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Isolated nucleic acid, and in particular, isolated high molecular weight DNA, has a variety of uses in molecular biology, biotechnology and clinical research. For example, isolated DNA is useful in a number of molecular biology techniques, including polymerase chain reaction (PCR), DNA hybridization, restriction enzyme digestion, DNA sequencing, and array-based experiments. 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 extracting
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 75Â° 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.
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.
High Purity and Molecular Weight DNA Result from gDNA Isolation Method
Materials and Methods
Approximately 200 ul of human blood was treated with
40 ul reconstituted Proteinase K and 350 ul of 3 M Ammo-
nium 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 pre-
served with heparin, sodium citrate and EDTA. The mixture
was vortexed on high for approximately 5 seconds and
incubated in a water Bath at 75Â° 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.
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 typi-
cally result in a higher percentage of total DNA being 23
kb 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 Â¿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.