Method for synthesizing DNA

Abstract

A DNA synthesis method with a shortened time period required for DNA synthesis by polymerase chain reaction (PCR), characterized in that a DNA polymerase is used in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 μl of a reaction mixture, when PCR is carried out under the following conditions (A) and (B): (A) reaction mixture: 50 μl volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from Escherichia coli, and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences of the primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerase; and (B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99° C., 1 second-66° C., 7 seconds; a kit for DNA synthesis usable for the DNA synthesis method; and an article of manufacture of a PCR agent. According to the present invention, the procedures in the genetic engineering studies and industries involved with PCR can be speeded up.

What is claimed:  
     
         1 . A kit for carrying out a DNA synthesis method with a shortened time period for DNA synthesis by polymerase chain reaction (PCR), comprising: 
 a PCR reagent mixture which is prepared in accordance with instructions of said kit comprising a DNA polymerase in an amount sufficient to provide more than 10 ng of amplified DNA fragments of about 2 kb per 50 μl of a reaction mixture,    wherein the amount of DNA polymerase added to said reaction mixture is pre-determined for each specific type of DNA polymerase by carrying out a test PCR reaction under the following conditions (A) and (B): 
 (A) reaction mixture: 50 μl volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from  Escherichia coli,  and 10 pmol each of primers Eco-1 (SEQ ID NO: 10) and Eco-2 (SEQ ID NO: 11); and having a composition suitable for said DNA polymerase; and  
 (b) reaction conditions: 35 cycles of PCR, wherein one cycle is at 99° C., 1 second-66° C., 7 seconds.  
   
     
     
         2 . The kit according to  claim 1 , comprising two or more kinds of DNA polymerases.  
     
     
         3 . The kit according to  claim 2 , wherein one DNA polymerase comprises 3′→5′ exconuclease activity, and the other DNA polymerase comprises substantially no 3′→5′ exonuclease activity.  
     
     
         4 . The kit according to any one of  claims 1  to  3 , wherein said PCR reagent mixture comprises an acidic substance and/or a salt thereof.  
     
     
         5 . The kit according to  claim 4 , wherein said acidic substance is an acidic macromolecular substance.  
     
     
         6 . The kit according to  claim 5 , wherein said acidic macromolecular substance comprises a sugar chain backbone.  
     
     
         7 . The kit according to  claim 4 , wherein said acidic substance and/or a salt thereof is selected from the group consisting of sulfated-fucose-containing polysaccharides, dextran sulfate, carrageenan, heparin, rhamnam sulfate, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, hyaluronic acid, alginic acid, pectin, polyglutamic acids, polyacrylic acids, polyvinyl sulfates, polystyrene sulfates, DNA and salts thereof.  
     
     
         8 . The kit according to any one of  claims 1  to  3 , wherein said PCR reagent mixture further comprises at least one substance selected from the group consisting of spergualins, degraded products thereof and salts thereof.  
     
     
         9 . The kit according to any one of  claims 1  to  3 , wherein said PCR reagent mixture further comprises a salt formed from an acidic substance and spergualins or degraded products thereof.