Higher DNA Yield Research Articles

High yield gold nanoparticle-based DNA isolation method for human papillomaviruses genotypes from cervical cancer tissue samples.

Gold nanoparticles (AuNPs) are commonly used in biosensors of various kinds. However, its application to extract DNA from cancer tissues has not been extensively studied. The purification of DNA from cancer tissues is an important step in diagnostic and therapeutic development. Almost, all cervical cancer cases are associated with high-risk human papillomavirus (HR-HPV) infection. Accurate viral diagnosis has so far relied on the extraction of adequate amounts of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Till now, no specific and sensitive DNA purification method has been introduced for the extraction of HR-HPV from FFPE tissue. Since the commercially available purification kits are not sensitive and specific enough for HR-HPV DNA targets, in this study, a DNA purification method was designed based on AuNPs to purify sufficient amounts of HR-HPV DNA from cervical cancer tissue samples. AuNPs were coated with a series of oligonucleotide probes to hybridize to specific DNA sequences of HR-HPV genotypes. Results showed that 733 out of 800 copies of type-specific HPV DNA were recovered with complete specificity, compared to 36 copies with a standard commercial kit (Qiagen FFPE). The high yield of DNA (91.6%) is the main advantage of the AuNPs-probe purification method.

Impact of swab material on microbial surface sampling

Efficient microbial sampling from surfaces for subsequent detection and quantification is crucial in fields such as food safety and hygiene monitoring. Cotton swabs are traditionally used for sample collection, but today there are numerous swabs of alternative material and different sizes available. Recovery efficiencies of swabs for different applications have been compared in several studies. However, the results are often contradictory. We have compared 15 different swabs made of cotton (n = 5), flocked nylon (n = 3) and foam (n = 7), for sampling of Listeria monocytogenes and mengovirus on small (4 cm2) and large (100 cm2) areas of window glass, ridged plastic and absorbing wood. Molecular quantification methods (qPCR and RT-qPCR) were applied, and all sampling and sample processing were standardized. Specific swabs gave higher DNA/RNA yields than others, depending on both the surface characteristics and the collected target. The highest DNA yields were achieved by applying Selefa or Puritan cotton swabs for Listeria sampling on 4 cm2 areas of window glass and ridged plastic. Certain foam swabs (Critical swab with medium head and Macrofoam) gave the highest yields when sampling Listeria on 4 cm2 areas of wood and on 100 cm2 areas of ridged plastic and wood. Most foam swabs, and especially Sigma Virocult, were advantageous for virus sampling, regardless of surface. Nylon-flocked swabs showed poor recovery regardless of surface characteristics. The recovery varied substantially between swabs made of the same material, suggesting that a single swab may not be representative for a certain swab material.

DNA Photocleavage in the Near-Infrared Wavelength Range by 2-Quinolinium Dicarbocyanine Dyes.

Here, we report the syntheses of two pentamethine cyanine dyes containing quinolinium rings and substituted with either hydrogen (3) or bromine (4) at the meso carbon. The electron withdrawing bromine atom stabilizes dye 4 in aqueous buffer, allowing complex formation to occur between the dye and double-helical DNA. UV–visible, CD, and fluorescence spectra recorded at low DNA concentrations suggest that dye 4 initially binds to the DNA as a high-order aggregate. As the ratio of DNA to dye is increased, the aggregate is converted to monomeric and other low-order dye forms that interact with DNA in a non-intercalative fashion. The brominated dye 4 is relatively unreactive in the dark, but, under 707–759 nm illumination, generates hydroxyl radicals that cleave DNA in high yield (pH 7.0, 22 °C). Dye 4 is also taken up by ES2 ovarian carcinoma cells, where it is non-toxic under dark conditions. Upon irradiation of the ES2 cells at 694 nm, the brominated cyanine reduces cell viability from 100 ± 10% to 14 ± 1%. Our results suggest that 2-quinolinium-based carbocyanine dyes equipped with stabilizing electron withdrawing groups may have the potential to serve as sensitizing agents in long-wavelength phototherapeutic applications.

The art of obtaining a high yield of cell-free DNA from urine.

Although liquid biopsies offer many advantages over tissue biopsies, they are not yet standard practice. An important reason for the lack of implementation is the unavailability of well standardized techniques and guidelines, especially for pre-analytical conditions which are an important factor causing the current sensitivity issues. To overcome these limitations, we investigated the effect of several pre-analytical conditions on the concentration of cell-free DNA (cfDNA) and cellular genomic DNA (gDNA) contamination. Urine samples from healthy volunteers (HVs) and cancer patients were collected and processed according to specific pre-analytical conditions. Our results show that in samples with a relatively small volume more than 50% of the cfDNA can be found in the first 50 mL of the urine sample. The total DNA concentration increased again when samples were collected more than 3.5 hours apart. Adding preservative to urine samples is recommended to obtain high concentrations of cfDNA. To remove the cellular content, high speed centrifugation protocols as 4,000g 10min or 3,000g 15min are ideal for urine collected in cfDNA Urine Preserve (Streck). Although this study was a pilot study and needs to be confirmed in a larger study population, clear trends in the effect of several pre-analytical conditions were observed.

Evaluation of DNA Extraction Methods for Detection of <i>Leishmania</i> by Polymerase Chain Reaction

There are several methods used to obtain DNA from cells; however, the quantity, integrity, and purity of DNA vary among the methods, which may interfere with the polymerase chain reaction (PCR) results. The objective was to determine the most efficient and cost-effective method that provides the best DNA yield and PCR results. Three methods of DNA isolation were compared: 20% sodium dodecyl sulfate (SDS), guanidine isothiocyanate-phenol-chloroform (GTPC), and DNA extraction using a commercial kit (GE Healthcare GenomicPrep Blood DNA Isolation KitTM). Human peripheral blood samples were inoculated with 104 promastigotes of Leishmania infantum. DNA was quantified and PCR was performed with 13A/13B primers. The results showed that a higher DNA yield was obtained using the GTPC technique (214.51 ng/μL), followed by SDS (26.16 ng/μL) and the commercial kit (10.99 ng/μL). We concluded that while all of the techniques were effective for obtaining DNA, the GTPC method provided the best yield and the brightest bands.

The need for automation is limited when using a quick and inexpensive one-tube DNA extraction protocol for crime scene samples

Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions. We have developed, validated and implemented a quick, inexpensive and efficient one-tube direct lysis DNA extraction protocol for the majority of our crime scene samples. The method includes one pipetting step, the addition of a lysis buffer based on Chelex beads, Proteinase K and Tween 20. After three incubation steps with vortexing in between, the sample is ready for downstream applications (DNA quantification and STR profiling). The amount of lysis buffer added varies between 200–1000 μL depending on the amount of carrier material in the tube. If needed the extract can be purified or concentrated using a filter device, in our case Amicon Ultra-2. Through in-house validation we show that the method is fit-for-purpose for application in casework for samples containing blood, saliva, shed cells and semen as it provides high DNA yields and high quality STR profiles. The method was implemented at the Swedish National Forensic Centre (NFC) in February 2018. During the first year more than 35,000 crime scene samples were extracted using the method, generating DNA yields equivalent to the previously used method. In conclusion, the need for automation of the DNA extraction process is limited when using a one-tube direct lysis protocol including only the addition of lysis buffer and no transferring steps. In addition it saves costs as there is no need for expensive pipetting robots or service fees and since the reagents used in this direct lysis protocol are relatively inexpensive.

Evaluation of sexual assault evidence collection kit: Comparison of the success rate of STR analysis of swabs versus rinses

In Belgium, it is a general recommendation to collect swabs and rinses in rape cases. We evaluated the need of both prelevations for successful DNA analysis based on historical data. DNA concentrations and success rate of STR analysis of a total of 213 PSA positive rinses (including 29 anal and 184 vaginal rinses) and 256 PSA positive swabs (including 55 anal and 201 vaginal swabs) were compared. The study includes 154 cases where both a swab and a rinse were analysed simultaneously. Considering all vaginal and anal swabs and all rinses together, the median of the DNA concentration of DNA isolates of swabs is approximately 2.5 times higher than those of the DNA isolates of rinses. Moreover the success rate of obtaining an autosomal STR profile useful to identify the assailant is 72% for swabs versus 65% for rinses for the cases where both a swab and a rinse were examined simultaneously. In conclusion, swabs were found to have a higher DNA yield and a higher success rate of STR analysis for identifying the alleged assailant than rinses.

Comparison of DNA Isolation and Dominant and Co-dominant Molecular Markers to Reveal the Genetic Sex of Gallus domesticus (Domestic Chickens)

Purpose: The quality of DNA and reliability of molecular markers are crucial for the success of Polymerase Chain Reaction (PCR) based genetic sex determination. This study was aimed at investigating the optimum conditions for isolation of DNA from chicken blood and the reproducibility of dominant and co-dominant sex markers to be validated as a tool for successful sexing in avian research.Research Method: Efficacy of six different extraction procedures including manual and solution based commercial purification kit were evaluated with different combinations of initial blood, lysis buffer and protein denaturant in relation to the DNA yield and purity. Three primer sets namely CHD1, HUR 0423 and HUR 0424 were evaluated by PCR. Findings: The study results showed that 10µl of initial blood volume yields a significantly high DNA yield with high purity. Dominant marker HUR0424 showed to be a reliable marker system for the genetic sexing of domestic chickens over co-dominant markers.Research Limitation: For the accuracy of the results, protocols had to be followed at the same time and using same sample to avoid any errors.Originality/ Value: PCR based sexing is considered, the most accurate and inexpensive method and hence validation of the method is important for success of future avian research.

Ekstraksi DNA total dari sumber jaringan hewan (Ikan Kerapu) menggunakan metode kit for animal tissue

DNA extraction is a series of processes to separate DNA from other cell components. Extraction is the most crucial early stage in molecular research. There are two methods in DNA extraction: traditional organic extraction and non-organic adoption extraction method. Traditional organic extraction method are used by many laboratories to obtain amount of high-yield DNA. However, in recent years it has been a tendency to adopt non-organic commercial protocols, known as extraction kit. This method was relatively faster, low times-consuming and avoid the toxicity due to phenol use. This study aims to determine the steps of DNA extraction to obtain total DNA from muscle tissue of fish grouper using Extraction Kit for Animal Tissue. The Kit extraction method has an advantages that is the processing time is shorter, simple, non-toxic and high-purity DNA yields. A total of 4 tissue samples from grouper muscle were successfully extracted using an Extraction Kit that was indicated by the visualization of the band on qualitative DNA analysis in 1% agarose gel.
 Keywords: DNA extraction, extraction kit, high-yield DNA, molecular method

DNA and RNA extraction from low amount of blood volume

Clinical and research-based tests in molecular biology require a substantial amount of DNA and RNA, unfortunately, a considerable number of cells is needed for this amount of sample. Blood is one of the best and easiest source of cells, but is not used due to its invasive drawing methods and the needed volume. Another considerable point is the low amount of samples detected in crime scenes. TRI reagent is one of the most available methods for DNA, RNA and protein extraction. However, based on unsuccessful results, this method has not been widely used on blood samples. In this study, for the first time, the use of TRI reagent on micro scale blood volume was reported, resulting in high yield of DNA and RNA with great quality.

Comparative assessment of DNA extraction procedures forAscarisspp. eggs

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

Comparative account of DNA extraction protocols in some freshwater prawns of Genus Macrobrachium (Bate, 1868) (Family Palaemonidae) from Jammu waters for PCR based applications

Genetic variations among prawns act as an important tool to characterize and differentiate between the species. Molecular and phylogenetic analysis of shrimps and prawns like any other organism rely on high yields of pure and better quality genomic DNA. In this regard isolation of DNA is the first and basic step. In spite of the availability of many protocols of DNA extraction from animal tissues, it is difficult to ascertain that which one would provide desired results for prawn tissue. In the present study, three different techniques of DNA isolation i.e., salting out, phenol-chloroform and Qiagen DNA extraction kit were performed and compared for their yield. Cephalothoracic tissue and muscle tissue of pleopods were used for isolation. Tissue samples from fresh specimens as well as from alcohol preserved specimens were employed for extraction. The quantity (µg/ml) and quality of isolated DNA were determined by UV spectrophotometry and agarose gel electrophoresis. Results showed that Phenol-chloroform method with slight modifications obtained higher yield of genomic DNA as compared to other methods. The present work also revealed that among fresh specimens cephalotoracic tissue yielded high concentration DNA than muscle tissue. However, among alcohol preserved specimens, the concentration of DNA was higher in muscle tissue of pleopods. The high quality DNA was then subjected to randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis. The DNAs produced clear, sharp and reproducible PCR (Polymerse chain reaction) product pattern.

Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study

Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7–164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at −80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and − 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method.

A mathematical model for a biphasic DNA amplification reaction.

Isothermal DNA amplification reactions are a prevalent tool with many applications, ranging from analyte detection to DNA circuits. Exponential amplification reaction (EXPAR) is a popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrated unexpected biphasic amplification and much higher DNA yield than EXPAR. This ultrasensitive DNA amplification reaction (UDAR) shows high-gain, switch-like DNA output from low concentrations of DNA input. Here we present the first mathematical model of UDAR based on four reaction mechanisms and show the model can reproduce the experimentally observed biphasic behaviour. Furthermore, we show that three of these mechanisms are necessary to reproduce biphasic experimental results. The reaction mechanisms are (i) positively cooperative multistep binding spurred by two trigger binding sites on the template; (ii) gradual template deactivation; (iii) recycling of deactivated templates into active templates; and (iv) polymerase sequestration. These mechanisms can potentially illuminate the behaviour of EXPAR as well as other nucleic acid amplification reactions.

Diff-Quik Cytology Smears from Endobronchial Ultrasound Transbronchial Needle Aspiration Lymph Node Specimens as a Source of DNA for Next-Generation Sequencing Instead of Cell Blocks

Background: Next-generation sequencing (NGS) in lung cancer specimens from endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) is usually performed on formalin-fixed paraffin-embedded cell block material. Objectives: Since DNA can be damaged by this process, we investigated the potential of using DNA extracted from Diff-Quik cytology smears made for rapid on-site evaluation during EBUS-TBNA. Methods: In a prospective study, 67 patients undergoing diagnostic EBUS-TBNA were ana­lysed. We compared cell blocks and smears for DNA yields and sequencing (TruSeq Amplicon Cancer Panel) outcomes. Smears were also evaluated for tumour cell fraction and overall cellularity (cell count). Results: Primary lung cancer was diagnosed in 64 patients and metastatic malignancy in 3 patients. The DNA yield from smears was significantly higher than that obtained from matched cell blocks (mean 1,740 vs. 434 ng; p = 0.001). For 33 cases with matched smears and cell blocks the mutation profiles were similar. Smears with abundant malignant cells (using a cut-off of > 25% tumour cell fraction and > 1,000 cells) accurately predicted high (> 50 ng) DNA yield and therefore success in triaging samples to sequencing. In terms of tissue workflow, using only smears as source DNA for sequencing was an improvement in the use of only cell blocks (54/67 [80.6%] vs. 41/67 [61.2%]); however, the use of cell blocks when smears were not available or did not yield sufficient DNA further improved the success rate to 62/67 (92.5%) cases. Conclusion: We recommend smears in laboratory workflows as the primary source of DNA for NGS following an EBUS procedure.

A Modified Method for Rapid Genomic DNA Extraction from Tuberose

The isolation of high yield and quality genomic DNA is crucial for studying the molecular genetics of plants. However, high contents of secondary metabolites, especially in medicinal and aromatic plants, interfere with the extraction of clean DNA, thereby rendering it useless for downstream analyses such as DNA amplification, restriction, sequencing and cloning. The chemotypic heterogeneity among plant species may not permit high quality DNA isolation with a single protocol, thus a species-specific extraction method is required for quality extractions. Here, we present a modified cetyltrimethylammonium bromide (CTAB) protocol for good quality DNA extraction from tuberose, which is an important plant for perfume and pharmaceutical industry due to its pleasant fragrance and essential oil content. In contrast to other CTAB methods, the modified procedure is rapid, omits the use of liquid nitrogen and phenol, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use ofPVP-10 to get rid of problems associated with polysaccharides and polyphenols, respectively. The yield and quality of extracted DNA were fairly good and amenable for downstream analyses. Moreover, to our knowledge, the described method is the first report of a modified DNA extraction protocol for tuberose.

New DNA Extraction Method for the Detection of Pneumocystis in Lung Tissue Samples of Colonized Individuals

Background: Pneumocystis jirovecii colonization in patients is associated with a low organism burden, which supports the need to use highly sensitive molecular techniques, such as nested-PCR to determine the presence of the organism. However, few studies have considered the effect of nucleic acid extraction methods on the detection of P. jirovecii. Here, we evaluate how pre-treatment affects microbial detection. Methods: Lung tissue samples from fifteen autopsied infants were processed using two different DNA extraction protocols: 1) the QIAamp DNA Mini kit (Qiagen) (method A) and the QIAamp DNA Mini kit (Qiagen) with additional steps including a pre-treatment, bead-beating and phenol-chloroform steps (method B). Detection of P. jirovecii was performed using the conventional nested-PCR in lung tissue samples. In addition, we estimated the bacterial, fungal and human DNA using qPCR method. Results: Higher DNA yields was obtained with DNA extraction method B compared to method A. P. jirovecii was detected in 10 out of 15 lung tissue samples for both methods at the first or second round of PCR. However, P. jirovecii was detected in 9 out of 10 at the first round with the DNA extraction method B, rather than those processed with DNA extraction method A that were detected in 5 out of 10. Moreover, the faint PCR band intensity observed in 2 out of 6 samples processed with DNA extraction method A was increased with method B. We also observed that DNA extraction method B increased significantly the bacterial and fungal DNA, but not the human DNA, compared to method A. Conclusions: Our findings show that the additional steps (i.e. pre-treatment, bead-beating and Phenol: Chloroform: Isoamyl alcohol steps) supplemented to the QIAamp DNA Mini kit (Qiagen) improves the detection of P. jirovecii in lung tissue samples.

Improving cost-efficiency of faecal genotyping: New tools for elephant species.

Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.

Higher DNA Yield for Epidemiological Studies: A Better Method for DNA Extraction from Blood Clot.

Blood clots can be used to extract DNA, but they are not as widely used as whole blood or buffy coats. This is due not only because of the relatively low DNA yields and quality obtained from blood clots, but also because sampling prior to DNA extraction is more difficult. To solve these problems, we compared several clot liquefaction methods, determined the four most feasible methods, and subsequently performed a comparative analysis among them. We compared the yields and optical density ratios of the resulting DNA samples and assessed their integrity using agarose gel electrophoresis, polymerase chain reaction, and next-generation sequencing (NGS). Each of the four methods has advantages and disadvantages. But in general, higher yields of DNA with better quality and integrity were obtained using the high-shear homogenization method than using the other three methods. Additionally, this method is cost-effective and feasible at large operational scales. The DNA yields and A260/280 ratios were optimal and stable, the operation time and labor costs were acceptable, and the success rate of NGS applications was 99.74%. Furthermore, we developed a simple and rapid method for cleaning the homogenizer head to remove residual samples. According to our experimental results, our cleaning method effectively eliminated the risk of cross-contamination caused by the homogenizer head. We recommend high-shear homogenization as a superior method for clot liquefaction. We believe that this method is worthy of large-scale application as it can improve the efficiency of DNA extraction from clots, thus reducing labor and economic costs.

Optimization of fermentation conditions for an Escherichia coli strain engineered using the response surface method to produce a novel therapeutic DNA vaccine for rheumatoid arthritis

BackgroundFermentation condition optimization and nutrients screening are of equal importance for efficient production of plasmid DNA vaccines. This directly affects the downstream purification and final quality and yield of plasmid DNA vaccines. The present study aimed to optimize the fermentation conditions for high-throughput production of therapeutic DNA vaccine pcDNA-CCOL2A1 by engineered Escherichia coli DH5α, using the response surface method (RSM).ResultsWe hypothesized that optimized fermentation conditions significantly increase the yield of pcDNA-CCOL2A1 therapeutic DNA vaccine, a novel DNA vaccine for treating rheumatoid arthritis (RA). Single-factor analysis was performed to evaluate the optimal basal culture medium from LB, 2 × YT, TB, M9 (Glycerol) and M9 (Glucose), respectively. Thereafter, the Plackett-Burman design (PBD) was used to ascertain the three most significant factors affecting the vaccine yields, followed by the paths of steepest ascent to move to the nearest region of maximum response. Initial screening through the PBD revealed that the most key factors were peptone, mannitol, and inoculum concentration. Subsequent use of RSM was further optimized for the production of therapeutic DNA vaccine pcDNA-CCOL2A1 through Box-Behnken design (BBD). The final optimized fermentation conditions were as follows: peptone, 25.86 g/L; mannitol, 8.08 g/L; inoculum concentration, OD = 0.36. Using this statistical experimental design, the yield of therapeutic DNA vaccine pcDNA-CCOL2A1 markedly increased from 223.37 mg/L to339.32 mg/L under optimal conditions, and a 51.9% increase was observed compared with the original medium.ConclusionsThe present results provide a basis for further production of high-quality and high-yield therapeutic DNA vaccine pcDNA-CCOL2A1 in pilot-scale and even industrial-scale.