CA2102264C - Boron silicates, aluminum silicates, phosphosilicates and purification of dna - Google Patents

Boron silicates, aluminum silicates, phosphosilicates and purification of dna Download PDF

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Publication number
CA2102264C
CA2102264C CA002102264A CA2102264A CA2102264C CA 2102264 C CA2102264 C CA 2102264C CA 002102264 A CA002102264 A CA 002102264A CA 2102264 A CA2102264 A CA 2102264A CA 2102264 C CA2102264 C CA 2102264C
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dna
molar percentage
reaction mixture
buffer
boron
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CA2102264A1 (en
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Daniel Lee Woodard
Adriann Jeanelle Howard
James Arthur Down
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Becton Dickinson and Co
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    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01BNON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00Silicon; Compounds thereof
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01BNON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B35/00Boron; Compounds thereof
    • C01B35/08Compounds containing boron and nitrogen, phosphorus, oxygen, sulfur, selenium or tellurium
    • C01B35/10Compounds containing boron and oxygen
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Abstract

The invention discloses boron silicates, aluminum silicates and phosphosilicates, how to make them, and processes for purifying DNA with them. The new composition is of the formula:
wherein X is Silicon, Boron, Aluminum or Phosphorus, and R is independently OH or a monomer unit, n is 3 when X is Si and n is 2 when X is Boron, Aluminum, or Phosphorus, and repeating units of the composition comprising the formula:

Description

r-G3U2f BORON SILICATES, ALUMINUM SILICATES
PHOSPHOSILICATES AND PURIFICATION OF DNA
FIELD OF THE INVENTION
The invention is in the field of molecular biology. In particular, the invention is in the area of deoxyribonucleic acid purification.
BACKGROUND OF THE INVENTION
The continued advances in molecular biology and related disciplines present continued needs for improvements in tools associated with fully appreciating and developing the advanced technology.
A wide range of technologies involve the use of deoxyribonucleic acids (DNA) in a variety of forms. For example, advances in the area of recombinant DNA technology continually require the use of DNA in the form of probes, genomic DNA, and plasmid DNA.
Advances in the area of diagnostics also continue to utilize DNA in a variety of ways. For example, DNA probes are routinely used in the detection and diagnosis of human pathogens. Likewise, DNA is used in the detection of genetic PATENT
disorders. DNA is also used in the detection of food contaminants. And, DNA probes are routinely used in locating, identifying and isolating target DNA of interest for a variety of reasons ranging from genetic mapping to cloning and recombinant expression.
In many instances DNA is available in extremely small amounts, and isolation and purification procedures can be laborious and time consuming. The often time consuming and laborious procedures can lead to loss of DNA. In the purification of DNA from specimens obtained from serum, urine, and bacterial cultures, there is the added risk of contamination and false-positive results.
Typical DNA purification protocols involve the use of caustic and poisonous compositions. The typical DNA
purification protocol uses high concentrations of chaotropic salts such as sodium iodine and sodium perchlorate.
There are numerous protocols for purifying DNA. As evidenced by recent activity in the area of DNA purification, there is a continued pursuit for optimal DNA purification protocols. U. S. Patent 4,923,978 discloses a process for purifying DNA in which a solution of protein and DNA is passed over a hydroxylated support and the protein is bound and the DNA is eluted. U.S. Patent 4,935,342 discloses purification of DNA by selective binding of DNA to anion exchangers and subsequent elution. U. S. Patent 4,946,952 discloses DNA
isolation by precipitation with water-soluble ketones. A DNA
purification procedure using chaotropes and dialyzed DNA is disclosed in U. S. Patent 4,900,677.
While the present protocols for purifying DNA are able to accomplish their goal, it is desirable to purify DNA without the use of such caustic and poisonous compounds such as the most often used chaotropes in addition to obtaining increased amounts of DNA.
SUMMARY OF THE INVENTION
The invention provides the monomer unit composition:
R Rn R- Si - X
O
-R , wherein X is Silicon, Boron, Aluminum or Phosphorus, and R is independently OH or a monomer unit, n is 3 when X is Si and n is 2 when X is Boron, Aluminum, or Phosphorus, and repeating units of the composition comprising the formula:
R Rn R- Si - O - X
R , 2~ p2264 PATENT
and compositions comprising the above monomer and repeating units thereof. Repeating units described above can include from about 2 up to infinity. Ranges include about 2 to about 100,000,000, and about 2 to about 100,000.
The invention can be used to purify DNA from a variety of sources and from a variety of forms. The process uses the composition of the invention and renders the use of binding buffers, such as chaotropes, optional. The DNA can be bound in aqueous solution such as TE buffer (lOmM Tris, 1mM EDTA) at l0 room temperature. In addition, the DNA can be eluted into water from the compositions of the invention by heating, or generally used elution buffers such as TE or 1 X TAE can be employed. Sources of DNA for purification include bacteria, bacteriophage, specimens, plants, animals, and the like. DNA
can be found in a variety of forms and includes single-stranded, double-stranded, circular, and linear. The invention can be practiced with DNA from any source in any form.

' 21 022 64 -4a-Thus, the present provides in one embodiment a process for purifying DNA which comprises binding the DNA to a DNA binding compound obtainable by a process comprising the steps of: a) preparing a reaction mixture consisting essentially of SiCl4 and a second component selected from the group consisting of:
a molar percentage of BC13 of about 33.5%, a molar percentage of BC13 of about 55.6%,a molar percentage of BC13 of about 83.30, a molar percentage of PC13 of about 50.0%-83.30, and a molar percentage of A1C13 of about 9.1%; b) cooling the reaction mixture; and c) adding water to the reaction mixture until evolution of gas is complete.
In another embodiment, the invention provides, a kit for purifying DNA, which comprises a binding buffer and a DNA binding compound obtainable by such a process.
In another embodiment of the invention, A DNA binding compound obtainable by a process comprising the steps of:
a) preparing a reaction mixture consisting essentially of SiCl4 and a second component selected from the group consisting of:
a molar percentage of BC13 of about 33.5%, a molar percentage of BC13 of about 55.6%, a molar percentage of BC13 of about 83.3%, a molar percentage of PC13 of about 50.0%-83.30, and a molar percentage of A1C13 of about 9.1%; b) cooling the reaction mixture; and c) adding water to the reaction mixture until evolution of gas is complete.
DETAILED DESCRIPTION
The invention provides the monomer unit composition:
R Rn R- Si - X
O
-R , ~ 10 2 2 6 4 PATENT
wherein X is Silicon, Boron, Aluminum or Phosphorus, and R is independently OH or a monomer unit, n is 3 when X is Si and n is 2 when X is Boron, Aluminum, or Phosphorus, and repeating units of the composition comprising the formula:
R Rn R- Si - - X
O

R

and compositions comprising the above monomer and repeating units thereof. Repeating units described above can include from about 2 up to infinity. Ranges include about 2 to about 100,000,000, and about 2 to about 100,000.
The surface provides for binding of DNA while also allowing easy recovery of DNA from the surface.
Also provided is a process for purifying DNA which comprises contacting DNA with a composition of the formula:
R Rn R- Si O X
- -R

wherein X is Silicon, Boron, Aluminum or Phosphorus, and R is independently OH or a monomer unit, n is 3 when X is Si and n is 2 when X is Boron, Aluminum, or Phosphorus, and repeating units of the composition comprising the formula:

r ~,~vo R Rn R-Si O X
- -R , and compositions comprising the above monomer and repeating units thereof. Repeating units described above can include from about 2 up to infinity. Ranges include about 2 to about 100,000,000, and about 2 to about 100,000.
Reaction products of a mixture of SiCl4 and BC13, or PC13, or A1C13 followed by the addition of water are also provided.
The invention also provides a method for making the composition of the formula:
R Rn R-Si O - X
-R , wherein X is Silicon, Boron, Aluminum or Phosphorus, and R is independently OH or a monomer unit, n is 3 when X is Si and n is 2 when X is Boron, Aluminum, or Phosphorus, and repeating units of the composition comprising the formula:
R Rn R-Si - - X
O

R , PATENT

-and compositions comprising the above monomer and repeating units thereof. Repeating units described above can include from about 2 up to infinity. Ranges include about 2 to about 100,000,000, and about 2 to about 100,000.
Generally, reaction products of water and a mixture of SiCl4 and PC13, or BC13 or A1C13 result in a bead like structure comprising repeating units of the above referenced monomer unit.
It is possible that the electronic nature of this polymer l0 is such that surface modifications can be made that are of a more conventional nature but are changed electronically due to the presence of this polymer being at the center of the bead (making it a more efficient surface for the purposes described in this disclosure). For example, the surface could be modified with SiCl4 followed by hydration which would result in a silanol coating on the surface. The exposure of the repeating unit is what interacts with the DNA, and thus surfaces comprising the repeating unit are also suitable for practicing the invention. Surfaces which can be designed to comprise compositions of the invention include dipstick configurations, tubes, vials, filtration devices, and the like.

PATENT

- ~~~~2~4 The procedure for obtaining the compositions of the invention generally comprises mixing SiCl4 with various amounts of PC13, or BC13, or A1C13, followed by cooling.
Water is then added until hydrogen chloride gas HC1(g) no longer elutes, then excess water is added to ensure complete reaction of SiCl4 and BC13, or A1C13 or PC13. Amounts of reactants are generally 15:1 to 1:15.
This resulting product is stirred for about thirty (30) minutes. The resultant product is filtered then washed and dried. Suitable washing reagents include acetone and the like. The product is now ready for use in purifying DNA.
The invention also provides a process for purifying DNA
which comprises contacting DNA with compositions of the invention.
The start of any DNA purification or isolation procedure requires obtaining the desired DNA from its source. Typical protocols for obtaining DNA from specimens such as serum, urine and bacterial cultures are well known and routinely carried out. Likewise, the ability to obtain DNA from genomic libraries and the like are routine. The key to the invention is the ability to purify DNA, once obtained from its source.
Typical procedures for obtaining DNA end with a suspension of the DNA in solution. References include those for isolation of DNA from biological samples, Harding, J.D., Gebeyehu, G., Bebee, R., Simms, D., Ktevan, L., Nucleic Acids Research ,17:6947 (1989), and Marko, M.A., Chipperfield, R., and Birnboim, H.C., Analytical Biochemistry, 121:382 (1982).
Procedures for isolation of plasmid DNA can be found in Lutze, L.H., Winegar, R.A., Nucleic Acids Research 20:6150 (1990).
Extraction of double-stranded DNA from biological samples can be found in Yamada, O., Matsumoto, T., Nakashima, M., Hagri, S., Kamahora, T., Ueyama, H., Kishi, Y., Uemura H., Kurimura, T., Journal of Viroloqical Methods 27:203 (1990). Most DNA
solutions comprise the DNA in a suitable buffer such as TE
(Tris-EDTA (lOmM:lmM)), TEA (40mm Tris-acetate, lmm EDTA) buffer, or a lysate.
Once the DNA is obtained in a suitable solution, a binding matrix is typically added to the solution. Generally used binding matrixes are silica in the form of glass or diatoms.
However, procedures using silica require high concentrations of chaotropes or alcohols for the DNA to bind to the surfaces.
Currently used chaotropes include sodium iodide (NaI), urea, guanidinium Hydrochloride, sodium perchlorate (NaC104), and potassium bromide (KBr). Chaotropes and alcohols can be toxic, caustic, flamable and/or expensive. The process of the present invention does not require the presence of chaotropes or alcohols for binding to surfaces of the invention. Processes PATENT

of the invention bind DNA in an aqueous solution at room temperature and elute the DNA in water at 37°C. However, if desired, chaotropes, alcohols and the like can be used with the process of the invention.
Typical procedures for using the process of the invention include the addition of the composition of the invention to a solution of DNA, which is generally followed by the addition of a binding buffer. At this point, it is advantageous that the process of the invention does not require a binding buffer.
l0 The solution can be incubated for a brief period at room temperature. After spinning, the supernatant can be discarded and the pellet washed. The DNA can then be eluted.
The composition of the invention is typically used in weight ranges from about 1:10 to 1:1 composition weight: water.
Preferably excess amounts of water are avoided and buffers such as TE can be used in place of water.
Next, a binding buffer is added, if used. After a brief incubation period at room temperature, although a range of about 20° to about 40°C is acceptable, from about 1 to about 20 minutes, preferably about 10 minutes, the container can be spun to obtain a pellet and supernatant fractions. The supernatant is separated and the pellet is washed with a reagent such as ethanol diluted with 50mM Tris. A preferred wash reagent concentration is 80% ethanol. DNA can then be eluted from the compositions of the invention by using elution buffers such as TE buffer, 1 X TAE buffer, and 1 X TBE buffer. More importantly, the use of elution buffers can be eliminated altogether, and DNA eluted in water by heating. For maximum yields the elution step can be repeated.
The chemical compositions of the invention can be conveniently assembled into a kit. A kit comprising the composition of the invention can include the composition in a container, such as a vial, with a suitable buffer, such as TE
buffer and TAE buffer and optionally include a container of a binding buffer such as chaotropes, a container of wash buffer, such as a solution of ethanol diluted with 50mM tris or 1 X
TAE, and a container of elution buffer, such as TE buffer, I X
TAE buffer, and 1 X TBE buffer. Such a kit would allow convenient purification of DNA.
The following examples illustrate the specific embodiments of the invention described in this document. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.

' ~ 1 0 2 2 6 4 pA2308 The purpose of this experiment is to synthesize l0 boronsilicate polymers. The polymers contain various amounts of boron because incorporation of boron, a less electropositive atom than silicon, into the polymer will change the electronic nature of the surface and may effect its ability to purify DNA
from an aqueous sample by solid phase extraction.
BC13 in CH2C12(1M) (Aldrich, Milwaukee, WI) SiCl4 (Petrarch*systems, Bristol, PA) Procedure: 10 experiments were set up the same way except the percentage of BC13 was varied as shown below.
BC13 SiCl4 Surface uL mMol g mMol 1 1.0 1.0 1.70 10.0 2 2.0 2.0 1.70 10.0 3 5.0 5.0 1.70 10.0 4 7.0 7.0 1.70 10.0 5 10.0 10.0 1.70 10.0 uL mMol 6 5.0 5.0 123.0 1.0 7 5.0 5.0 247.0 2.0 8 0 5.0 370.0 3.0 . 5.0 493.0 4.0 9 5.0 10 5.0 5.0 0.0 0.0 * Trademark .

PATENT

The SiCl4 and BC13 are mixed and cooled to about 5°C
for 20 minutes. With stirring, H20 is slowly added until HCl(g) no longer elutes. 5mL excess H20 is added to ensure complete reaction. Stirred 1 hour.
Filter, wash 3X10 mL H20 then 3X10mL acetone, air dry 25 minutes and heat dry 1 hour.
Phosphorus has approximately the same electronegativity as Boron. Boronsilicates have been shown to work very well for DNA purification. Therefore, if polarization of the surface is important in DNA adhesion/elution then Phosphosilicates should work as well as boronsilicates for DNA purification.
r , ., , ,.. .
SiCl4 (Petrarch Systems) PC13 - 2M in CH2C12 (Aldrich) l0 experiments were done exactly the same way except that the amount of PC13 was different in each experiment. The following table describes each of the 10 experiments.

SiCl4 PC13 Experiment mL mMol ~ mL mMol 1 1.340 10.0 0.1 0.5 1.0 2 1.340 10.0 0.3 1.5 3.0 3 1.340 10.0 0.5 2.5 5.0 4 1.340 10.0 0.7 3.5 7.0 5 1.340 10.0 1.0 5.0 10.0 6 0.65 5.0 1.5 3.75 7.5 7 0.65 5.0 2.0 5.0 10.0 8 0.65 5.0 3.0 7.5 15.0 9 0.65 5.0 4.0 10.0 20.0 10 0.65 5.0 5.0 12.5 25.0 In the typical experiment, the SiCl4 is added to a 25 mL
erlenmeyer flask and cooled to 0°C in an ice bath for about IO
minutes. The PC13 is then added, cool for 5 minutes. H20 was added very slowly, about 2 drops per minute, until white gas no longer elutes. Stir 5 minutes and add 3mL in 1mL
increments. Stir at room temperature for 15 minutes. Filter, wash the solid with 3X10 mL H20 then 3*10 mL acetone. Air dry 15 minutes, heat dry one (1) hour. Store in a desicator.
The purpose of the following experiment is to synthesize aluminum silicate polymers containing various amounts of aluminum. Aluminum is more electropositive than PATENT

silicon and, therefore, as the amount of aluminum in the polymer increases so should the amount of DNA adhering to that polymer.
Starting Materials A1C13, 1M in nitrobenzene (Aldrich) SiCl4 (Petrarch System) 8 experiments were performed the same way except that the amount of A1C13 was varied from one to the next.
SiCl4 A1C13 Experiment mL mMol mL mMol 1 1.34 10.0 1.0 1.0 2 1.34 10.0 3.0 3.0 3 1.34 10.0 5.0 5.0 4 1.34 10.0 7.0 7.0 5 1.34 10.0 10.0 10.0 6 0.67 5.0 7.5 7.5 7 0.67 5.0 10.0 10.0 8 0.67 5.0 15.0 15.0 In a typical experiment the A1C13 and SiCl4 are mixed together and cooled in an ice bath for 15 minutes. Water is added dropwise, with vigorous stirring. Very slowly (5 drops every 2 minutes), water is added until no HC1(g) elutes from the reaction vessel.
Add about 3 mL H20 to ensure complete reaction. Stir at room temperature for 15 minutes.

. 21 022 64 Filter, wash 3X10 mL acetone, 3X20 mL H20, 3X10 mL
acetone. Air dry for 20 minutes, heat dry one (1) hour. Store in a dessicator.
This experiment describes how the DNA binding capacity of SUPER
FINE SUPER FLOSS CELITE*(the industry standard, Manville) was determined and what that capacity is. It was determined that SUPER FINE SUPER FLOSS CELITE strongly binds and elutes DNA at 2.5M with NaC104 as the binding buffer.
Materials:
Super Fine Super Floss (SFSF) (Sample from Manville, Denver, CO (1:5 w/w in H20)) aDNA (BRL Cat. 56125A) 50mM Tris pH7.0 (Dilute from 1M stock) BRL Cat. 5505UA
(PREP-A-GENE*KIT (Bio-Rad* Richmond, CA)) Binding Buffers (Diluted from 6M stock) NaC104 Fisher Cat. 5490-500 Wash Buffer 80% Ethanol in 50mM Tris, pH7.0 Elution Buffer Milli Q*H20 Ethidium Bromide (lOmg/ml) Sigma Cat. E-8751 1% agarose BRL Cat. 5510UA
1X TAE (from 50X stock) Tris Base-Sigma CAT T-1503 Acetic Acid - Fisher A38-500 EDTA - Sigma CAT ED2550 * Trademarks Type II Loading Dye (25o Ficoll 400, 0.250 Bromophenol Blue, 0.25% xylene cyanol Ficoll*400 - Sigma*CAT F4375, Bromophenol Blue - BIO-RAD*CAT 161-0404 Xylene Cyanole*- Sigma CAT X-4126 Type 57 and 55 POLAROID*Film METHODS
1. Two groups of reactions are set up, one for each surface type. Each surface has 8 tubes containing 50u1 of the DNA solution. This solution is 0.5u1 0 7~DNA in 50u1 50mM Tris, pH7.0 for 31~g DNA/reaction. The titration ranges from OM NaC104 to 6M NaC104.
2. Add 20u1 of each surface to the reaction mixes.
3. Add 400u1 Binding Buffer according to the titration. For Prep-A-Gene this was O.OM, 2. OM, 2.5M, 3.OM, 3.5M, 4. OM, 4.5M, and 6. OM NaC104. For SFSF, the titration is OM, 1.OM, 1.5M, 2. OM, 2.5M, 3. OM, 3.5M, and 4. OM NaC104.
4. Incubate for 10 minutes, with rocking, at room op temperature.
5. Spin and discard supernatant.
6. Wash pellet 2 times with 80o ethanol/50mM Tris, pH7Ø
7. Elute DNA in 20u1 H20, 37°C, 10 minutes.
* Trademarks A

PATENT
210~~~4 P-2308 8. Spin and remove supernatant to a separate tube.
Repeat elution step and combine supernatants for ~40u1 total.
9. Add 2u1, Type II loading dye to each tube.
10. Load onto a 1% agarose, 1 X TAE gel. Run for ~25 minutes at 100-130 volts in 1 X TAE buffer.
11. Stain with ethidium bromide in H20 (1:1000) for ~15 minutes. Destain for ~20-30 minutes.
12. Photograph over UV light with Type 57 Polaroid film.
If possible, take negatives with Type 55 film.
Results and Conclusions Prep-A-Gene shows no elution of DNA until 3. OM NaC104, whereas SFSF binds DNA in its native state and elutes strongly at 2.5M NAC104. Clearly SFSF performs better than Prep-A-Gene.
This experiment describes the DNA binding capacity of baron silicates, phosphosilicates, and aluminum silicates.
Electrophoresis shows that many of these surfaces give good recovery of DNA down to 1M NaC104 as the binding buffer.
This exceeds the Super Fine Super Floss Celite which gives good recovery only down to 2.5M NaC104. It would also appear from PATENT

gel electrophoresis analysis that some of these surfaces give equal or greater recovery of DNA down to these lower levels of NaCl04 as the binding buffer, and under native conditions.
Material of composition described for Boronsilicates in substantial accordance with examples 1, 2, and 3.
Phosphosilicates or aluminum silicates.
SUPER FINE SUPER FLOSS (Manville) 1:5 weight: water Methods l0 Eight reaction groups are tested for each surface shown below. The binding buffer concentrations are 1.OM, 1.5M, 2. OM, 2.5M, 3.OM, 3.5M, 4.OM with SFSF at 3.OM NaC104 as the standard used. See Example 4 for procedure.

Results Table 1 Results DNA Binding udies - of St Molar percentage DNA Binding of BC1 AlCl3 or 1M-4M
, i PC1~

n the [NaC1041 Bindinq Surface mixture reaction in water 1 9.1 ++ 1.5M

2 16.6 -3 33.5 +++ Yes 4 42.2 ++ 1.5M

5 50.0 -6 55.6 +++ Yes 7 62.5 ++ 1.5M

8 71.4 ++ 2.OM

9 83.3 +++ Yes 10 9.1 -11 16.6 ++ 1.5M

12 33.3 ++ 1.5M
13 42.2 +
14 50.0 +++ Yes 15 62.5 +++ Yes 16 66.6 ++
17 75.0 +++ Yes 18 80.0 +++ Yes 19 83.3 +++ Yes 20 9.1 +++ Yes 21 23.1 +
22 33.3 +
23 41.2 -24 50.0 -25 60.8 -26 66.6 -27 75.0 -2 1 p22 6,~

- if DNA binding occurred, the DNA didn't elute off.
+ Trace amounts of DNA elute across the titration.
++ Near complete elution down to the indicated Molarity of binding buffer +++ Strong elution of DNA across the titration +, ++, and +++ were determined by visualization of ethidium bromide stained gel.
1-9 boron silicates 10-19 phosphosilicates 20-27 aluminium silicates Conclusion Several surfaces out-perform SFSF Celite both in the amount of DNA recovered from solution and the concentration of binding buffer required to bring about this recovery. According to agarose gel electrophorsis analysis, near 1000 recovery of DNA
from solution even to 1.OM NaCl04 as the binding buffer were achieved, with several of the surfaces.
Although the invention has been described with respect to specific modifications, the details thereof are not to be construed as limitations, for it will be apparent that various equivalents change and modifications may be resorted to without departing from the spirit and scope thereof, and it is understood that such equivalent embodiments are to be included therein.

Claims (8)

1. A process for purifying DNA which comprises binding the DNA to a DNA binding compound obtainable by a process comprising the steps of:
a) preparing a reaction mixture consisting essentially of SiCl4 and a second component selected from the group consisting of:
- a molar percentage of BCl 3 of about 33.5%, - a molar percentage of BCl 3 of about 55.6%, - a molar percentage of BCl 3 of about 83.3%, - a molar percentage of PCl 3 of about 50.0%-83.3%, and - a molar percentage of AlCl 3 of about 9.1%;
b) cooling the reaction mixture; and c) adding water to the reaction mixture until evolution of gas is complete.
2. The process of claim 1, in which DNA is bound to the DNA binding compounds in the presence of an aqueous solution.
3. The process of claim 1, which further comprises elution of DNA from the DNA binding compounds.
4. The process of claim 3, in which elution of DNA is obtained by heat and water.
5. A kit for purifying DNA, which comprises a binding buffer and a DNA binding compound obtainable by a process comprising the steps of:
a) preparing a reaction mixture consisting essentially of SiCl4 and a second component selected from the group consisting of:
- a molar percentage of BCl3 of about 33.5%, - a molar percentage of BCl3 of about 55.6%
- a molar percentage of BCl3 of about 83.3%, - a molar percentage of PCl3 of about 50.0%-83.3%, and - a molar percentage of AlCl3 of about 9.1%, b) cooling the reaction mixture; and c) adding water to the reaction mixture until evolution of gas is complete.
6. The kit of claim 5, further comprising a wash buffer.
7. The kit of claim 6, further comprising an elution buffer.
8. A DNA binding compound obtainable by a process comprising the steps of:
a) preparing a reaction mixture consisting essentially of SiCl4 and a second component selected from the group consisting of:
- a molar percentage of BCl3 of about 33.5%
- a molar percentage of BCl3 of about 55.6%, - a molar percentage of BCl3 of about 83.3%, - a molar percentage of PCl3 of about 50.0%-83.3%, and - a molar percentage of AlCl3 of about 9.1%;
b) cooling the reaction mixture; and c) adding water to the reaction mixture until evolution of gas is complete.
CA002102264A 1992-11-13 1993-11-02 Boron silicates, aluminum silicates, phosphosilicates and purification of dna Expired - Fee Related CA2102264C (en)

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CA2102264A1 (en) 1994-05-14
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US5525319A (en) 1996-06-11
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DE69304495D1 (en) 1996-10-10
AU5042593A (en) 1994-05-26
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JP2714529B2 (en) 1998-02-16
JPH06217775A (en) 1994-08-09

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