Introduction For Dna Isolation Dna From Blood Biology Essay


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

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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 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.

Starting Material

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

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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.

Wash Buffer

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 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.

Results and Discussion

As illustrated in Figures, isolation of gDNA from blood

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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 ¿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.