RESUMEN
A series of novel water-soluble, bioreducible poly(amido amine)s containing disulfide linkages in their amino units (SS-PAAs) was synthesized by Michael addition polymerization of N,N'-dimethylcystamine (DMC) with various bisacrylamides. The synthetic route allows large structural variation in the bisacrylamide segments and is complementary to the earlier developed route to SS-PAAs in which the disulfide bond is incorporated in cystamine bisacrylamide units. The physicochemical and biomedical properties of the novel DMC-based polymers were evaluated for their application as non-viral gene delivery vectors and compared with analogs lacking the disulfide moieties. DMC-based SS-PAAs show high buffer capacities in the pH range pH 5.1-7.4, a property that may favorably contribute to the endosomal escape of the polyplexes. The polymers are capable to condense DNA into nanoscaled (<250 nm) and positively charged (>+20 mV) polyplexes which are relatively stable in medium mimicking physiological conditions but rapidly disintegrate in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment. Polyplexes from DMC-based SS-PAAs are capable to transfect COS-7 cells in vitro with transfection efficiencies up to 4 times higher than those of pDMAEMA and PEI, with no or only very low cytotoxicity at the polymer/DNA ratios where the highest transfection is observed. The results show that DMC-based SS-PAAs have very promising properties for the development of potent and non-toxic polymeric gene carriers.
Asunto(s)
Materiales Biocompatibles/química , Disulfuros/química , Sistemas de Liberación de Medicamentos/métodos , Técnicas de Transferencia de Gen , Poliaminas/química , Animales , Materiales Biocompatibles/efectos adversos , Materiales Biocompatibles/síntesis química , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , ADN/administración & dosificación , Diaminas/química , Disulfuros/efectos adversos , Disulfuros/síntesis química , Luz , Peso Molecular , Poliaminas/efectos adversos , Poliaminas/síntesis química , Dispersión de Radiación , Relación Estructura-Actividad , TransfecciónRESUMEN
To evaluate the effect of quaternary nicotinamide pendant groups on gene delivery properties, a series of poly(amido amine) (co)polymers were synthesized by Michael addition polymerization of N, N'-cystaminebisacrylamide with variable ratios of 1-(4-aminobutyl)-3-carbamoylpyridinium (Nic-BuNH(2)), and tert-butyl-4-aminobutyl carbamate (BocNH-BuNH(2)), yielding poly(amido amine)s (NicX-NHBoc) with X=0, 10, 30, and 50 % of quaternary nicotinamide groups in the polymer side chains. Deprotection of the pendant Boc-NH groups afforded an analogous series of polymers (NicX-NH(2)) with higher charge density (due to the presence of protonated primary amino groups in the side chains) and subsequent acetylation yielded a series of polymers (NicX-NHAc) of lower hydrophobicity than the Boc-protected polymers. The polymers with the Boc-protected or the acetylated amino groups showed high buffer capacity in the range pH 5.1-7.4, which is a property that can contribute to endosomal escape of polyplexes. The presence of quaternary nicotinamide groups has distinct beneficial effects on the gene vector properties of these polymers. The polymers containing >or=30 % of quaternary nicotinamide groups in their side chains condense DNA into small, nanosized particles (
Asunto(s)
ADN/metabolismo , Técnicas de Transferencia de Gen , Niacinamida/farmacología , Poliaminas/farmacología , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Chlorocebus aethiops , ADN/química , Etidio/química , Sustancias Intercalantes/química , Espectroscopía de Resonancia Magnética , Nanopartículas/química , Niacinamida/química , Poliaminas/química , Espectrometría de Fluorescencia , Electricidad Estática , TransfecciónRESUMEN
A group of bioreducible poly(amido amine)s containing multiple disulfide linkages in main chain and oligoamines in side chain (SS-PAOAs) were prepared by Michael-type polyaddition of N-tert-butyloxycarbonyl (N-Boc) protected oligoamine to the disulfide-containing cystaminebisacrylamide, followed by deprotection of the Boc-protective groups. These linear polymers show strong DNA condensation capability at low N/P ratios. The chemical structure of oligoamine side chains (i.e. amine type and amino spacer length) in the SS-PAOAs has a distinct effect on their buffer capacity, transfection efficiency and toxicity profile. The SS-PAOAs containing secondary amino functions in the side chain show high buffer capacities and are able to transfect COS-7 cells in vitro at low N/P ratios, with transfection efficiencies similar or even higher than those of 25 kDa branched pEI, along with very low cytotoxicity as determined by XTT assay. Increase of the alkyl spacer from ethylene to propylene between the amino units in side chains results in significant lower transfection and increased toxicity. This study presents detailed factors influencing the relationship between structure and gene delivery properties and may provide helpful insights for the further development of safe and efficient non-viral vectors.
Asunto(s)
Técnicas de Transferencia de Gen , Nylons/química , Poliaminas/química , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Chlorocebus aethiops , Nylons/farmacología , Poliaminas/administración & dosificaciónRESUMEN
Brushed polymers composed of a backbone of poly(hydroxyethyl methacrylate) (pHEMA) onto which poly(2-(dimethylamino)ethyl methacrylate)s (pDMAEMAs) was grafted via a hydrolyzable linker were synthesized and evaluated as nonviral gene delivery vectors. Both pDMAEMA and pHEMA polymers with controlled molecular weights and narrow distributions were synthesized by controlled atom transfer radical polymerization (ATRP). The azide initiator was used to ensure complete and monoazide functionalization of the pDMAEMA polymer chains. Click reaction between pHEMA with alkyne side groups and the azide end group in the pDMAEMA resulted in a high-molecular-weight polymer composed of low-molecular-weight constituents via an easily degradable carbonate ester linker. The length of the pDMAEMA grafts as well as the number of grafts of the brushed pHEMA-pDMAEMA can be easily varied. At physiological conditions (pH 7.4 and 37 degrees C), the brushed polymer degraded by hydrolysis of the carbonate ester with a half-life of 96 h. The molecular weights of the formed degradation products was very close to that of the starting pDMAEMA, which is likely below the renal excretion limit (<30 kDa). It was shown that the degradable brushed pHEMA-pDMAEMAs were able to condense plasmid DNA into positively charged nanosized particles. The resulting polyplexes were able to transfect cells efficiently in the presence of the endosomal membrane disrupting INF-7 peptide, and all these degradable polymers showed lower cellular toxicity compared to a high-molecular-weight pDMAEMA reference. On the other hand, the low-molecular-weight pDMAEMA used for the grafting to pHEMA was neither able to condense the structure of DNA nor able to transfect cells. This study demonstrates that grafting a low-molecular-weight cationic polymer via a hydrolyzable linker to a neutral hydrophilic polymer is an effective approach to modulate the transfection activity and toxicity profile of gene delivery polymers.
Asunto(s)
Metacrilatos/química , Nylons/química , Polihidroxietil Metacrilato/química , Animales , Células COS , Chlorocebus aethiops , ADN/química , ADN/genética , Radicales Libres/química , Hidrólisis , Cinética , Metacrilatos/síntesis química , Estructura Molecular , Nylons/síntesis química , Tamaño de la Partícula , TransfecciónRESUMEN
Poly(amido amine) (SS-PAA) random and block copolymers having bioreducible disulfide bonds in the main chain and amino groups with distinctly different basicity in the side chain were designed and synthesized by Michael addition polymerization between N, N'-cystaminebisacrylamide (CBA) and two amine monomers, i.e., histamine (HIS) and 3-(dimethylamino)-1-propylamine (DMPA). Copolymers containing variable HIS/DMPA ratios show higher ability to bind DNA than p(CBA-HIS) homopolymer and condense DNA into the polyplexes with particle sizes (<150 nm) that are smaller than polyplexes of p(CBA-HIS) ( approximately 220 nm). The buffer capacities of the copolymers increase with increasing HIS/DMPA ratio. These copolymers are able to transfect COS-7 cells in vitro with efficiencies that increase with increasing HIS/DMPA ratio. The random and block copolymers at a HIS/DMPA ratio of 70/30 combines optimal DNA condensation capability and buffer capacity, thereby inducing higher transfection efficiency in the absence and presence of serum as compared to p(CBA-HIS) homopolymer. Moreover, random and block copolymers show a similar transfection capacity, but both have higher capacity than the physical mixtures of p(CBA-HIS) and p(CBA-DMPA) homopolymers. XTT assay reveals that the polyplexes of the SS-PAA copolymers have essentially low cytotoxicity when the highest transfection activity is observed.
Asunto(s)
ADN/genética , ADN/metabolismo , Técnicas de Transferencia de Gen , Poliaminas , Transfección/métodos , Animales , Tampones (Química) , Células COS , Chlorocebus aethiops , ADN/administración & dosificación , Lactatos/metabolismo , Poliaminas/administración & dosificación , Poliaminas/metabolismo , Polietilenglicoles/metabolismoRESUMEN
A series of novel bioreducible poly(amido amine)s containing multiple disulfide linkages (SS-PAAs) were synthesized and evaluated as nonviral gene vectors. These linear SS-PAAs could be easily obtained by Michael-type polyaddition of various primary amines to the disulfide-containing cystamine bisacrylamide. The SS-PAA polymers are relatively stable in medium mimicking physiological conditions (pH 7.4, 150 mM PBS, 37 degrees C), but are rapidly degraded in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment (pH 7.4, [R-SH] = 5 mM, 37 degrees C). The polymers efficiently condense DNA into nanoscaled (<200 nm) and positively charged (>+20 mV) polyplexes that are stable under neutral conditions but are rapidly destabilized in a reductive environment, as was revealed by both dynamic light scatting measurement and agarose gel assays. Moreover, most of the poly(amido amine)s possess buffer capacities in the pH range pH 7.4-5.1 that are even higher than polyethylenimine (pEI), a property that may favorably contribute to the endosomal escape of the polyplexes. Polyplexes of four of the seven SS-PAAs studied were able to transfect COS-7 cells in vitro with transfection efficiencies significantly higher than those of branched pEI, being one of the most effective polymeric gene carriers reported to date. Importantly, also in the presence of serum, a high level of gene expression could be observed when the incubation time was elongated from 1 h to 4 h. XTT assays showed that SS-PAAs and their polyplexes possess essentially no or only very low cytotoxicity at concentrations where the highest transfection activity is observed. The results indicate that bioreducible poly(amido amine)s have excellent properties for the development of highly potent and nontoxic polymeric gene carriers.
Asunto(s)
Poliaminas/química , Poliaminas/metabolismo , Transfección/métodos , Animales , Supervivencia Celular/efectos de los fármacos , ADN/química , Disulfuros/química , Estructura Molecular , Peso Molecular , Tamaño de la Partícula , Poliaminas/toxicidad , VolumetríaRESUMEN
A group of novel poly(amido amine) homo- and copolymers (PAAs) containing secondary and tertiary amine groups in their main chain and different structures in the bisacrylamide segments were synthesized and evaluated as non-viral gene delivery vectors. Among these, also the disulfide-containing cystaminebisacrylamide was employed as a (co)monomer, yielding PAAs with variable amounts of bioreducible disulfide linkages in the main chain. Michael addition the trifunctional 1-(2-aminoethyl) piperazine to equimolar amounts of the appropriate bis(acrylamide) yielded linear polymers as was elucidated by their (13)C NMR spectra. The polymers possess buffering capacities between pH 5.1 and pH 7.4 higher than branched polyethylenimine (pEI) and are able to efficiently condense DNA into nanosized (<150 nm) and positively charged complexes. Transfection experiments with COS-7 cells showed that polyplexes from PAAs with disulfide linkages give significant higher transfections than those from PAAs lacking the disulfide linkage, and XTT assays showed that these polymers are essentially non-toxic. Variation of the disulfide content revealed that polyplexes of PAA copolymers with appropriate disulfide content have largely improved biophysical properties, yielding enhanced levels of gene expression along with low toxicity. The results demonstrate that bioreducible poly(amido amine)s are a very promising class of polymers for safe and efficient gene delivery.
Asunto(s)
Disulfuros/síntesis química , Poliaminas/síntesis química , Polímeros/síntesis química , Transfección/métodos , Acrilamidas/química , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , ADN/metabolismo , Disulfuros/química , Disulfuros/toxicidad , Genes Reporteros , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Operón Lac , Espectroscopía de Resonancia Magnética , Metacrilatos/metabolismo , Metacrilatos/toxicidad , Estructura Molecular , Nylons/metabolismo , Nylons/toxicidad , Tamaño de la Partícula , Piperazinas/química , Poliaminas/química , Poliaminas/toxicidad , Polietileneimina/metabolismo , Polietileneimina/toxicidad , Polímeros/química , Polímeros/toxicidadRESUMEN
Water-soluble polymers with hydrolyzable cationic side groups (structure of the monomers are shown in Figure 1) were synthesized and evaluated as DNA delivery systems. The polymers, except for pHPMA-NHEM, were able to condense plasmid DNA into positively charged nanosized particles. The rate of hydrolysis at 37 degrees C and pH 7.4 of the side groups differed widely; the fastest rate of hydrolysis was observed for HPMA-DEAE (half-life of 2 h), while HPMA-DMAPr had the lowest rate of hydrolysis (half-life of 70 h). In line with this, pHPMA-DEAE-based polyplexes showed the fastest destabilization of the polyplexes at 37 degrees C and pH 7.4. Polyplexes based on pHPMA-DEAE, pHPMA-DMAE, and pHPMA-MPPM showed release of intact DNA within 24, 48, and 48 h, respectively, after incubation at 37 degrees C and pH 7.4. PHPMA-DEAE and pHPMA-MPPM based polyplexes showed the highest transfection activity (almost twice as active as pEI). Importantly, the pHPMA-DEAE, pHPMA-MPPM, and pHPMA-BDMPAP polyplexes preserved their transfection activity in the presence of serum proteins. All polymers investigated showed a substantial lower in vitro cytotoxicity than pEI. In conclusion, pHPMA-based polyplexes are an attractive class of biodegradable vectors for nonviral gene delivery.
Asunto(s)
Acrilamidas/química , Técnicas de Transferencia de Gen , Polímeros/química , Animales , Células COS , Cationes , Chlorocebus aethiops , ADN/administración & dosificación , Hidrólisis , Espectroscopía de Resonancia Magnética , Plásmidos , Espectrometría de Fluorescencia , TransfecciónRESUMEN
The ability to efficiently produce hundreds of proteins in parallel is the most basic requirement of many aspects of proteomics. Overcoming the technical and financial barriers associated with high throughput protein production is essential for the development of an experimental platform to query and browse the protein content of a cell (e.g. protein and antibody arrays). Proteins are inherently different one from another in their physicochemical properties; therefore, no single protocol can be expected to successfully express most of the proteins. Instead of optimizing a protocol to express a specific protein, we used sequence analysis tools to estimate the probability of a specific protein to be expressed successfully using a given protocol, thereby avoiding a priori proteins with a low success probability. A set of 547 proteins, to be used for antibody production and selection, was expressed in Escherichia coli using a high throughput protein production pipeline. Protein properties derived from sequence alone were correlated to successful expression, and general guidelines are given to increase the efficiency of similar pipelines. A second set of 68 proteins was expressed to investigate the link between successful protein expression and inclusion body formation. More proteins were expressed in inclusion bodies; however, the formation of inclusion bodies was not a requirement for successful expression.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Proteómica , Proteínas Recombinantes/genética , Clonación Molecular , Codón/genética , Codón/metabolismo , Genoma Humano , Humanos , Cuerpos de Inclusión/genética , Ingeniería de Proteínas , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos/química , Polietileneimina/química , Polietileneimina/síntesis química , Polímeros/química , Animales , Fenómenos Bioquímicos , Bioquímica , Células COS , Bovinos , Supervivencia Celular , Células Cultivadas , Quitina/análogos & derivados , Quitina/química , Chlorocebus aethiops , Coloides/química , ADN/química , ADN/genética , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Células Endoteliales/citología , Endotelio Vascular/citología , Genes Reporteros , Luciferasas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Químicos , Peso Molecular , Plásmidos/metabolismo , Poliaminas , Polietilenglicoles/química , TransfecciónRESUMEN
A variety of degradable hyperbranched poly(ester amine)s containing primary, secondary and tertiary amino groups, were synthesized and evaluated as non-viral gene carriers. The polymers were obtained in high yields through a Michael-type conjugate addition of diacrylate monomers with trifunctional amine monomers. Analysis of degradation products using liquid chromatography-mass spectroscopy (LC-MS) demonstrated that all poly(ester amine)s had a hyperbranched structure with a degree of branching of approximately 0.30. These poly(ester amine)s were readily water-soluble and degradable under physiological conditions (pH 7.4, 37 degrees C), in which more than 10% ester bonds were hydrolyzed within 4 h. Moreover, these hyperbranched poly(ester amine)s showed high buffering capacities between pH 5.1 and 7.4. Three out of nine synthesized polymers, i.e. p(HDDA-AEP), p(HDDA-AMP), and p(BDDA-AMP), were shown to effectively condense plasmid DNA into small-sized (approximately 94-135 nm) and positively charged complexes. Polymer/DNA complexes ('polyplexes') based on these three polymers, and larger complexes of p(BDDA-AEP) (approximately 497 nm) were able to transfect COS-7 cells in vitro. Importantly, the transfection activity of polyplexes was preserved in the presence of serum proteins. The highest transfection level was observed for p(HDDA-AEP) polyplex which had a transfection efficiency higher than or comparable to that polyplexes of polyethylenimine (PEI) and poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA). Furthermore, these poly(ester amine)s revealed no or low cytotoxicity. These results demonstrated that hyperbranched poly(ester amine)s can be applied as safe and efficient gene delivery polymers.
Asunto(s)
Portadores de Fármacos , Terapia Genética/métodos , Poliésteres/química , Algoritmos , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , ADN/administración & dosificación , ADN/química , Electroquímica , Espectroscopía de Resonancia Magnética , Tamaño de la Partícula , Plásmidos/genética , TransfecciónRESUMEN
A membrane-disrupting peptide derived from the influenza virus was covalently linked to different polymethacrylates (pDMAEMA, pDAMA and the degradable pHPMA-DMAE, monomers depicted in Fig. 1) using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as coupling agent to increase the transfection efficiency of polyplexes based on these polymers. It was shown by circular dichroism (CD) measurements that the polymer-conjugated peptide was, as the free peptide, able to undergo a conformational change of a random coil to an alpha helix upon lowering the pH to 5.0. This indicates that the property of the peptide to destabilize the endosomal membrane was preserved after its conjugation to the cationic polymers. In line herewith, a liposome leakage assay revealed that the polymer-bound peptide has comparable activity as the free peptide. The DNA condensing properties of the synthesized polymer-peptide conjugates were studied with dynamic light scattering and zeta-potential measurements, and it was shown that small (100 to 250 nm), positively charged (+15 to +20 mV) particles were formed. In vitro transfection and toxicity was tested in COS-7 cells, and these experiments showed that the polyplexes with grafted peptide had a substantially higher transfection activity than the control polyplexes, while the toxicity remained unchanged. Cellular uptake of the polyplexes was visualized with confocal laser scanning microscopy, and no differences in cellular uptake could be determined between the peptide containing systems and the control formulation. This shows that the increased transfection activity is indeed due to a better endosomal escape of the peptide grafted polyplexes. This study demonstrates that it is possible to covalently conjugate an endosome disruptive peptide to cationic gene delivery polymers with preservation of its membrane destabilization activity, making these conjugates suitable for in vivo DNA delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , Técnicas de Transferencia de Gen , Ácidos Polimetacrílicos/administración & dosificación , Animales , Células COS , Membrana Celular/metabolismo , Supervivencia Celular , Dicroismo Circular , Concentración de Iones de Hidrógeno , Metacrilatos/administración & dosificación , Nylons , Ácidos Polimetacrílicos/química , TransfecciónRESUMEN
A cationic polymethacrylate with a guanidinium side group was designed in order to create a polymer with cell membrane-penetrating properties such as Tat or other arginine-rich peptides. The polymer, poly(3-guanidinopropyl methacrylate), abbreviated as pGuaMA, was synthesized by free radical polymerization. The DNA-condensing properties of pGuaMA (Mw 180 kDa) were investigated via dynamic light scattering and zeta potential measurements, and small, positively charged particles (110 nm, +37 mV) were found. It was shown that polyplexes based on pGuaMA were able to transfect COS-7 cells efficiently in the absence of serum, while under the same conditions poly(arginine) (pArg) polyplexes did not show detectable transfection levels. Addition of a membrane-disrupting peptide, INF 7, derived from the influenza virus, to preformed pGuaMA polyplexes did result in approximately 2 times increased transfection levels. DLS, zeta potential measurements, gel electrophoresis, and ethidium bromide displacement measurements indicated that serum induced aggregation of the polyplexes at high polymer/plasmid ratios, while at low polymer/plasmid ratios the polarity of the polyplexes reversed likely due to adsorption of negatively charged proteins on their surface. Likely, the unfavorable interactions of pGuaMA polyplexes with serum proteins is the reason for the absent transfection activity of these polyplexes in the presence of serum. Confocal laser scanning microscopy indicated cellular internalization via endocytosis of both polyplexes and free polymer. Thus, pGuaMA polyplexes enter cells, as reported for other polyplexes, by endocytosis and not, as hypothesized, via direct membrane passage.
Asunto(s)
Marcación de Gen/métodos , Guanidina/síntesis química , Polímeros/síntesis química , Ácidos Polimetacrílicos/síntesis química , Animales , Células COS , Cationes , Chlorocebus aethiops , ADN/administración & dosificación , ADN/química , ADN/genética , Sistemas de Liberación de Medicamentos/métodos , Guanidina/administración & dosificación , Polímeros/administración & dosificación , Ácidos Polimetacrílicos/administración & dosificación , Transfección/métodosRESUMEN
A set of 1438 human exons was subjected to nested PCR. The initial success rate using a standard PCR protocol required for ligation-independent cloning was 83.4%. Logistic regression analysis was conducted on 27 primer- and template-related characteristics, of which most could be ignored apart from those related to the GC content of the template. Overall GC content of the template was a good predictor for PCR success; however, specificity and sensitivity values for predicted outcome were improved to 84.3 and 94.8%, respectively, when regionalized GC content was employed. This represented a significant improvement in predictability with respect to GC content alone (P < 0.001; chi(2)) and is expected to increase in relative sensitivity as template size increases. Regionalized GC was calculated with respect to a threshold of 61% GC content and a sliding window of 21 bp across the target sequence. Fine-tuning of PCR conditions is not practicable for all target sequences whenever a large number of genes of different lengths and GC content are to be amplified in parallel, particularly if total open reading frame or domain coverage is essential for recombinant protein synthesis. Thus, the present method is proposed as a means of grouping subsets of genes possessing potentially difficult target sequences so that PCR conditions can be optimized separately in order to obtain improved outcomes.