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DNA was isolated and studied in order to understand how it works and 'read' and decipher its mysterious sequence. However most think that DNA is a new discovery, it had been isolated, analyzed and characterized as a macromolecule more than a century ago in 1869 by Friedrich Miescher (1844-1895). Friedrich graduated Joh.Friedrich Miescher-Rüesch 1844 - 1895
handing in an outstanding thesis in 1868 in Basel where he attended lectures by both his father and his uncle.
The discovery was made in Hoppe-Seyler's laboratory in Tubingen where his mentor, who was interested in body fluids, mainly blood, and has recently discovered the hemoglobin-oxygen binding in erythrocytes, has asked Miescher to work on lymphocytes. However, Miescher prepared leukocytes from pus and his isolation of pure nuclei was a success. In December, 1869, Miescher isolated a substance that is acid and alkali soluble with high phosphorus content from the nuclei and submitted a manuscript to his mentor, Hoppe-Seyler, writing about this new substance and he called it "nuclein", which was then called nucleic acid, today known as DNA. George Wolf, 2003 [Friedrich Miescher, The Man Who Discovered DNA]
Watson and Crick
Originally called James D. Watson and Francis Crick, used x-ray diffraction data provided by Rosalind Franklin to observe the double helix structure of the DNA molecule in 1953.
As described earlier, DNA was first identified in the 1860's by Friedrich Miescher and not by James Watson and Francis Crick like many people believe. After Miescher's discovery, a long chain
Watson and Crick
of research efforts were carried out by several other scientists, like Phoebus Levene and Erwin Chargaff to reveal more details about the molecule of DNA, its primary chemical components and the mechanism by which they joined each other.
Surely, without this basic foundation built by previous scientists, Watson and Crick could have never reached their memorable conclusion in 1953 - The three-dimensional structure of the double helix.
After Miescher's discovery, other scientists continued examining the 'nuclein' or the DNA molecule. The Russian biochemist Phoebus Levene, a former physician before being a chemist, was in particular a prolific researcher who published over 700 papers about biological molecules' chemistry along his career.
Long before the discovery of the sugar and phosphate backbone, in the beginning of Levene's career, no one knew the arrangement of nucleic acids in space. Levene's idea was a tetranucleotide structure with a constant oreder of sequence, for example G C T A G C T A G C T Aâ€¦.etc.. The high variability of the order of nucleic acids though proved his idea as over simplistic. His idea, however, is accurate in a way that the DNA nucleotides are serially ordered in codons and made up of three components, a phosphate group, a single nitrogen-containing base and a ribose (in RNA) or a deoxyribose (in DNA) sugar molecule along the stretch of DNA or RNA. Also, two basic nitrogenous bases categories, purines (adenine(A) and guanine(G)) with two fused rings and pyrimidines (thymine(T), cytosine(C) and uracil(U)) with a single ring. Furthermore, RNA contains only A,C,G and U instead of T, while DNA contains A,C,G and T instead of U as illustrated in the following figure.
Nature.com [Purines and Pyrimidines] The classification of DNA nitrogen-containing bases and their structure
Erwin Chargaff's discovery that A=T and C=G and crucial X-ray data revealed by Rosalind Franklin and Maurice Wilkins supported Watson and Crick's proposal of the three-dimensional double helix of the DNA. Also the advanced technique developed by Linus Pauling, a biochemist, that allows the prediction of three-dimensional structure of molecules provided the molecular distances and bond angles helped Watson and Crick's discovery. Pauling proposed another model of three-dimensional DNA structure just months before Watson and Crick proposed theirs. Which worried them a little until his model was proved incorrect.
Like putting together a puzzle, Watson and Crick placed cardboard cutouts representing the different chemical components of the bases and nucleotide subunits on their desktops, trying to predict the correct structure that would flawlessly fit. This was made possible after they fully understood how thymine and guanine's different elements were configured, upon the idea of an American scientist, Jerry Donohue. Their proposed structure, however, also reflected Chargaff's rule.
Over the years, scientists made some changes to Watson and Crick's model, or modified it, yet the four major features stayed the same today:
DNA is a double helix held together by hydrogen bonds. A=T and C=G.
All DNA, except Z-DNA, are right handed double helices.
The DNA double helix is anti-parallel.
The helix exterior of nitrogen bases bonded with each other internally are exposed and capable of further hydrogen bonding.
Leslie A. Pray, 2008 [Discovery of DNA Structure and Function: Watson and Crick]
The discovery of DNA leads us to understanding human genetics. It represents studying the inheritance factors that happen through generations of human beings. It holds several of connected topics including: classical genetics, cytogenetics, molecular genetics, biochemical genetics, genomics, population genetics, developmental genetics, clinical genetics, and genetic counseling, which are all based on using DNA isolation in the first place. Most inherited traits are connected to a common factor, genes. Studying human genetics explains a lot about human nature, inherited diseases and developing more accurate disease treatment and understand genetics of human life.
Studying human genetics leads to a better understanding of the nature and origins of human genetic variation. It opens the doors for the possibilities of probing deep into common and rare disease genetic basis. This study is shining the light down the path of new and effective diagnostic approaches, as well as clarifying human evolution. The rapid developing technology of studying human genetics is expected to bring major health benefits and put aside today's use of medicine.
Nucleic Acids (DNA/RNA)
The huge amount of information, detailing the specific structure of the proteins inside of our bodies, is stored in a set of molecules called nucleic acids - large molecules with a backbone of alternating sugar and phostphate molecules bonded together in a long chain.
Isolation of DNA
In any field of biotechnology and any other field engaging in the experiments of molecular biology, DNA isolation and purification technique is the primary routine to carry out to collect DNA for study, restriction digestion analysis, sequencing, genetic engineering, cloning, ligation, PCR amplification, genetic transformation, in vitro transcription, RFLP, RAPID, DNA fingerprinting, nick translation and radio labeling, preparing genomic DNA library and cDNA library and many other experimental purposes. A number of DNA isolation techniques have been developed, but all of them work the same way to a certain extent to extract DNA.
DNA extraction primarily includes, first, rupturing the cells from which the DNA of interest is to be extracted to expose the DNA within, which is commonly done by putting the cells under sonication of by simply grinding them. Coming next is the removal of membrane lipids which is done by adding a detergent. Third, removing proteins, done by adding a protease such as proteinase K. Fourth, precipitating down the DNA which is done by adding ice-cold alcohol like ethanol or isopropanol. DNA insolubility in such solvents causes it to coagulate giving a pellet of insoluble DNA after centrifugation.
Many different ways have been employed for breaking cells down. A rather common method used in lysing bacterial cultures is the alkaline lysis method. Animal cells can be lysed by hypotonic solutions or by simple detergents. Plant tissues can be treated by heating and using strong detergents like SDS.
With regard to biotechnology, isolated DNA is useful in the development of genetically engineered recombinant proteins and in identifying potential new therapeutic targets. In the clinical setting, isolated DNA is useful in the identification of genetic disorders and in the diagnosis of bacterial and/or viral infections. As such, there is a need for simple and reliable methods for isolating DNA, and in particular, for isolating high quality, high molecular weight DNA.
The most commonly used method for isolating DNA from a DNA source, e.g., blood, saliva, bacterial cultures, etc., involves lysing the DNA source with a combination of a
proteolytic enzyme and a detergent followed by extractin the mixture with an organic solvent, e.g., phenol and chloroform, so that the DNA enters the aqueous phase and the hydrolyzed products enter the organic phase. The DNA in the aqueous phase is then precipitated by the addition of alcohol. However, these organic extraction methods are laborious and time consuming and require the use of phenol
(or other organic solvents), which are typically toxic and, therefore, a safety hazard.
In another approach, the DNA is isolated by lysing the DNA source with a chaotropic substance, for example guanidinium salt, urea and sodium iodide, in the presence of
a DNA binding solid phase. The released DNA is bound to the solid phase in a one step reaction, where the beads are washed to remove any residual contaminants. Although these methods have proven to be less time consuming and toxic, they have resulted in a moderate level of DNA shearing and some level of contamination.
In a further approach, the DNA is isolated from a starting source by mixing the starting source with a cationic detergent, which forms a hydrophobic complex between the DNA and detergent. The hydrophobic complex is separated
from the solubilized contaminants and the DNA recovered by addition of a salt. As above, this approach has proven to be much less time consuming, but does result in some level of DNA shearing and contamination. Against this backdrop
the present invention has been developed.
One embodiment of the present work is a method includes mixing the starting material with a lysing and denaturing substance to release the DNA. The mixture is preferably vortexed for at least 5 seconds and allowed to incubate at at 75° C. for 10 minutes. The sample should be vortexed periodically.
Isolated nucleic acid DNA may be analyzed by any well known means within this method, including taking A260/A280 ratios, separating the nucleic acid via gel electrophoresis, etc. In addition, the isolated nucleic acid from the present method
is suitable for use in any number of molecular biology reactions, including PCR, DNA ligation, etc.
In preferred embodiments of the present method the nucleic acid is genomic DNA, and in more preferred embodiments of the present method the isolated nucleic
acid is genomic DNA having at least 60%, more preferably 70%, and most preferably 80%, of the isolated gDNA at least above 23 kb in molecular weight. In a most preferred embodiment of the present method the isolated nucleic acid is genomic
DNA having at least 60%, and more preferably 70%, of the isolated gDNA from above 23 to 75 kb in molecular weight.
Alkaline Lysis Method: Many variations to this protocol exist according to the type of bacterial cultures.
Followed up by phenol/chloroform extraction, this method is used for the large-scale plasmid/cosmid DNA isolation. First, the target cells containing either the plasmid or the cosmid DNA are harvested by centrifugation, incubated in a resuspension buffer and then treated with alkaline detergent. The chemical decomposition of the alkali causes the breakdown of the cells and the release of DNA and proteins into the medium which are solubilized by the detergent. The proteins, along with membranes, are precipitated by sodium acetate. This precipitate is centrifuged at a higher RPM and the DNA is pushed into the supernatant. 95% ethyl alcohol or propanol precipitates the DNA in a pellet that is resuspended in a Tris EDTA buffer. The DNA sample still is contaminated by some DNA binding proteins such as histones that have to be removed. Here is where phenol/chloroform extraction is employed.
Genomic DNA isolation from Blood: This method is based on the standard protocol by Federal Bureau of Investigation, USA.
The blood sample is stored at -70°C in EDTA vacutainer tubes and then thawed. A standard citrate buffer is mixed with the sample and the tubes are centrifuged. The supernatant's top part is discarded and more of the buffer is added, mixed and centrifuged. Supernatant is also discarded and the pellet is resuspended in a solution containing proteinase K and SDS detergent. This mixture is incubated for one hour at 55°C and then phenol-extracted with a phenol/chloroform/isoamyl alcohol solution. The sample is then centrifugated and an aqueous layer formed is removed to a fresh microcentrifuge tube. Ethanol precipitates the DNA which is then resuspended in buffer and ethanol precipitated again. The pellet is left to dry and then buffer is added to resuspend DNA overnight by incubation at 55°C. PCR is then used to assay the genomic DNA solution.
DNA isolation from Plant Tissues: Due to plant cells' rigid cell walls and extracellular toxic metabolites that may interact with the DNA undesirably, several problems are brought up. Metabolites form complexes with the DNA causing it not to function for further experimental purposes. So, the extraction buffer (e.g. CTAB) used should be supplemented by certain components that protect the DNA.
Detection of DNA
After isolating DNA, confirming its presence is crucial before any other routines to take place. Many ways of detection have been developed including detection in solution and in gels.
Two of the standard approaches to detecting DNA are UV Spectroscopy at 260 nm and Ethidium Bromide fluorescence.
UV Spectroscopy depends on the absorbency of a substance to detect it. A pure uncontaminated DNA sample would have an absorbency at 260 nm, a contaminated one reaches 280 nm. An absorbency of one A260 unit per ml represents the presence of 50 micrograms of DNA per ml, which gives a quantitative measure to a sample. However, in eukaryotes, RNA and DNA as well as proteins also absorb so it is hard to tell how large your DNA is. And in bacteria, plasmids and DNA have absorbency values causing them to both be detected. So using UV spectroscopy wouldn't make much use of the portion of the sample used, because there is a huge probability of cross-contamination.
The other method is about staining DNA and RNA by ethidium bromide in a 1ug/ml ethidium bromide solution. Mixing ethidium bromide staining with gel electrophoresis gives a high resolving power able to detecting 10 to 50 ng of DNA in a single band. The size of DNA can also be measured using the DNA's electrophoretic mobility in the gel, giving DNA identity. However, the DNA must be arranged in bands to be easily detected. If the sample is comprised of a mixture of different DNA sizes, the fluorescence would be spread among a bigger area of the gel. And if the sample is pure enough, spotting the DNA sample on a piece of plastic wrap and comparing its fluorescence to a standard of known concentration is enough for detection (no need for gel).
The PCR cycle
Extracting genes of interest to insert them in vectors or hosts follows up DNA isolation. One of the ways of extracting genes of interest from DNA is using thermal cyclers that carry out polymerase chain reactions (PCR). This reaction depends mainly on the same enzyme that is responsible for DNA replication, DNA polymerase. DNA polymerase's mechanism of action is synthesizing new DNA strands in a 5' to 3' direction from a DNA template that is single stranded. But DNA polymerase also can be polyfunctional in that it is able to remove nucleotides from either ends of the DNA strand.
Thermal cyclers use DNA polymerases to amplify DNA strands' regions of interest in PCR. This amplification is able to output as much as a microgram of DNA from only a few molecules of a target nucleic acid that are too few to be used or analyzed. Like the pattern of normal DNA replication process, PCR uses a DNA polymerase, thermostable ofcourse because of the high temperature used in PCR, like Taq DNA polymerase. Two primers are designed and used to define the target sequence to be generated.
The PCR cycle includes the several repeat of three steps: denaturation, annealing and extension. This repetitive technique is the source of thermal cyclers' amplifying power. The DNA template used produces copies during each cycle that double after each cycle and serve as templates for every next cycle. A million copies of the target DNA are produced from only 20 cycles. Roche Diagnostics GmbH, Mannheim, 2006 [PCR Application Manual].
The present research relates to an improved process for isolating DNA from biological samples, particularly from human whole blood.
The extraction of DNA represents an essential role in molecular biology. The extracted DNA and other things are considered as starting materials for diagnosis based on nucleic acids, for constructing genetic profiles and in the field of pharmacogenomics. Thus, the extraction of DNA/RNA from biological samples, like blood, body secretions, tissue samples, urine, stools etc., for further use in genetic analysis is of certain importance. In the methods known before for recovering nucleic acids, DNA is isolated from cells and tissues by destroying the starting materials under certain conditions to facilitate denaturation and reducing conditions, partly using protein-degrading enzymes, purifying the nucleic acid fractions obtained by means of phenol/chloroform extraction processes and recovering the nucleic acids from the aqueous phase by dialysis or ethanol precipitation [Sambrook, J., Fritsch, E. F. in T. Maniatis, C S H, "Molecular Cloning", 1989].
Along with these long-established methods of extracting DNA from cells or tissues comes a considerable amount of disadvantages, such as huge time consumption and a lot of experimental effort. These protocols are also connected with an acute risk of health harming to the researchers carrying out the purification techniques, such as chloroform or phenol.
The several amount of cons aforementioned leads to the production of newer alternative methods for extracting DNA from different starting materials, by which the dangerous and health risking phenol/chloroform method of extracting DNA could be avoided and the time taken shortened.
All these proposed methods known from the past researchers are based on the method developed and first described by Vogelstein and Gillespie [Proc. Natl. Acad. Sci. USA, 1979,76, 615-619] for preparing and analytical extraction of DNA fragments from agarose gels. The method includes the dissolving of the agarose containing DNA fragments in a chaotropic saline solution with the attachment of the DNA to carrier particles. The fixed DNA is then washed with a washing solution (20 mM EDTA; 50% v/v ethanol) and then separated from the carrier particles.
Up till now this method undergoes a number of modifications and is utilized for different processes for extracting and isolating nucleic acids from various sources
(Marko, M. A., Chipperfield, R. and Birnboim, H. G., 1982, Anal. Biochem. 121, 382-387). Also, there are several methods that have been developed globally primarily for purifying DNA from agarose gels and for the extraction of plasmid DNA from bacterial lysat. In addition to that, for isolating long chain nucleic acids (genomic DNA, total RNA) from tissues, blood or cell cultures.
Alot of these internationally available purification systems are derived from the well known principle of attaching nucleic acids to mineral carriers in the presence of solutions with different chaotropic salts. In these methods, suspensions of finely ground glass powder or silica gels are used as carrier materials.
Many patents, for DNA isolation technology, relate to the processes of nucleic acids extraction that can be utilized in a number of different applications. This discloses a process for isolating nucleic acids from starting materials which contain nucleic acids by incubating the starting material with a chaotropic buffer and a DNA-binding solid phase. The chaotropic buffers facilitate the lysing of starting materials and the binding of nucleic acids to the solid phase. The process is used to isolate nucleic acids from small samples and is particularly used in practice for isolating viral nucleic acids.
The physicochemical principle of the prior-art systems currently used and commercially available for isolating nucleic acids based on the binding of nucleic acids to the surfaces of mineral carriers is supposed to consist of the disruption of the higher structures of the aqueous medium by which the nucleic acids are adsorbed on to the surface of mineral materials, particularly glass or silica particles.
The disruption of the higher structures of the aqueous medium is always effected in the presence of chaotropic ions and is virtually quantitative when high concentrations of these ions are present. On the physicochemical basis described above a number of commercially available systems for isolating nucleic acids contain buffer compositions with chaotropic salts having high ion intensities which are regarded as necessary for the binding of nucleic acids to a nucleic acid-binding solid phase.
However, among the serious disadvantages of the process are the fact that the lysing effected by the chaotropic buffers is by no means suitable for all materials or is extremely inefficient for larger quantities of starting materials and is extremely time-consuming. In addition, different concentrations of different chaotropic buffers have to be used for different situations, which proves very complicated.
On the other hand, the isolation of nucleic acids based on a salting out step is known before[L.A. Salazar et al., Clin. Chem. 44 (1998) 1748; S. A. Miller et al. Nucleic Acid Res., 16(3) (1988) 1215]. These processes has the disadvantage, however, that during the process the reaction vessel has to be changed at least once, with the risk of mixing up the samples. Furthermore, the salted out proteins can only be separated from the nucleic acid which is to be purified by using the experimentally laborious method of centrifugation.
Another process for purifying DNA obtained from blood is disclosed by T. A. CiuUa [Analytical Biochemistry, 174 (1988) 485]. In this, first of all the cell nuclei are
liberated by a lysing step. The DNA contained therein is isolated using guanidium-iso-thiocyanate and P-mercapto- ethanol and subsequent precipitation with 2-propanol. How- ever, this process includes relatively time-consuming steps for resuspension and centrifuging. Moreover, this process uses a lysing buffer which is susceptible to microbial growthand has to be used in a nine-fold excess in relation to the
blood. Furthermore, P-mercaptoethanol is classed as a toxic substance.
The aim of the present research is therefore to provide a process which overcomes the disadvantages of the processes known previously.
In particular, the problem of the present research is to avoid the use of toxic or corrosive substances in the isolation of DNA. Furthermore, the present research sets out to propose a less time-consuming and less laborious process for isolating DNA.
A further aim of the present research is to provide a process which can be carried out without changing the reaction vessel-as a so-called "one-pot reaction"-and thereby minimises the risk of confusion or contamination of the samples. The above problems are solved according to the research by mixing the biological sample with a lysing reagent in a reaction vessel. In the next step the cell components which contain DNA are separated from the other cell components. This separation may be done by centrifuging, for example. The DNA contained in the sediment is then separated from other impurities such as proteins, for example. This separation is achieved by resuspending the sediment in a saline solution. Optionally, the contaminants may also be separated from the DNA by heating and/or by the use of enzymes. The DNA is subsequently precipitated by the addition of alcohol and the precipitate is separated from the solution. This may be done, for example, by centrifuging or by rinsing the DNA on a glass hook. After washing with a washing fluid in which salts dissolve but not the DNA, followed by drying, the DNA is resuspended in a suitable buffer. Examples of biological samples containing nucleic acid may be cell-free sample material, plasma, body fluids, such as blood, Buffy coat, cells, leukocyte fractions, crustaphlogistica, sputum, urine, sperm or organisms (single-or multi-celled organisms; insects etc) or eukaryotes or prokaryotes.
In particular, blood (human whole blood), Buffy coat, leukocyte fractions and cell cultures are suitable for carrying out the process according to the research. Blood (human whole blood) is most particularly suitable for carrying out the process according to the research.
Suitable lysing reagents for carrying out the process according to the research are the detergent-containing lysing reagents known from the prior art. Large numbers of suitable detergents are known before.
Lysing reagents which are particularly suitable for solving the problem according to the research are those which contain detergents selected from among the groups Triton, Tween, NP-40 or mixtures of substances from these groups. Among these groups, the Triton group is particularly suitable, while Triton X-100 (octylphenol polyethoxylate) is particularly preferred.
According to the research, lysing reagents based on 0.5 to 7.5% by volume of detergent are preferably used, while a content of 1.0 to 5.0% by volume is particularly preferred. If desired, salts of mono- or dihydric cations and complexing agents may be added, individually or in combination, to the lysing reagent.
Suitable saline solutions for resuspending the sediment containing the nucleic acid in order to carry out the process according to the research are the solutions known before for this purpose, based on high salt concentrations, particularly using chaotropic salts. By chaotropic salts are meant according to the research salts which have a high affinity for water and therefore form a large hydrate shell; suitable chaotropic salts are known in large numbers before.
Denaturing solutions of this kind may optionally contain other substances such as complexing agents or buffers. The pH of the denaturing buffer may vary over a wide range in order to carry out the process according to the research; it preferably has a pH in the range from 3 to 10and most preferably in the range from 7.5 to 9.5. The buffer systems known to the skilled man may be used to adjust the pH. According to the research, buffer systems based on tris(hydroxymethyl)aminomethane are preferably used.Most preferably, a buffer system based on 0.5 to 250 mM of
tris(hydroxymethyl)aminomethane hydrochloride is used.
In order to assist the denaturing activity of the denaturing buffer the sample may optionally be heated. The reaction temperature of this optional step of the process according to the research is within the range from 15 to 95° C, preferably in the range from 30 to 85° C. and this step is particularly preferably carried out at a temperature of 75° C. In order to further separate the DNA from impurities the sample may optionally be treated with enzymes.
The process according to the research may conveniently be carried out using proteases, lipases, amylases and other breakdown enzymes known in the art or mixtures thereof.
The DNA may expediently be precipitated in order to carry out the process according to the research by means of alcohols known before such as straight-chain or branched C1-C4 alkanols. Propan-2-ol (iso-propanol) and ethanol have proved particularly suitable.
The washing fluid used is, in particular, a mixture of a straight-chain or branched C-^-C^ alkanol with water, while aqueous solutions of ethanol are particularly preferred. A 70% by volume solution of ethanol in water is particularly preferred.Suitable "low salt solutions" for resuspending the DNA precipitates include all liquids capable of dissolving DNA. Solutions or suitable buffers are also known in large numbers from the prior art. According to the research, low salt solutions (solutions with a low ion intensity) are preferred, this category including water, according to the research.
Such solutions may optionally contain other substances such as complexing agents or buffers. The pH of the buffer may vary over a wide range and is preferably in the range pH 6 to 10 and more preferably in the range pH 7.5 to 9.5. The buffer systems known in the art may be used to adjust the pH. According to the research, buffers based on tris(hydroxymethyl)aminomethane are preferably used. A
buffer system based on 0.5 to 150 mM tris(hydroxymethy-l)aminomethane hydrochloride is particularly preferred. Most particularly preferred as a hydrogenation buffer is a solution containing 10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 8.5.
A process for isolating DNA, wherein a biological
sample is mixed with a lysing reagent, then the DNA-
containing cell components are separated from the other cell
components, the DNA-containing cell material is resuspended in a saline solution which contains at least salt, any contaminating
ingredients are removed by chemical, thermal and/or enzymatic treatment, the DNA is precipitated, washed, dried and
optionally resuspended in a low salt solution, characterized in that the isolation is carried out in a reaction vessel.
The process is characterised in that the lysing reagent may contain in addition to the detergent salts, mono- or polyhydric cations and/or one or more
The process is characterised in that the lysing reagent may additionally contain one or more buffer substances.
The process is characterised in that in order to remove contaminants the sample is heated to a temperature in a range from 15° C. to 95° C.
The process is characterised in that enzymes are used to break down contaminants.
The process ischaracterised in that the nucleic acid is precipitated by the addition of a branched or unbranched Ci-C4-alkanol, or mixtures of these alcohols.
The process is characterised in that a mixture of a branched or unbranched Cj-C4-alkanol and water is used as the washing liquid.
The process is characterised in that a solvent or a saline solution in which the DNA dissolves is used as the low salt solution.
The is characterised in that water is used as solvent.
The process is characterised in that the low salt solution or the solvent of the other buffer substances preferably contains a buffer based on tris(hy-droxymethyl) aminomethane and/or optionally complexing agents.
The process is characterised in that human whole blood is used as the biological sample.
A prospective kit containing reagents, also in concentrated form,
for final mixing by the user, containing the following aqueous solutions:
a detergent as well as salts of mono- or dihydric cations
and/or one or more complexing agents, and optionally
a buffer system based on tris(hydroxymethyl)ami-
nomethane-hydrochloride, sodium acetate, glycine-hy-
drochloride, lysine-hydrochloride or omithine-hydro-
a salt solution for resuspending the DNA-containing
material which comprises as chaotropic salts, at least
guanidinium hydrochloride which may optionally be
used in admixture with
optionally enzymes for breaking down impurities;
a branched or unbranched C1-C4 alcohol or mixtures of
these alcohols for precipitating the nucleic acids;
optionally a washing liquid consisting of a mixture of
a branched or unbranched Cj-C4-alkanol and water;
The process is where in, in order to remove contaminants, the sample is heated to about 30° C.
The process is wherein said enzymes for breaking down contaminants are selected from the group consisting of proteases, lipases, amylases, or mixtures thereof.
The process is wherein the nucleic acid is precipitated by the addition of propan-2- ol(iso-propanol).
The process is wherein said mixture is comprised of a solution of ethanol in water.
The process is wherein said mixture is comprised of 70% by volume ethanol in water.
The process is, wherein said buffer is 150 mM tris (hydroxymethyl) aminomethane hydrochlo- ride.
The process is wherein the low salt buffer is from about pH 7.5 to about 9.5.
The kit is wherein said enzymes for breaking down contaminants are selected from the group consisting of proteases, lipases, amylases, or mixtures
The prospective kit is wherein said C1-C4 alcohol is propan-2-ol(iso-propanol).
The kit is wherein said washing liquid is comprised of a solution of ethanol in water.
The propective kit is wherein said solution is comprised of 70% ethanol by volume in water.
Starting materials have a target nucleic acid for isolation, for example blood, buffy coat, saliva, cell cultures, etc, where the most preferred starting material for use with the present invention is blood. In preferred embodiments, the starting material is a liquid about 5-10ml, and preferably from about 35-40 ml Lysing and Denaturing Substance.
The lysing and denaturing substance of the invention causes the release of the nucleic acids from the intact cells of the starting material. Typically, the lysing and denaturing substance includes a buffering agent, a salt, a detergent and
a protease. The combination of ingredients causes the digestion of proteins, inhibition of nucleases, and the solubilization of lipids and proteins .
Typically, the lysing and denaturing substance is added to the starting material to achieve a salt concentration in the range of about 2 to 4 M, and preferably in the range of about 2.5 to 3.5 M; a detergent concentration in the range of about
0 to 4%, and preferably in the range of about f .5 to 2.5%; and a protease concentration in the range of about 20-50 ul/ml, and preferably in the range of about 40 ul/ml.
The buffering agent, typically Tris-HCL, is included at a concentration of about 10 mM, so as to maintain a pH of the mixture in the range of 7 to 8.5 and preferably in the range of 7.9 to 8.5.
Alcohol and Detergent Substance
The alcohol and detergent substance of the invention causes the precipitation of the nucleic acid from solution. Typically the alcohol and detergent substance contains an
alcohol and a detergent, although in some circumstances the substance may only include an alcohol.
Typically the alcohol and detergent substance is added to the nucleic acid containing solution to achieve an alcohol concentration of from about 60 to 100%, and preferably from about 70 to 95%, and a detergent concentration of from 35
about 0 to 40%, and preferably from about 20 to 35%.
In preferred embodiments of the alcohol and detergent substance, the alcohol is Isopropanol, ethanol, and the like, and the detergent is Tween 20 and the like.
The wash buffer of the invention serves to gently separate the precipitated nucleic acid trapped on the membrane from associated protein, lipids and cell debris in general. Typically, the wash buffer includes a buffering agent, a salt, EDTA and can contain alcohol. Preferably, the salt is from 400 to 600 mM NaCl or the like, and the buffering agent is approximately 1 0 mM Tris-HCL. Typically the volume of
wash buffer passed over the trapping membrane is sufficient to remove contaminants, but not of a volume to substantially effect the yield of the isolated nucleic acid.
High Molecular Weight Nucleic Acid Isolation method
Embodiments of the present work provide kits for the performance of the above described nucleic acid isolation methods. In one embodiment of the present work, the kit includes a lysis and denaturing substance, an alcohol and detergent substance, a wash buffer, a re-suspension buffer, and a trapping membrane. In a preferred embodiment the kit further includes molecular biology grade water and collection tubes. The kits of the present invention may also include any of the following:, pipette tips, a water bath, a heat block, blood collecting equipment, i.e., syringes, needles, anticoagulant, protective gloves, etc, and a microcentrifuge.
For maximum stability, the kits contain lyophilized protease, for example Proteinase K. Kits are believed to be stable for at least six months.
Preparation of Solutions
DNase free stock solution is prepared as follows
Dissolve solid RNase A(Sigma Aldrich, USA) with concentration 10mg/ml in 10 mM Tris-HCl pH7.5 15mM NaCl
Heat to 100 C for 15 mins
Cool slowly to room temperature
Store in aliquots at -70 C until used
Lysis Buffer composition
0.01M Tris-HCl pH 7.5
1% Triton x 100
Washing powder (Persil)
Prepare 35g/L (35mg/ml) concentration and store at 4 0C
Preparation of Immersed solid Glass Beads(4mm diameter)
Glass beads are immersed in absolute Ethyl alcohol 99%
Store in laminar flow under UV lamp overnight to remove greases from beads surfaces, eliminate any contaminations if present and to activate beads surfaces to attract more proteins in sample.
Dry beads by air direr and then autoclave for further sterilization.
[Glass Beads] Beads should be
sterilized with 99% Ethyl Alcohol before
ready to use.
Approximately 200 ul of human blood was treated with 40 ul reconstituted Proteinase K and 350 ul of 3 M Ammonium chloride, 2% w/v cetyltrimethylammonium bromide, 4% polyvinypyrrolidone and f 0 mM Tris-HCL, pH 8 in a f .5 ml microfuge tube. Blood samples were obtained and preserved with heparin, sodium citrate and EDTA. The mixture was vortexed on high for approximately 5 seconds and incubated in a water Bath at 75° C, for 10 minutes.
Genomic DNA samples were also isolated, for comparison sake, using the Qiagen kit.
Purity and concentration of the isolated gDNA was deter- mined taking a ratio of sample absorbance at 260 nm to 280 nm, noting that a Absorbance ratio of 1 .6 to 1 .8 illustrates a highly purified sample of DNA.
Analysis of gDNA integrity was performed by running isolated gDNA on a 0.6% agarose gel.
Blood samples were collected from "Demerdash Hospital" patients and on stored in EDTA tube (5ml volume) for anticoagulation on ice.
Equal volumes of the blood samples are added to the DNase free stock solution and heated to 75 0C for 10 mins to rupture Red Blood Cells ' surface easily.
[Red Blood Cells] biosciencetechnology.com
10ml of treated blood is transferred to a falcon tube and 35ml of lysis buffer (kept at 4 0C) is added.
[Falcon Tube] stage.itp.nyu.edu
The mixture is immediately vortexed vigorously for 5 mins.
Centrifuge at 4000 rpm for 5 mins.
[Centrifuged Blood Sample] celebritydiagnosis.com
The centrifuge causes blood to separate as shown in the figure. The plasma represents 55% of blood containing 91% water, 7% Blood Proteins and 2% Nutrients and is lightest therefore is up the tube in the supernatant. White blood cells come next, the "buffy coat", and is mixed with platelets and not very far from the red blood cells at the bottom of the tube.
Discard supernatant and 1ml of 0.01 mM Trist pH 8 is added to pellet to dissolve the sediment.
Sediment transferred to a 2ml eppendorf.
1ml of DNase free stock solution is added and the mixture is vortexed vigorously. Ya Karim please search for the chemical kills or destroys the DNAse in the solution. Pronase (http://www.mpbio.com/product_info.php?open=HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"&HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"cPath=HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"&HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"selecttab=HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"&HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"family_key=02180108HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"&HYPERLINK "http://www.mpbio.com/product_info.php?open=&cPath=&selecttab=&family_key=02180108&country=223"country=223company, and the source of publication) [Before use, a solution of 20 mg/ml pronase was incubated at 370C for 60 min to destroy contaminating DNase.]
Centrifuge for 1 minute at 1500 rpm
Discard supernatant (leave in the bottom of tube about 0.5ml of sediment solution) and add 340 ul of 10mM Tris-HCl pH 8 to pellet.
Add 40ul/ml of proteinase K (Fermentas, Lu) to confirm the removal of any present proteins in the sample. Incubate at 55 0C for 3 hours
localhire.co.nz [Proteinase k in eppendorf tube]
Add 0.5ml of fresh prepared washing powder (Persil) solution to the sample.
mycleaningsupply.us [Persil Washing Powder] Washing powder contains Lipase enzyme which acts on destroying any lipid contaminants in the sample.
By a pair of forceps, transfer the glass beads to the 2ml eppendorf containing the sample and vortex for 1 min at maximum speed then remove beads by forceps.
By a forceps , remove the glass beads from the 2ml eppendorf tube
Add 500 ul of 7.5M Ammonium Acetate (MB grade,Sigma, USA) (Fresh prepared) to ensure dissolving proteins
ehow.com [Ammonium Acetate]
Vortex vigorously for 1 min at maximum speed
Centrifuge at 10,000 rpm for 5 mins.
Transfer the supernatant to the new tube.
Add 0.5ml of 6M NaCl (MB grade-Sigma ,USA)to the tube and vortex for 30s vigorously
Centrifuge at 14,000 rpm for 5 mins.
Transfer supernatant into new eppendorf tube 1 ml and add 1ml absolute Isopropanol alcohol (Sigma Aldrich, USA).
Incubate at -70 0C for 2 hours
Centrifuge at 20,000 rpm for 1 min
Discard supernatant and add 1ml of 70% ethanol (twice times)
If the ppt is little , Store in -700C for 1h and centrifuge at 14,000 rpm for 5 mins.
Discard supernatant and let the pellet completely dry by air stream under tissue paper in laminar flow suction (to ensure completely no residue of alcohol present in the tube).
Add 100 ul of 10 mM Tris HCL pH 8 and incubate for over night at 550C in water bath to ensure the DNA is completely dissolved kept at -200C until used in the experiments.
Results and Discussion
As illustrated in Figures, isolation of gDNA from blood using the methods of the present invention resulted in a vast majority of the gDNA being isolated in molecular weight sizes above 23 kb (lanes2-?). In fact, the data shows that only approximately 20% of the total density of DNA in the samples are 23 kb, indicating that the isolation technique did not generate a high percentage of shearing. Further, the isolated gDNA is of a high purity, having an absorbance ratio of f.6 to f.8.
In contrast, conventional gDNA isolation techniques typically result in a higher percentage of total DNA being 23kb size range. In a side-by-side comparison, gDNA isolated using the Qiagen kit resulted in approximately 40% of the total density of DNA being 23 kb.
This data illustrates that the gDNA isolation method provides a powerful tool for isolating high molecular weight gDNA that minimizes the shearing of the DNA, and that the isolated gDNA has a minimal amount of contaminants.