![]() ![]() The products of the thermostable Taq DNA Polymerase (Perkin-Elmer, Norwalk, CT) reactions contain “ragged ends” that are caused by non-template, nucleotide-addition reactions and interfere with the blunt-end cloning of the DNA fragment and other amplimers ( 1). Treatment of the PCR product with Klenow enzyme did not make the DNA fragment compatible for the ligation. We have had difficulties in subcloning the PCR fragment of extracellular ligand binding domain of GC/ANF-R into the SmaI site of the pGEX-3X prokaryotic expression vector in order to maintain the open reading frame for the synthesis of fusion protein in Escherichia coli. The restriction endonuclease digestion of the linker-added DNA fragment (to generate the flanking BamHI restriction sites) and ligation to the pGEX-3X plasmid vector (Pharmacia Biotech, Piscataway, NJ) were performed according to the published protocols ( 5). The pGEX-3X plasmid vector was linearized by digestion with BamHI and then dephosphorylated using calf intestinal phosphatase (Promega). The blunt-ended DNA fragment was purified using the Geneclean ® Kit (Bio 101, La Jolla, CA), and then phosphorylated BamHI linkers (GGCATCCC Boehringer Mannheim, Indianapolis, IN) were added by the routine procedures ( 5). After 30 min at room temperature, the reaction mixture was loaded onto a low melting point agarose gel to remove the primers. After the kinase reaction, the DNA was subjected to a fill-in reaction that contained 5 mM deoxyribonucleoside triphosphates (dNTPs) and one unit of the Klenow fragment of DNA Polymerase (Promega). The reaction was allowed to proceed at 37☌ for 1 h, and then the tubes were heated to 75☌ for 10 min and cooled at 4☌. The 40 μl kinase reaction mixture contained 1–2 μg DNA, 66 mM Tris-HCl, pH 7.6, 10 mM MgCl 2, 10 mM dithiothreitol, 0.2 mg/ml bovine serum albumin (BSA), 20 mM ATP and 2.0 units T 4 polynucleotide kinase (Promega, Madison, WI). The amplified product was ethanol precipitated following routine procedures ( 5). The PCR mixture was treated with 150 μl of chloroform, and the upper aqueous phase was recovered. The extracellular ligand-binding domain of the murine GC/ANF-R was amplified from its cDNA clone ( 6). ![]() In our attempt to subclone the extracellular ligand-binding domain of the murine guanylate cyclase coupled atrial natriuretic factor receptor (GC/ANF-R) ( 6, 7), we have developed an efficient subcloning procedure to ligate the PCR fragments by adding desired linkers. A number of reports have appeared to circumvent the undesired problems in subcloning the PCR-generated DNA fragments. Another remedy is to add the restriction enzyme to the blunt-end ligation reaction to check the vector self-ligation ( 4). One of the solutions to this problem is the treatment of the PCR product with proteinase K, thus providing access to the restriction endonuclease to efficiently cut and create the sticky ends that are inserted into the DNA fragment through predesigned primers flanking the amplified product ( 2). We and others have experienced certain difficulties in subcloning the PCR products in desired vectors ( 9). PCR has become a routine tool for amplifying the desired piece of DNA from various sources of templates ( 8). This intermediate kinase reaction was found to be the critical step that enhanced the blunt-end cloning of the PCR products and increased the efficiency of the linker-addition to a desired amplified DNA fragment. A brief kinase reaction was performed using T 4 Polynucleotide kinase and ATP before the “fill-in” reaction by Klenow enzyme to blunt end the DNA fragment. The PCR-amplified DNA was purified first by chloroform extraction to remove the mineral oil and then ethanol precipitated. In this report we provide evidence that inserting an additional step (kinase reaction) between the purification of polymerase chain reaction (PCR) products and the blunt-end cloning or restriction site creation by linker-addition greatly increases the efficiency of the PCR-product subcloning. ![]()
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