Why purification of dna

Article Views Altmetric -. Citations Abstract A microchip solid-phase extraction method for purification of DNA from biological samples, such as blood, is demonstrated. Cited By. This article is cited by publications. Analytical Chemistry , 90 9 , Analytical Chemistry , 89 6 , Schlappi , Stephanie E. McCalla , Nathan G. Schoepp , and Rustem F. Analytical Chemistry , 88 15 , Marco , and Erkin Seker.

Journal of the American Chemical Society , 24 , Biondi , Jordan J. Feld , and Warren C. ACS Nano , 10 4 , Analytical Chemistry , 87 2 , Root , Carmen R. Reedy , Jeffrey A. Hickey , Orion N. Scott , Joan M. Bienvenue , James P. Landers , Luc Chassagne , and Philippe de Mazancourt. Analytical Chemistry , 86 16 , Analytical Chemistry , 84 14 , Analytical Chemistry , 84 11 , Leslie , Jingyi Li , Briony C.

Strachan , Matthew R. Begley , David Finkler , Lindsay A. Bazydlo , N. Scott Barker , Doris M. Haverstick , Marcel Utz , and James P. Journal of the American Chemical Society , 12 , Duarte , Carol W. Price , Brian H. Augustine , Emanuel Carrilho , and James P. Analytical Chemistry , 83 13 , Analytical Chemistry , 83 9 , Root , Abhishek K. Agarwal , David M. Kelso , and Annelise E. Analytical Chemistry , 83 3 , Estes , John P. Haley , Colin R.

Analytical Chemistry , 82 16 , Reedy , Kristin A. Hagan , Briony C. Strachan , Joshua J. Higginson , Joan M. Bienvenue , Susan A. Greenspoon , Jerome P. Ferrance and James P. Analytical Chemistry , 82 13 , Marshall and Juan G. Analytical Chemistry , 81 22 , Hagan , Whitney L.

Meier , Jerome P. Analytical Chemistry , 81 13 , Analytical Chemistry , 81 11 , Analytical Chemistry , 81 9 , Langmuir , 25 5 , Hagan , Joan M. We checked for cell lysis due to the sDNA extraction procedure by epifluorescence microscopic enumeration Methodological details in SOM. To assess how other sDNA extraction methods compared to ours, we performed cell counts on sediments from which sDNA had been extracted using protocols by Ogram et al.

Nucleic acid extraction efficiency was assessed by fluorescence spectroscopic measurements and quantitative real-time PCR qPCR assays. DNA or RNA solutions were kept on ice until addition of dye, then maintained at room temperature until measurement.

Each run included extraction and PCR negative controls. PCR inhibition was checked using and dilutions of extracts.

All standards, controls, samples, and sample dilutions were run in duplicate or triplicate. Table 3. Using the online statistical software program Wessa.

This test was chosen after confirming—using the D'Agostino skewness test, Anscombe-Glynn kurtosis test, and Jarque-Bera Normality Test on a subset of data—that DNA yields within treatments and treatment permutations did not deviate significantly from normality. P -values below 0. We present the tests, which were instrumental to the design of this modular protocol for nucleic acid extraction.

During the protocol development, progress in extraction yields was not always linear, with outcomes of downstream treatments resulting in modifications to upstream treatments late in the development. For the sake of clarity, we have organized the test results in the chronological order of the extraction protocol. We here only discuss effects of bead-beating and homogenizer treatment.

We compared effects of bead-beating on gene copy numbers across organic-rich coastal sediment, oligotrophic subglacial lake sediment, drilling mud, and subseafloor sediment cuttings Figure 1. No effect was observed by contrast in subglacial lake sediment Figures 1C,D. Figure 1. BB indicates sample was bead-beaten prior to freeze-thawing and chemical lysis, no BB indicates no bead-beating prior to freeze-thawing and chemical lysis.

White bars indicate bacterial copy numbers, gray bars archael copy numbers. Only bacterial qPCR checks were performed on drilling mud and cuttings. Solid error bars indicate ranges of two replicate extractions, dashed error bars indicate ranges of PCR replicates on the same extract. Spillage and overheating of sediment extracts occurred during the use of homogenization probes. Overheating also occurred when these probes were directly applied to basalt rock.

We conclude that this physical disruption method is not compatible with our extraction method. Typically these cycles were preceded by freeze-thawing. Similarly, neither the combination of proteinase K and lipase nor the combination of proteinase K, lipase, and lysozyme increased DNA yields relative to treatments where enzymes were omitted.

Figure 2. Effects of lysis buffer composition on DNA yields. Drilling mud was used for the tests shown in E. D Effect of SDS 0.

The two detergents were added alone or in combination to the TE-based extraction buffer containing guanidium hydrochloride.

We kept the proteinase K treatment and examined how changes in extraction buffer composition affected DNA yield. The variables tested were pH 5. Parallel to increasing the pH from 5. Adding guanidium hydrochloride and Triton X had no significant effect, and neither did reducing the SDS concentration from 4 to 0.

For both detergents, we observed positive concentration-dependent effects when they were added alone. Yet, Triton X produced higher bacterial copy numbers when the same volumes of detergent were added 0. Interestingly, increasing the amount of Triton X from 0.

Thus, we—contrary to the results based on drilling mud—opted for a final lysis solution consisting of 30 mM Tris-HCl, 30 mM EDTA, mM guanidium hydrochloride, and—unless specified otherwise—0. We termed this solution lysis solution I. We investigated possible benefits of adding a second aqueous lysis solution, consisting of 2.

PVPP was added because it binds polyphenolic substances. We termed this solution lysis solution II. However, neither chemical significantly affected DNA yields data not shown. Over the course of our tests, three different chemical lysis protocols were established, each suited for a different sample type or extraction criterion. For sediment from Subglacial Lake Whillans, archaeal copy numbers increased more than twofold compared to LP I Figure 3D , but there was no difference in bacterial copy numbers Figure 3C.

In certain highly oxidized samples, e. Figure 3. Error bars indicate data ranges for samples where extractions were duplicated A—D. From then on, a pH of Figure 4. Error bars in C indicate data ranges of duplicate DNA extractions. We tested how adding different phosphate species prior to cell lysis affected nucleic acid recovery. In organic-rich sediment from Aarhus Bay Station M5, addition of pyrophosphate prior to cell lysis caused an average increase in DNA yield at intermediate phosphate additions, but this effect was not statistically significant Figure 4B.

Similarly, adding two different concentrations of dNTPs to Greenland glacial lake sediment had no effect on bacterial or archaeal gene copies Figure S3B. These elevated archaeal gene copy numbers at the high PO 4 treatment were reproduced in a second extraction test 5. Yet, the opposite trend, i. DNA yields in several other samples, e. By comparison, addition of small amounts of dsDNA 0.

We also tested addition of salmon sperm DNA. The results were inconclusive, however, as viewing qPCR products on agarose gels revealed unspecific amplifications of both bacterial and archaeal 16S rRNA genes. This suggested that—without further treatment of salmon sperm DNA to eliminate this unspecific amplification—reliable quantifications of bacterial and archaeal gene copy numbers were not possible.

After the various lysis and adsorption prevention treatments, we washed nucleic acid extracts with CI or PCI. Initially, we had also tested washes with phenol and chloroform; however, DNA yields were lower with pure phenol, and chloroform alone failed to produce a sharp interface between the aqueous and organic phase. In the absence of a sharp interface, clean transfers of aqueous supernatants containing nucleic acids were more difficult. Due to the stabilizing effect of isoamylalcohol on aqueous-organic interfaces, we only used CI or PCI from then on.

Several observations were made nonetheless: on one hand, washes with PCI more efficiently removed color, detergent and precipitates than the initial CI wash.

Thus, in some cases, three CI washes were necessary to remove precipitates and obtain the same visual purity as after one PCI and one CI wash. On the other hand, phenol oxidation, indicated by pinkish to bright red color, often occurred after vortexing PCI with DNA extracts. This discoloration was—as mentioned earlier—most prominent in oxidized sediments, such as red clays. However, even in organic-rich, anoxic sediment from Aarhus Bay Station M1 there was an increase in phenol oxidation, from being virtually absent in surface sediments to being pronounced in deeper layers, especially with extracts from the terrestrial soil layer.

Figure 5. In two separate experiments, two different concentrations of bp DNA ladder were prepared by dilutions with water or lysis solution I. Error bars indicate standard deviations of tests that were run in triplicate. In further tests, we examined effects of lysis solution I carryover on DNA recovery. We used DNA ladder instead of DNA extracts as a template, and mixed this ladder with water or lysis solution I prior to precipitation with polyethylene glycol solution. Figure 6.

This ensured that nucleic acids in solution were exposed to LPA and added salt. Without this prior homogenization step, LPA was immediately precipitated without going into solution. Due to light sensitivity of LPA, all precipitations were for 2 h in the dark. Error bars in A-C indicate standard deviations of tests that were run in triplicate, error bars in D indicate data ranges of tests that were run in duplicate.

We also checked the influence of precipitation temperature—i. DNA pellets were smallest when precipitation was conducted at room temperature, possibly due to less co-precipitation of residual detergent, and with the lower NaCl concentration, due to less co-precipitation of salt. These smaller pellets more readily dissolved after drying. Consequently, we opted for precipitations at room temperature and 1.

The size of DNA pellets differed markedly between different precipitation methods. In fact, after PEG precipitation, DNA pellets were frequently invisible and often did not stick to centrifuge tube walls.

To further improve our precipitation methods, we examined whether interactions between the amount of PO 4 added and the precipitation method affected the DNA yield and purity after precipitation Figure S4C. Based on dilutions of extracts, there was no difference in bacterial 16S rRNA gene copy numbers at different amounts of PO 4 added 15 vs.

However, based on undiluted extracts, PCR inhibition was more pronounced in high PO 4 treatments, and highest in high PO 4 treatments that had been precipitated with ethanol. Furthermore, effects of centrifugal force 14, vs. With overall DNA recovery being very high and virtually no DNA loss compared to non-precipitated original solutions, none of these treatments performed significantly better than the others.

Since extract dilution reduces the detection sensitivity of nucleic acids by spectrofluorometry or qPCR. Figure 7. Triplicate DNA extracts from Station M5 were divided into equal parts for these tests, with each half of the extract purified by a different kit.

Subsequently, the same general protocol that was the outcome of the tests outlined in the previous section was used. The sDNA fraction is extracted by a 1-h incubation of 0. Throughout the incubations, samples are kept at room temperature and gently mixed, e.

Figure 8. D Comparison of cell counts on sediments that had undergone different methods of sDNA extraction. These methods were by Ogram et al. Controls consisted of sediment that had not undergone sDNA extraction.

No effect was seen. There was no difference in cell numbers after our sDNA extraction protocol compared to controls without sDNA extraction. To assess how other sDNA extraction methods compared to ours, we also performed cell counts on sediments after sDNA extraction following the protocols by Ogram et al. We observed no cell loss after treatment by the Ogram et al. Further tests, in which we omitted SDS, which had been included at low concentration in the original sDNA extraction method by Corinaldesi et al.

We then examined possible sources and particle size-associations of sDNA. Our results indicate that nearly all sDNA passes through a 0. Ratios of the amount of DNA passing through 0. In a buried terrestrial soil layer We then examined if repeated freezing of sediment samples affected the extracted nsDNA pool size Figure S6.

One-time freezing had virtually no effect on the size of the nsDNA pool. Even two freeze-thaw cycles yielded no statistically significant change in extracted nsDNA. We conclude these tests by examining size distributions of sDNA and nsDNA pools by gel electrophoresis, using two different precipitation and cleanup methods Figure S7. We examined the compatibility of our protocol with RNA extraction.

Figure 9. Error bars in A indicate standard deviations of extractions that were run in triplicate. We tested possible variations of the manufacturers protocol, including longer digest times 2 h instead of 30 min or addition of bovine serum albumin to improve the efficiency of the DNA digest, but found that neither resulted in improvements Eickenbusch, DNA yields obtained with this extraction protocol were significantly higher than those obtained with three widely used DNA extraction kits.

The only exceptions were bacterial copy numbers in the soil layer, which were nearly identical across all three methods Figure 10E. Figure Therefore, any automatic system must include not only automatic equipment for each extraction step but also equipment for automating the transfer of liquid between machines. Automation has aided in increasing the throughput and improving the reliability of the process, but these systems are still designed for use in a laboratory environment only.

Some of the nucleic acid extraction system that are available in the market are large and require manual pre-processing stages by laboratory staff with technical expertise [ 54 ]. A combination of all-in-one biomolecules extraction solution and method with fully automated extraction system can be a prospective invention in the future. The purification of DNA, RNA or protein from various organisms can be performed simultaneously using this type of extraction system with just a single extraction method.

It is often inconvenient that targeted biomolecules sample from an animal, plant or even a clinical sample must be sent to a laboratory for it to be extracted and analyzed [ 54 ]. The samples, especially clinical sample such as blood, need to be refrigerated and transferred to the nearest laboratory for extraction and analyzing. Hence, a portable biomolecules extraction system, which brings several advantages such as reduced labour, reduced waste and increased speed of extracting process, can be a potential development in the future [ 54 ].

The combination of portable extraction system with DNA, RNA, or protein analyzer can be build up in the future to help researchers in reducing working time and increasing the work efficiency. Continued improvement in miniaturization will be the future trend of robotic automation in the laboratory [ 28 ]. Many clinical laboratories are performing workflow analysis and finding that smaller systems with lower throughput are more consistent with clinical laboratory workload.

Besides, this automation system can be implemented at relatively low cost, improving the turnaround times and also reduce the labor costs [ 55 ]. Since the first DNA isolation was successfully done by Friedrich Miescher in and the initial DNA extraction developed from density gradient centrifugation strategies by Meselson and Stahl in , many techniques for biomolecules purification has been developed. From guanidinium thiocyanate-phenol-chloroform extraction to the column-technology that is widely used in DNA and RNA extraction, and chromatography purification method to immunoblotting that used to extract proteins, biomolecules extraction has helped researchers and scientists in manipulating subsequent molecular biology analysis in order to have a better understanding in the biological materials of the earth.

The automated nucleic acid extraction system has been developed due to the influence of rapid growth of automation technology nowadays.

Automating nucleic acid extraction process is potentially beneficial for a number of reasons including to reduce working time, decrease labor costs, increase worker safety and at the same time provides opportunity in increasing reproducibility and quality of results. However, improvement of the weaknesses for some of the instruments needs to be conducted all the time. In the mean time, an all-in-one biomolecules extraction system, or the invention of a miniature and portable extraction system can become a prospective development in the future.

This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors. Read the winning articles. Journal overview. Special Issues. A Erratum for this article has been published.

Academic Editor: Joakim Lundeberg. Received 01 Jul Accepted 05 Nov Published 30 Nov History 2. Protein Extraction In the eighteenth century, proteins were known as a distinct class of biological molecules by Antoine Fourcroy and others. Current Tendency After the fated event where Miescher managed to obtain DNA from cell, many others have followed suit which lead to further advancement in the DNA isolation and purification protocol. Type of Nucleic Acid Extraction 3.

Solid-phase Nucleic Acid Extraction Solid-phase nucleic acid purification can be found in most of the commercial extraction kits available in market. Type of Protein Extraction Method The first step in protein purification is cell lysis. Ion Exchange Chromatography Ion exchange chromatography separates proteins based on their surface ionic charge using resin that are modified with either positively-charged or negatively-charged chemical groups [ 4 , 7 ].

Gel Filtration Chromatography Gel filtration chromatography, also called size-exclusion or gel-permeation chromatography, separates proteins according to molecular sizes and shape and the molecules do not bind to the chromatography medium [ 39 ].

Affinity Chromatography Affinity chromatography depends on a specific interaction between the protein and the solid phase to affect separation from contaminants. Table 1. Typical biological interactions used in affinity chromatography [ 42 ]. References M. Buckingham and M. Cseke, P. Kaufman, G. Podila, and C. Kojima and S. View at: Google Scholar J. Watson, T. Baker, S. Bell, A. Gann, M. Lecine, and R. View at: Google Scholar S. Smarason and A.

Sambrook and D. Chomczynski and N. Birnboim and J. Esser, W. Marx, and T. Gjerse, L. Hoang, and D. Smith, D. Holmes, D. Simpson, J. Kayzhendler, R. Bitner, and J. View at: Google Scholar V. Padhye, C. York, and A. View at: Google Scholar D. Woodard, A. Howard, and J. View at: Google Scholar K. View at: Google Scholar T. Arnold, M. Meyering, and R. Dederich, G.

Okwuonu, T. Garner et al. View at: Google Scholar M. Beckman Coulter, Inc. Thermo Scientific Inc. Selingson and E. Fido, E. Mills, N. Rigby, and P. Bowen, J. Steinberg, U. Laemmli, and H. Handen and H. Loeffler, K. Schmidt, H. Hebart, and H. View at: Google Scholar G. Sadarangani, B. MacDonald, A. Rodrigo, and D. View at: Google Scholar A. View at: Google Scholar.

More related articles. There are a number of techniques used in purifying genomic and plasmid DNA samples. These include the following:. Since there are a lot of techniques to choose from, you should take the following criteria into consideration to determine the most suitable purification method for your sample:. To make the process less time-consuming, biochemical companies developed genomic isolation kits that will suit a wide array of applications.

There are also a number of products available for the isolation, transformation and high throughput screening of plasmids.