{'fraction_non_alphanumeric': 0.040032025620496396, 'fraction_numerical': 0.02942353883106485, 'mean_word_length': 4.878823529411765, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
"Megaprimer" method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor.

The technique of site-directed mutagenesis (SDM) is widely used in the field of molecular biology to introduce mutations into DNA (5). The development of polymerase chain reaction (PCR)-based SDM has eliminated the need to clone DNA, therefore simplifying the technique and enabling the desired results to be achieved more quickly (3). The “megaprimer” method of PCR-based SDM incorporates three primers and two amplification steps (2,4). The first PCR incorporates an internal mutagenic primer (containing the mutation) and a reverse primer. In a second PCR, the product of the first reaction is used as a megaprimer together with a forward primer annealing upstream of the mutagenic site, resulting in the amplification of the final mutagenic product. Most studies have obtained very poor or no final PCR product when very large megaprimers (>300 bases) are used (1,6). This is probably due to the inefficient priming of template DNA by megaprimer because of self-annealing of megaprimer strands. Datta (2) reported efficient amplification using a megaprimer, by utilizing five cycles of asymmetrical PCR with only the megaprimer, before adding the forward primer and completing the final amplification. Using this method, we have obtained successful amplification with an extremely large 980-base megaprimer. However, successful amplification required a slight modification to the protocol. Datta (2) used 25 ng of megaprimer and 5 ng of template per 50 μL PCR, while our PCR used a much higher concentration of megaprimer (up to 6 μg of megaprimer per 5 ng of template). This modification produced an extremely high yield of mutagenic product. Barik and Galinski (1) reported that increasing the template (supercoiled plasmid DNA) concentration from nanogram to microgram quantities increased the yield of PCR product. In our study, we amplified the entire template (2200 bp), and therefore a high concentration of template DNA would have led to a significant “contamination” of the mutagenic product. Our modification to the technique only requires minute quantities of template DNA (5 ng), and there was therefore negligible “contamination” of the mutagenic product. A penicillin-binding protein (PBP) 1A gene from Streptococcus pneumoniae was our DNA template in the PCR (GenBank® Accession No. X67866). A 2200-bp product was amplified using the forward (CGGCATTCGATTTGATTCGCTTCT) and reverse (GTCGTACTATTATTTGTGCTTGGAGTGGTT) primers. The mutagenic primer had the sequence GACTGGGGTTCTACTATGAAACCAA, with the underlined nucleotide (A) substituted for the wild-type nucleotide (G). Fifty-microliter PCRs were set up. The first PCR contained 5 ng of PBP 1A DNA, 10 mM Tris-HCl (pH 8.85), 25 mM KCl, 5 mM (NH4)2SO4, 2 mM MgSO4, 1 μM mutagenic primer, 1 μM reverse primer, 200 μM deoxynucleoside triphosphates (dNTPs) (Boehringer Mannheim, Mannheim, Germany) and 2 U of Pwo DNA Polymerase (Boehringer Mannheim). The PCR was performed in a Model 480 DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT, USA), with 25 cycles of denaturation at 93°C for 1 min, primer annealing at 60°C for 1 min and primer extension at 72°C for 2 min. The 980-bp PCR product (megaprimer) was purified from agarose with GENECLEAN (Bio 101, Vista, CA, USA) and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). The second PCR contained 5 ng of PBP 1A DNA, 10 mM Tris-HCl (pH 8.85), 25 mM KCl, 5 mM (NH4)2SO4, 2 mM MgSO4, 200 μM dNTPs, 2 U of Pwo DNA polymerase and varying concentrations of megaprimer DNA (100 ng to 6 μg) with 5 thermal cycles at 93°C for 1 min and 72°C for 3 min. While at 72°C, 1 μM of forward primer was added and gently mixed with the reaction, and thermal cycling continued for 25 times at 93°C for 1 min, 60°C for 1 min and 72°C for 2 min. Agarose gel electrophoresis of 5 μL of PCR mixture revealed the successful amplification of a 2200-bp product (Taq DNA polymerase produced the same results, however Pwo or Pfu DNA polymerases are preferred enzymes because of their proofreading activity). Nucleotide sequencing of the product confirmed the introduction of the desired mutation. As shown in Figure 1, the 2200-bp PCR product only started appearing in PCRs that contained 2 μg of megaprimer. Reactions containing up to 6 μg of megaprimer produced the strongest product. This PCR worked equally well incorporating similarly sized megaprimers in the amplification of PBP 2B and PBP 2× genes. This study has demonstrated that the concentration of megaprimer in a PCR is a critical factor. A high ratio of megaprimer (6 μg) to template (5 ng) produced the best results. This study therefore extends the “megaprimer” method of asymmetric PCR-based SDM, as described by Datta (2), to show that the technique is extremely efficient in generating mutagenic PCR products when incorporating a megaprimer as large as 980 bases, provided that the megaprimer is used in a high concentration.