Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates
© Farell and Alexandre; licensee BioMed Central Ltd. 2012
Received: 10 April 2012
Accepted: 15 May 2012
Published: 24 May 2012
While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds.
Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR.
BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.
Ever since the introduction of the Polymerase Chain Reaction , it has been one of the most often used tools in molecular biology, and has played a role in many of the major advances in Biology including cloning , mutagenesis , even with small amounts of DNA target . This technique is not without its limitations though, as some DNA templates have proved difficult to amplify. The most common reason for troublesome amplification lies in target DNA sequences that have high GC content (GC content >60%) . Many studies have been undertaken to identify experimental modifications that would alleviate or eliminate this problem altogether, with most studies focusing mainly on primer design [5–7], altering the cycling parameters [8, 9], and the use of PCR additives [10–17]. PCR additives most often employed are organic co-solvents such as DMSO and formamide [12, 13, 15, 16]. DMSO has been found to significantly increase the yield of a PCR reaction on GC-rich DNA templates, by preventing the formation of secondary structures . The effects of formamide are less clear and still debated, with some studies indicating that formamide greatly increases specificity of amplification of GC-rich DNA templates and others failing to detect any effect [14, 16]. Formamide also appears to be effective only within a narrow concentration range  which may be related to the fact that formamide is postulated to bind to the grooves in DNA, thus destabilizing the double helix and perhaps improving initial melting . Bovine serum albumin (BSA) has been applied to many laboratory molecular techniques, including restriction enzyme digestions of DNA to increase the thermal stability and half-life of the restriction enzymes in the reactions . For this reason, its effects have also been investigated in PCR and several studies have demonstrated that BSA have a beneficial effect on the yield of PCR (and qPCR) amplification of ancient DNA or of DNA found in extracts from feces, freshwater, or marine water [19, 20]. The beneficial effects of BSA were observed in the absence of any other additive. Since, most of the PCR inhibitors in the samples analyzed in these experiments were also substances that BSA can bind to, the beneficial effects of BSA were proposed to prevent these inhibitors from interacting with DNA (Taq) polymerase . When used in PCR amplification from genomic DNA that is free of any PCR inhibitors, BSA has not been shown to have a significant effect on specificity or amplification yield . In fact, the effect of BSA on PCR has not been systematically analyzed. Here, we use BSA in conjunction with organic solvents, DMSO or formamide, to amplify NA templates of high GC content. Our results demonstrate that when used with organic solvents, BSA acts as a powerful co-enhancer of PCR amplification of these DNA templates. We also provide evidence that supports the notion that one of the reasons that its effects have gone unnoticed is due to the fact that BSA is sensitive to high temperatures of PCR, and rapidly loses its enhancing abilities. Adding BSA to PCR reactions in presence of organic solvents also allows high PCR yields of GC-rich DNA of various sizes to be obtained while reducing the concentration of solvent used. Using the genomic DNA of the alphaproteobacterium Azospirillum brasilense Sp7  which has a GC content above 65% , we have tested various cycling parameters and combination of additives to amplify DNA fragments ranging from 392 to 7,103 base pairs, (with each having a GC content of 66% or greater). For this study, the DNA sequences corresponding to regions of interest were retrieved from the draft genome sequence of Azospirillum brasilense (http://genome.ornl.gov/microbial/abra/19sep08/) and from sequences available in the NCBI GenBank database. The DNA templates were a 392 base pair fragment with a GC content of 66% (che1P) (NCBI GI 17864024), a 798 base pair fragment with a GC content of 68% (tlp5) (contig 115, or2365), a 1,641 base pair fragment with a GC content of 73% (cheA4) (contig 120, or3019), a 2,638 base pair fragment with a GC content of 66% (tlp2) (contig 213, or4271), a 3,389 base pair fragment with a GC content of 68% (cheA1) (NCBIGI17864025), and a 7,103 base pair fragment with a GC content of 68% (cheOp1) (NCBI GI 17864024). We also applied this protocol in “Touchdown” PCR, as well as in an overlap extension PCR and in combination with a widely used commercialized site-directed mutagenesis kit (Stratagene Quickchange Site Directed Mutagenesis Kit, Stratagene) with primers designed to introduce a single amino acid change. Our results highlight a strategy and experimental conditions for using BSA as a co-enhancer that significantly increases PCR yields when used with solvent additives in various PCR applications.
Target DNA fragments and primers used in this study
Target DNA Fragment
ATM_CFP OL For
ATM_CFP OL REV
While BSA is a powerful co-enhancer of PCR yield when used in combination with DMSO or formamide, its yield-promoting effect seems to act by increasing the range at which the organic solvent used is effective as a PCR additive (formamide or DMSO). BSA used as a co-enhancer with DMSO also increases overall PCR yield. The BSA PCR step protocol evaluated here demonstrate that high yield of traditionally difficult to amplify DNA fragments can be obtained, with a combination of primers of different sequence complementarity to the template they target and of different nucleotide length. The positive effects of BSA, a cost-effective co-enhancer for PCR amplification of GC-rich DNA templates, are not specific to particular DNA sizes or methods as they could be obtained for fragment over 7 kb in length, in overlap extension PCR and site directed mutagenesis applications.
Materials and reagents
PCR reactions were carried out in 2x Go Taq Colorless Master Mix (Promega), 1.00 ng/μl of template DNA. Reactions were also carried out with Failsafe PCR Buffer C and D (Epicentre), in which BSA demonstrated a similar enhancing effect (data not shown). Site directed mutagenesis reactions were carried out in supplied reaction buffer, dNTP mix and Pfu Turbo DNA polymerase, according to the manufacturer’s instructions (Stratagene). The genomic DNA of Azospirillum brasillense Sp7 was used as the template for all PCR reactions and obtained using the Wizard Genomic DNA Purification Kit (Promega), according to the manufacturer’s recommendations. After extraction, DNA concentration was determined on an Eppendorf Biophotometer and 100 μg/ml were used in each PCR. For amplification of the CFP-encoding gene (overlap extension PCR protocol), the pANT579 vector (a gift from A. Nebenfuehr, University of Tennessee, Knoxville) that carries the CFP gene cloned in a pBSKII (Pharmacia, Biotech) vector derivative was used as a template. The pBBRcheATM template (Bible and Alexandre, upublished) was used to amplify the ATM fragment. Additives used included bovine serum albumin (BSA), dimethylsulfoxide (DMSO), formamide, and glycerol. BSA was obtained from New England Biolabs, DMSO from Acros Organics, and formamide and glycerol were from Fisher Bioreagents (ThermoFischer, Waltham MA). The primers were synthesized and purified by HPLC (Integrated DNA Technologies, IA) and are listed in Table 1.
Polymerase Chain Reactions were carried out in a Mastercycler ep from Eppendorf, in 500 μl thin-walled PCR tubes. The cycling parameters consisted of an initial denaturation step of 95°C for five minutes, followed by a three step cycle comprised of a denaturation step of 95°C for 1 minute, an annealing step of 45 seconds, and an extension step of 72°C for 1 minute per kilobase of target gene, unless otherwise noted this cycle repeated 30 times. This cycling step was followed by a final extension time of 72°C for 10 minutes, and then the reaction was cooled down to 4°C. The reactions that were supplemented with BSA at intermediate steps followed the same cycling parameters as listed above, except that the cycle was manually paused after every tenth steps to allow BSA addition. In control reactions, these same cycling parameters were carried out with the addition of the same volume of sterile distilled water or glycerol added instead of BSA. For the “Touchdown” PCR, the cycling parameters consisted of an initial denaturation step of 95°C for 5 minutes followed by a three step cycle comprised of a denaturation step of 95°C and annealing step for 1 minute in which the temperature was initially set 10°C above the predicted annealing temperature and decreased one degree per cycle, and an extension time of 72°C for 1 minute per kilobase of target gene. Another three step cycle followed this one in which there was an initial denaturation step of 95°C for 1 minute, an annealing step of 45 seconds, and an extension step of 72°C for 1 minute per kilobase of target gene. Unless otherwise noted, the initial cycling step was repeated for fifteen cycles and the second cycling step was repeated for 20 cycles. The final cycling step was followed by a final extension time of 72°C for 10 minutes and was cooled down to 4°C. The reactions that were supplemented with BSA at intermediate steps followed the same cycling parameters as listed above, except that the cycle was manually paused after every five steps to allow BSA addition. The mutagenesis PCR was carried out using the protocol supplied by the manufacturer (Stratagene) with primers Tlp2-SDM3-For2 and Tlp2-SDM3-Rev2 and pUCTlp2 that contains the tlp2 gene cloned in pUC18 (total size of 5324 bp) as a template (Table 1). Cycling conditions for site-directed mutagenesis included an initial 95°C denaturation step, followed by a three-step cycle that included 95°C for thirty seconds, 55°C for 1 minute, and 68°C for 1 min/kb of template. The cycle was repeated 16times. For the overlap extension PCR, CFP was amplified from the plasmid pAN579 and the TM region of cheA was amplified from the plasmid PBBRCheATM. The reaction parameters for each of these was an initial denaturation step of 95°C for five minutes followed by a three step cycle comprised of a denaturation step of 95°C for 1 minute, an annealing step of 45 seconds, and an extension step of 72°C for 1 minute per kilobase of target gene, unless otherwise noted this cycle was carried out 30 times. Following this step, there was a final extension time of 72°C for 10 minutes, and then the reaction was cooled down to 4°C. Results were then viewed on an agarose gel and the bands corresponding to the target genes were cut out and extracted using the QIAquick Gel Extraction Kit (Qiagen). The extracted fragments were then added in a 1:1 ratio to a reaction that was carried out using the same parameters that were used to amplify the individual fragments. This same reaction was carried out following the parameters of the previously detailed BSA PCR step plus Touchdown PCR.
The resulting PCR products loaded into a 1.0% agarose gel stained with ethidium bromide. The gel was then photographed, and densitometric quantification of amplification products was carried out using the NIS Elements Br 2.30 (Nikon) program as described in . Each PCR was repeated at least 3 times and an average value and standard deviation was recorded from the image analysis. PCR yield increase was calculated by subtracting the yield obtained in control PCR without any additives from the yield obtained in presence of the additive tested. The resulting number was then divided by the yield obtained in PCR without any additives to give the percentage of PCR yield increased due to a certain additive .
Polymerase chain reaction
Bovine serum albumin
Cyan fluorescent protein.
This work was supported by a NSF award (MCB-0919819) to G.A
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