Title of Invention

PROCESS FOR EXTRACTION OF SUPERIOR-QUALITY PLASMID DNA

Abstract

(57) Abstract: 1. An improved process for the extraction of superior quality plasmid DNA, said process comprising the steps of: a.. harvesting bacterial cells by centrifugation, b. suspending the harvested cells in solution I such as herein described, c. lysating the suspended cells by adding solution II such as herein described, d. neutralising the lysed cells by adding solution ITI such as herein described, e. centrifuging the neutralised cells and thereby precipitating cell debris tind proteins and to obtain a supermatant, f. adding NaCI stock, into the supernatant to increase concentration of Na-+- ions in the supermatent, g. adding a silica suspension such as herein described to the aupernatant to obtain 11 silica solution, h. centrifuging the silicu solution of step e bound to silica pellets, i- w'ashing the silica pellets -with a buffer to remove impurities and J. eluting the bound plasmid DNA with distiled water or flow unit buffer and therby obtaining the superior quality plasmid DNA. PRICE: THIRTY RUPEES

Full Text

FIELD OF THE INVENTION The invention relates to the field of molecular biology and genetics. More particularly, the invention relates to methods used for DNA purification. The present invention is directed to a novel process for the extraction of superior quality plasmid DNA, a composition for DNA purification and a kit prepared in accordance with the said process. BACKGROUND Preparation of plasmid DNA from bacterial vectors on a small scale is a basic requirement in laboratories engaged in molecular biology research. Alkaline lysis of the bacterial cells is one of the common methods used for preparation of plasmid DNA from bacterial cultures. This is a very simple and rapid method wherein bacterial cells are lysed using SDS, chromosomal DNA, and the proteins are precipitated and removed by pheno[-chloroform extractions. The plasmid DNA is eventually recovered by precipitation using alcohols. However, various problems are associated with this process. For instance, the DNA extracted by this method is often crude and the quality is inadequate for subsequent molecular manipulations. Although phenol is efficient in denaturing proteins, mpDNA (mini-prep DNA) made by standard alkaline lysis has several impurities, especially proteins which are of bacterial origin. The residual protein impurities left in the plasmid DNA preparation inhibits subsequent enzyme manipulations. Additionally, the organic impurities associated with phenol oi chloroform preparations also inhibits enzyme digestion. Whenever mpDNA is extracted by the conventional alkaline lysis method, higher quantities of restriction enzymes are required for digestion. Further, with several not-so-good-cutters, it is often essential to perform a second round of phenol chloroform extraction of the DNA to ensure enzyme digestion. Another problem associated with the alkaline lysis method is that this method is time consuming. Good quality mpDNA could also be prepared using a number of commercial kits (Geneclean II, BIO 101; Ion-exchange resins, Qiagen elc). Although the quality of mpDNA extracted using these commercial kits is superior, the standard alkaline lysis protocol continues to be popular, as the cost of the kits are unaffordable for routine use. Commercial kits as well as several previous reports employed silica or silica derivatives as principal matrices for specific isolation of nucleic acids. The primary objective of several of these protocols, however, is to purify DNA fragments from agarose gel. PRIOR ART REFERENCES:- There are several other protocols that employ silica or its derivatives for extraction of plasmid DNA. The kits for these processes are expensive. For instance, Marko et al describe a procedure for "the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder" in Analytical Biochemistry, 121 382-7(1982) wherein the source of silica is giass-powder, a different form of silica. The process employs chaotropic agents such as Na-perchlorate to facilitate DNA binding to silica. The said agent is expensive, chemically unstable, hazardous. SDS is sued for lysis of bacterial cells. This protocol is extremely complicated. It also uses a chaotropic ion (sodium perchlorate) instead of NaCl, which is expensive, hazardous and unstable. Bacterial cells are lysed by standard alkaline lysis method using SDS instead of Triton-XlOO. Boyle & Lew in 'An inexpensive ahemative to glassmilk for DNA purification'. Trends in Genetics. U 8, 1995 describe another method wherein a chaotropic ion Nal (sodium iodide) was used instead of NaCl. Nal is unstable and relatively more expensive. They also used Gaimidine which is also a chaotropic ion. No mention is made of how the bacterial cells were lysed, hence they must have used the standard alkaline lysis method (using SDS instead of Triton X-100). Boom et al. in 'Rapid and simple method for purification of nucteic acids' J. Clinical Microbiology, 28 495-503 (1990), wherein SDS was used instead of Triton for the lysis of bacteria. The chaotropic ion Guanidine thiocyanate was used here for DNA binding to silica instead of NaCl. Carter and Milton 'An inexpensive and simple method for DNA purifications on silica particles'. Nucteic Acids Research. 21 1044 (1993) used diatomaceous earth (different tprm of silica, also available from sigma and others commercially). Silica has much more binding capacity for DNA than diatoraaceous earth. In this process, SDS was used for lysis of the bacterial cells instead of Triton X-100. Also, guanidine thiocyanate, which is a chaotropic salt (unstable, expensive and hazardous) has been used instead of NaCl. U.S. Patem No.5,503,816 relates to a process for DNA purification using silicate compounds. The conventional processes are used for isolation of DNA, Chaotropes such as Sodium perchlorate and Nal are used along with NaCi, isopropanol, guanidine HCI. The process is not directed towards use of NaCl as main ingredient, which is mentioned as passing reference. Most commercial kits in the market use silica based derivatives. Most of these are confidential or proprietary. All are expensive. Hence, these cannot be used routinely in laboratories. Accordingly, in order to obviate the drawbacks of the said prior art processes, the applicants have developed a novel process for the extraction of superior quality plasmid DNA and a composition for DNA extraction in accordance with the said process. OBJECTS OF THE INVENTION It is an object of the invention to provide a cheap, economical and quality process for the extraction of superior quality plasmid DNA for routine use in labs; Another object of the invention is to provide a process, which avoids harmful effects of the use of phenol. Yet another object of the invention is to provide a process, which eliminates the step of RNAase digestion. it is a further object of the invention to provide a process, which uses half the time, required for conventional alkaline lysis method. Another object of the invention is to provide a process wherein the re-agents used are inexpensive and stable at room temperature. It is a further object of the invention is to provide a process, which enables the preparation of superior quality mpDNA in the laboratory on a routine basis without the need for a commercial kit, expensive, unstable, hazardous re-agents or sophisticated equipment. It is another object of the invention to provide a novel and good quality composition, which can be used for the extraction of plasmid DNA routinely in labs. SUMMARY OF THE INVENTION The present invention relates to a novel process for the lysis of bacterial cells using Triton X-100 {t-Octyl phenoxy polyethoxyethanol) Mid NaOH, wherein the pH is neutrali2ed using K-acetate, plasmid DNA is purified ftom celt lysate using silica-suspension in the presence of high sodium concentration, and is eluted using sterile distilled water or low salt buffer following alcohol washes. The invention also provides a novel composition, which can be used, routinely for the purification of plasmid DNA in labs, comprising 10 ml of cell suspension buffer, 20 ml of Bacterial lysis solution, 15 ml of neutralization buffer 45 ml of DNA bind-solution and 10 ml 10 times wash buffer concentrate. In addition, the invention provides a kit prepared in accordance with the said process. DETAILED DESCRIPTION: In accordance with the above and other objectives, the applicant has devised an improved process for the extraction of superior quality plasmid DNA^^said process comprising the steps of: An improved process for the extraction of superior quality plasmid DNA, said process comprising the steps of: a. harvesting bacterial cells by centrifugation, b. suspending the harvested cells in solution I such as herein described, c. lysating the suspended cells by adding soJution II such as herein described, d. neutralising the lysed cells by adding solution III such as herein described, e. centrifiiging the neutralized cells and thereby precipitating cell debris and proteins and to obtain a supernatant, f. adding NaCl stock into the supernatant to increase concentration of Na+ ions in the supernatant, g. adding a silica suspension such as herein described to the supernatant to obtain a silica solution, h. centrifuging the silica solution of step g bound to silica pellets, i. washing the silica pellets with a buffer to remove impurities and j. eluting the bound plasmid DNA with distilled water or low salt buffer and thereby obtaining the superior quality plasmid DNA. In one embodiment, solution I comprises a mixture of 50mM glucose, 25 mM Iris. CI of pH 8.0, and lOmM EDTA (pH 8.0). In another embodiment, solution II comprises a mixture of 0.2N NaOH, and any non-ionic detergent. In a preferred embodiment, the non-ionic detergent is selected from 3-5% t-Octyl phenoxy polyethoxyethanol (Triton-X-100), NP-40, iVlonolaurate {Tween-20), Monooleate (Tween 80), IgePal CA-630, Polyoxyethylene thers (Brij) etc. In yet another embodiment, solution III comprises a mixture of 5M potassium acetate and glacial acetic acid (pH 4.8). In a further embodiment, the centrifugation may be carried out in a microcentrifuge, at a speed preferably more than 8,000 rpm. In yet another embodiment, the concentration of Na* ions in the sample after addition of NaCl stock may be 2-4 M. In a ftirther embodiment, 15 [il of silica suspension used in the process is taken from the stock prepared by suspending 6 gms of silica in 50 ml of distilled water, decanting the fine silica particles and resuspending the sedimented particles in 50 ml of sterilized water. In yet another embodiment, the wash buffer may comprise 50-70% ethanol, 1 OmM TrisofpHS.O, 100 mM NaCl, and ImMEDTA. The invention is described in detail with reference to the following examples and drawings. Various modifications of the process described that would be apparent to those in the art are intended to fall within the scope and teachings of the present invention. The applicant presents a very simple and economic strategy to prepare mpDNA from bacterial cultures. Tliis strategy, with an important modification to the standard alkaline lysis protocol and in combination with the silica matrix, is simpler and faster and yields superior quality mpDNA. In addition, this strategy neither requires extractions with hazardous organic compounds such as phenol or chloroform nor enzyme digestion with RNAse. The present invention relates to a simple and reproducible protocol for extracting plasmid DNA from bacterial cultures on a smaller scale for routine use in the laboratory. The triton/silica protocol, of the invention is technically simpler and faster than the standard alkaline lysis method and the quality of the plasmid DNA isolated is superior. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 represents the extraction of mpDNA by increasing concentrations of triton X- 100. Figure 2 represents a comparison of mpDNA preparation by three different methods. Figure 3 represents a comparison of restriction enzyme patterns of mpDNA prepared by three different methods. Figure 4 represents a comparison of the purity of the mpDNA isolated using triton silica method with that of alkaline lysis method. Figure 1: Extraction of mpDNA by increasing concentrations of Triton X-IOO. Bacterial cells (DH5a) transformed with pBC!2/PL/SEAP (9) were harvested from 1.5 ml of over¬night cultures. Following re-suspension of the pellet in 100^1 of Solution-l, the cells were lysed by adding 200 ^1 of Solution-2 containing 0.2 N NaOH and 1 % SDS, or 0.2 N NaOH and varying concentrations of Triton X-100 as shown at the top of the lanes. The subsequent steps for the isolation of the mpDNA are as described in the protocol. Five \i\ (10%) of the mpDNA were resolved on a 1% agarose gel and stained with ethidium bromide. rigure i.: comparison of mpDNA preparation by three different methods. Bacterial cells transformed with pBC12/PL/SEAP were harvested from 1.5 ml of over-night cultures, Plasmid DNA was isolated by standard alkaline lysis method (lanes 1 and 2), or using a combination of 1% SDS in Solution-2 and siiica (lanes 3 and 4) or 4% Triton X-IOO in Solution -2 and silica (lanes 5 and 6). Following the addition of 150fil of 5 M potassium acetate and transfer to fresh vials, samples were incubated with (lanes 2, 4 and 6) or without (lanes 1, 3 and 5) RNAse-A at SeX for ! hr. One \\S. of RNAse-A (10 mg/ml) was added to SDS/silica and Triton/silica mpDNA preparations (lanes 2 and 4), whereas 25)J1 of the enzyme were added to alkaline lysis preparation (lane 6). Following the incubation, phenol-chloroform extraction and alcohol precipitation were used to extract mpDNA by standard protocol (1). Plasmid DNA by the modified procedure was prepared as described in the protocol. Five fil (10%) of the mpDNA were resolved on a 1% agarose gel and stained with ethidium bromide-Figure 3: Comparison of the restriction enzyme patterns of mpDNA prepared by three different methods. Plasmid DNA was isolated from bacterial cells using three different protocols as described in the legend for figure 2- RNAse-A treated mpDNA preparations were restricted with several enzymes. Results with three representative enzymes are shown. Fivefil of mpDNA were incubated with 1.0 or 10 units (as shown at the top of the lanes) of the indicated enzyme at 37°C for one hour. Following the incubation, the samples were resolved on a 1% agarose gel and stained with ethidium bromide. Following restriction patterns were expected after successful enzyme-digestion: BamH 1 = 1,695 and 3,532 kb; Kpn 1 = linearization; Pst 1 = 1,687, 1,834 and 1,914 bp. M= 1 kb ladder (GibcoBRL). Figure 4: Comparison of the purity of the nipDNA isolated using triton/silica method with that of alkaline-lysis. Bacterial cells transformed with pBC12/PL/SEAP were harvested from 1.5 ml of over-night cultures- Plasmid DNA were isolated by standard alkaline lysis or a combination of 4% Triton X-100 in Solution -2 and silica as described in the protocol. Fivefil of the mpDNA preparation concentrations of Hind 111 (top panel) or EcoR 1 (bottom panel) for one hour at 37"C. Concentration were incubated with varying of the enzyme (units/reaction) is shown at the top of the lanes. Following restriction patterns were expected after successful enzyme digestion: Hind III = linearization; EcoR ! = 801 and 4,425 bp. M= 1 kb ladder (GibcoBRL). Several protocols employed silica for extracting DNA fragments from agarose gel. A block of gel containing the DNA fragment is dissolved in excess solution of a chaotropic salt and the DNA fragment is rescued from the solution using silica suspension. The potential of silica, however, has not been exploited for developing a similar inexpensive strategy for routine preparation of good quality mpDNA. At the outset the applicant wanted to develop an analogous approach to prepare mpDNA by combining the standard alkaline lysis method using 1% SDS and the silica matrix. Although mpDNA free of bacterial proteins or other impurities could be recovered by this method, the DNA was completely resistant to restriction (see below). The applicant reasoned that SDS being negatively charged could also bind the silica particles, persist through the washing steps and co-elute with the plasmid DNA. SDS could inhibit enzyme activities. To test for this possibility, the applicant replaced SDS with non-ionic detergents in Solution 2. Plasmid DNA obtained with 1% Triton X-100 or IGEPAL CA-630 was readily restricted with different enzymes while that of 1% SDS was not. Non-ionic detergents lacking a negative charge, probably do not interact with the silica particles and are removed during the washing step. Although 1% Triton X-100 could lyse the bacterial cells, the yield of mpDNA was considerably lower than that of 1% SDS, indicating incomplete lysis. SDS bemg an ionic detergent is very efficient in lysing bacteria! cells. To improve the level of lysis, The applicant tested gradually increasing concentrations of Triton X-IOO and compared with that of 1% SDS (Figure 1). Lysis by Triton X-100 up to 2% concentration appeared incomplete. In addition, the species of the mpDNA obtained at this concentration were different from those of 1% SDS; alkaline supercoiled DNA was the major species isolated. Triton X-!00 at 3% concentration or above completely lysed the bacterial cells. The pattern of DNA species obtained was also identical to that of SDS, with covalently closed DNA molecule being more abundant than the other forms (Figure 1). Based on these results The applicant used 4% Triton X-100 in the subsequent experiments. IGEPAL CA-630 also appeared to function equally efficient (results not shown). Using the triton/silica protocol, most of the plasmid DNA from the bacterial lysate could be isolated with 15 fil of silica suspension. Addition of fresh silica to bacterial lysate, already extracted once, or re-elution of silica particles, by adding fresh distilled water yielded insignificant quantities of additional mpDNA. One advantage of using the triton/silica protocol is that only trace levels of bacterial RNA are co-purified with the mpDNA (Figure 2, compare lane 1 with 5) obviating the need for RNAse treatment. Presence of this RNA did not interfere with restriction of the mpDNA using several enzymes. This RNA, however, could be readily eliminated by adding a few units of RNAse-A to the bacterial lysate (Figure 2, compare lanes 3 and 5 with 4 and 6, respectively). Plasmid DNA prepared by the standard alkaline lysis contained not only large quantities of bacterial RNA but also this RNA was partially resistant to enzyme digestion (Figure 2, lanes 1 and 2). In the standard alkaline lysis protocol, lysis of the bacteria is achieved with 1% SDS in Solution 2. In addition to dissolving the bacterial membrane, SDS would also denature and precipitate bacterial protein. Addition of potassium acetate to the lysate would neutralize sodium hydroxide and any residual SDS. The optimized protocol presented here is analogous to the standard alkaline lysis method so far as the lysis of the bacterial cells is concerned. Lysis in our protocol, however, is accomplished with Triton X-100, not SDS. Unlike SDS, Triton X-100 may not be capable of denaturing bacterial protein or chemically interacting with potassium acetate. Our strategy, however, deviates from the standard protocol in the manner the plasmid DNA is recovered from the bacterial lysate. The applicant employed silica matrix for efficient purification of the plasmid DNA from the bacterial lysate. Since silica demonstrates strong affinity for nucleic acids but not proteins, the possible presence of intact bacterial proteins and other impurities in the lysate may not be of concern. In the triton/silica protocol, chromosomal DNA is removed from the bacterial lysate by alkali-denaturation and precipitation whereas bacterial proteins and other impurities are eliminated during the washing steps. Binding of DNA to silica requires the presence of high salt concentration, Tlie applicant used sodium chloride rather than sodium iodide (Geneclean II. BIO lOl). guanidium thiocyanate (4). or sodium perchlorate (3) to facilitate DNA binding to silica. The applicant tested varying concentrations of sodium chloride ranging from 1 to 4 M and found that 2 M and above allow maximum binding (results not shown). Importantly, use of sodium chloride instead of a chaotropic salt to promote DNA binding to silica offers the advantage that sodium chloride is inexpensive and the solution is stable at room temperature. Moreover, when compared in parallel, sodium chloride co-eluted the least quantity of bacterial RNA than sodium iodide or guanidium thiocyanate (results not shown). Restriction enzymes vary in the efficiency to tolerate the levels of impurities present in the mpDNA preparations. While enzymes such as BamH I, Hind III and Bgl II are relatively tolerant to the presence of impurities, others such as Kpn I and Dra HI are often sensitive. It is important to ensure the quality of the DNA when such sensitive enzymes are used. To test for the quality of mpDNA isolated by the triton/silica method, the applicant used restriction analysis as a direct measure of purity. The applicant digested mpDNA with several restriction enzymes at two different concentrations of the enzyme. Plasmid DNAs isolated by standard alkaline lysis and 1% SDS/silica method were also included for comparison. Results obtained with three representative enzymes are shown (Figure-3). BamH I at 10 or 1 units readily restricted plasmid DNA isolated by standard alkaline lysis method. Pst 1 restricted this DNA only at higher enzyme concentration whereas, Kpn 1 failed to restrict at either concentration. Kpn I, however, digested the same DNA preparation following an additional phenol-chloroform extraction indicating that it is possibly the presence of bacterial protein impurities that inhibited this enzyme. Phenol used in these experiments was distilled to ensure the quality and frozen in aliquots at -gCC. It is unlikely that impurities present in phenol inhibited Kpn I digestion as the same DNA preparation was readily restricted by BamH I and several other enzymes. Although Kpn 1, in this particular experiment failed to restrict the mpDNA isolated by alkaline lysis, on other occasions, the applicant observed digestion. Experimental variation in lestriciion paiiein is coniiuon with sensitive enzymes such as Kpn I when mpDNA is prepared wilh alkaline lysis cxtiaclion. iirespeclive of what enzyme used, all the enzymes, at both liic concentiations readily restricted mpDNA prepared by the Iritoii/silica method. The applicant used ten or more different enzymes in these experiments. Tritoa'silica extraction consistently and reproducibly yielded very high quality DNA that was digested by all the enzymes used. Plasmid DNA extracted by SDS/silica method, on the other hand, was not digested by any of the enzymes used (Figure 3). Reducing SDS concentration from 1% to 0.4% in soiulion-2, however, did not inhibit subsequent enzyme-restriction, if the concentration of SDS was made a limiting factor, 0.4% in this experiment, probably all the SDS was precipitated by potassium acetate leaving mpDNA free of residual SDS. The purity of mpDNA prepared by triton/silica was compared further with that of standard alkaline lysis method. Plasmid DNA preparations were incubated with serial ten-fold dilutions of two restriction enzymes (Figure 4). Plasmid DNA prepared by alkaline lysis was restricted with Hind lU and EcoR I at 10 and 1.0 units. Reducing the enzyme concentration ftiither resulted in failure of restriction. Plasmid DNA prepared by triton/silica method, on the other hand, was restricted at enzyme concentrations much lower; up to 0.1 and 0.01 unit with EcoR I and Hind III, respectively. Although EcoR I did not release the 801 bp fragment at 0.01 unit enzyme concentration, a condition which requires restriction at two different sites, much of the plasmid in this reaction was restricted at least at one site (Figure 4. last lane). This result provided evidence that mpDNA isolated by triton/silica method is of superior quality as compared to standard alkaline lysis. The applicant routinely use restriction enzymes at O.iunit concentration in our laboratory for plasmid digestion thus significantly reducing the consumption of these expensive enzymes. Plasniid DNA isolated by triton/silica method is also amenable for manipulation using other enzymes such as Klenow, T4-ligase and Taq- DNA polymerase. Although The applicant presented here results obtained with one strain of the bacterial host (DH5a) and one plasmid vector. The applicant obtained identical results with a few more bacterial hosts (XL-1 blue, DH-1 and DHlOp) and a variety of plasmids that varied in size. Tlie applicant repoiis here a simpie and reproducible protocol for small-scale isolation of plasmid DNA from bacterial hosts. This protocol is safer than the standard alkaline lysis method as it eliminates the need for organic reagents. Silica matrix used here is inexpensive and a 100 gm quantity is sufficient for a moderate size laboratory for a few years (approximately 50,000-55,000 individual mini-preps could be prepared). In addition, digestion of the plasmid preparation with RNAse is also not necessary. Elimination of organic extraction, ElNAse digestion and alcohol precipitation, combined together, not only renders this protocol very rapid but also highly economical. Using the triton/silica protocol it is possible to prepare mini-preps in half the time required for the standard alkaline lysis method. An added advantage of the modified protocol is that, the current protocol requires only two sets of plastic vials as opposed to four or more for standard alkaline lysis. Importantly, the quality of plasmid DNA isolated by this protocol is comparable to that of the commercial kits. This was analyzed by restriction digestion and spectrophotometry (results not shown). All the reagents used in this protocol are inexpensive and stable at room temperature. Using this protocol it is possible to prepare superior quality mpDNA in the laboratory on a routine basis without the need for a commercial kit, expensive/unstable/hazardous reagents or sophisticated equipment. The applicant believe that the tritorb'silica protocol presented here is the simplest and quickest of several protocols reported previously to prepare good quality mpDNA from bacterial cultures, with or without employing silica. Materials and methods Chemicals and enzymes: Silicon dioxide (S 5631), Igepal CA-630 (I 3021), Ribonuclease-A (R 6513) are from Sigma, Triton X-100 (22686) and all the restriction enzymes are purchased from Amersham International, England. Agarose and Sodium Chloride are from Life Technologies. Sodium dodecyl sulfate is from United States Biochemicals. Preparation of the Silica suspension: auica suspension was preparea as aescrmea ^^] wim minor modifications. Six grams of silica were suspended In 50 ml of sterile distilled water in a 50 ml plastic tube. The tube was left undisturbed at room temperature for 24 hours for the coarse silica particles to settle. Fine silica particles suspended in the top 43 ml were decanted and the volume was made up to 50 ml by adding fresh sterile distilled water. Contents were mixed and left for 5 hrs. Forty four ml of the supematant from the top were discarded, 60 i^l of concentrated HC! were added, contents mixed and autoclaved. Finally, the volume was made up to 50 ml with sterile distilled water, contents thoroughly mixed and the suspension stored in refrigerator in small aliquots. The washing procedure described above removed approximately 0.6 gm of fme silica. Fifteen |il of the final silica suspension (approximately 1.6 mg silica) were used per 1.5 ml bacterial culture as described in the protocol. Plasmid DNA extraction: The plasmid vector, pBC12/PL/SEAP, used in these experiments was a kind gift form Dr. Brian R. Cullen (9). Bacterial hosts were transformed by Calcium Chloride method as described (10). Single colonies were picked up from selection plates and grown over¬night in Luria-Bertani medium (1) at 37'C with shaking. Plasmid DNA was prepared by three different methods from 1.5 ml over-night bacterial cultures. Alkaline lysis was performed according to the standard protocol (I). The DNA pellet, followmg alcohol precipitation was dissolved in 50 pi of sterile distilled water or TE and stored at -20°C. A detailed protocol for preparing nijiDNA by the triton/silica procedure has been presented in the protocol. For extracting the mpDNA with SDS/silica, essentially the same protocol was employed except that Solulion-2 consists of 1% SDS instead of Triton X-100. Following the alcohol washes, DNA from the silica was eluted in 50 p.1 sterile distilled water or TE and stored at 4°C. Protocol: Preparation of mini-prep DNA bv the triton/silica method. 1) Harvest over-night grown bacterial culture in to 1,5 ml polypropylene vials. To the bacterial pellet add 100^1 of Solution-1 (50 mM glucose, 25 mM Tris, pH 8.0 and 10 mM EDTA). Suspend the cells thoroughly. 2) To each sample add 200^1 of modified Solution-2 {0.2 N NaOH and 4% Triton X-100). Close the cap and invert the vial a few times. The contents become clear indicating lysis of the bacterial cells. 3) Add 150^1 of So!ution-3 (5 M Potassium acetate, pH 4.8), close the caps and mix contents thoroughly. Incubate the vials for 5 min at room temperature, spin in a micro-centrifuge at full speed for 10 min. Transfer the supernatant to fresh vials. 4) To each vial add 450|.il of 5 M NaCl and mix. Add 15p,l of silica suspension (prepared as described in the materials and methods), close the cap and mix the contents with continuous agitation for ten minutes. Spin the vials at full speed for 10 seconds. Decant the supernatant. 5) Add 250|il of the wash solution (60% ethanol, 10 mM Tris, pH 8.0, 100 mM NaCl and 1 mM EDTA). Resuspend silica by vortexing or lapping. Spin and decant the wash solution. Repeat the washing a couple of times. 6) After the third wash, spin the silica pellet at full speed for 10 seconds and remove any traces of alcohol using a micropipette-tip. Leave the vials for 5 min at room temperature with caps open to allow residual alcohol evaporate. 7) Add 50|il of autoclaved distilled water or TE. Mix silica by gentle tapping and leave the vials for 5 min at 37°C. Spin at full speed for 10 seconds and collect the supernatant. Store the eluted DNA at 4°C. Preparation of the composition for DNA Purification: The invention provides a novel composition for the purification of plasmid DNA, comprising 10 ml of cell suspension buffer, 20 ml of bacterial lysis solution, 15 ml of neutralization buSTtv, 45 ml of DNA bind-solution and \0 ml 10 times wash buffer concentrate; The ingredients of the said novel composiiioii are as under: 10 mi of cell suspension buffer : 50 mM Glucose, 25 mM Tris pH 8.0 and lOmMEDTA, 20 ml Bacterial lysis solution : 0.2 N NaOH and 4% Triton X-100, 15 ml Neutralization buffer: 5M Potassium acetate pH 4.8, 45 ml DNA bind-solution: 5 M NaCl and silica suspension 3.5 mg/ml. 1 u 1111 1 u mil es Wash buffer concentrate: 100 niM Tris pH 8.0, I M NaCland lOmMEDTA, The kit is designed to provide reagents in separate containers to prepare piasmid PNA from bacterial cultures. The reagents are sufficient to prepare 100 individual piasmid isolation from 1 to 5 ml of bacterial culture (mini-prep scale).The same reagents may also be used to isolate piasmid DNA at a higher scale, for instance from 10 to 100 nfil of bacterial cultm-e. In the later case, the reagents will be sufficient for 10-25 individual piasmid preparations. Depending on the user's requirement, the kit provides flexibility as per the scale of piasmid isolation within a range of 1-100 ml bacterial culture per isolation. 1. An improved process for the extraction of superior quality piasmid DNA, said process comprising the steps of: a. harvesting bacterial cells by centrifugation, b. suspending the harvested cells in solution I such as herein described, c. lysating the suspended cells by adding solution II such as herein described, d. neutralising the lysed cells by adding solution III such as herein described, e. centriftiging the neutralized cells and thereby precipitating cell debris and proteins and to obtain a supernatant, f. adding NaCl stock into the supernatant to increase concentration of Na+ ions in the supernatant, g. adding a silica suspension such as herein described to the supernatant to obtain a silica solution, h. centrifuging the silica solution of step g bound to silica pellets, i. washing the silica pellets with a buffer to remove impurities and j. eluting the bound plasmid DNA with distilled water or low salt buffer and thereby obtaining the superior quality plasmid DNA. 2. A process as claimed in claim 1, wherein solution I comprises a mixture of 50mM glucose, 25 mM Tris. CI of pH 8.0, and lOmM EDTA. 3. A process as claimed in claim 1, wherein solution II comprises a mixture of 0.2N NaOH, and a non-ionic detergent. 4. A process as claimed in claim 3, wherein the ionic detergent is selected from 3-5% Triton-X-100, ND-40, Tween-20, Tween 80, IgePal CA-630, and Brij. 5. A process as claimed in claim 1, wherein solution III is carried out on a microcentrifuge, at a speed more than 8,000 rpm. 6. A process as claimed in claim 1, wherein the centrifugation is carried out on a microcentrifuge at a speed more than 8,000 rpm. 7. A process as claimed in claim 1, wherein the final concentration of Na+ ions in the supernatant is 2-4 M. 8. A process as claimed in claim 1, wherein about 15 p,l of silica suspension is prepared by suspending 6 gms of silica in 50 ml of distilled water, decanting the fine silica particles and resuspending the sedimented particles in 50 ml of sterilized water. 9. A process as claimed in claim 1, wherein the wash buffer comprises 50- 70% ethanol, lOmM Tris of pH 8.0, 100 mM NaCl, and ImM EDTA.

Documents:

0614-mas-1999 abstract.pdf

0614-mas-1999 claims.pdf

0614-mas-1999 correspondence-others.pdf

0614-mas-1999 correspondence-po.pdf

0614-mas-1999 description (complete).pdf

0614-mas-1999 drawings.pdf

0614-mas-1999 form-1.pdf

0614-mas-1999 form-13.pdf

0614-mas-1999 form-26.pdf

0614-mas-1999 form-5.pdf

0614-mas-1999 form-9.pdf

0614-mas-1999 others.pdf

0614-mas-1999 petition.pdf