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Chromatographic process for the separation of nucleic acids

United States Patent 4699717
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A process for the chromatographic separation of nucleic acid using a chromatographic carrier material is
described in which the surface of the carrier material is specially modified.
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US Patent References:
Filler for liquid chromatography and method for separating water-soluble substances using said filler
Kamithma et al. - May, 1984 - 4447328

Separation agent
Glad - September, 1983 - 4406792

Anion exchange chromatographic separation of polyfunctional compounds

Abbott - September, 1981 - 4290892

Solid support for liquid chromatography

Inmura et al. - February, 1979 - 4140653 begin_of_the_skype_highlighting 1979 - 4140653
end_of_the_skype_highlighting

Quaternized siliceous supports for gel permeation chromatography

Tallet et al. - October, 1978 - 4118316 begin_of_the_skype_highlighting 1978 - 4118316
end_of_the_skype_highlighting

Inventors:
Riesner, Detlev (Eichenwand 15, 4000 Dusseldorf 12, DE)
Colpan, Metin (Christoph-Strasse 67, 4000 Dusseldorf 1, DE)
Application Number:
06/830708
Publication Date:
10/13/1987
Filing Date:
02/14/1986
Export Citation:
Click for automatic bibliography generation
Primary Class:
536/25.400
Other Classes:
502/401, 502/439, 210/502.100, 210/656, 536/26.730, 514/44, 210/635, 210/198.200
International Classes:
B01D15/08; B01J20/32; B01J20/30; B01D15/08
Field of Search:
210/635, 210/656, 210/659, 210/198.1, 210/502.1, 514/44, 536/27, 502/401, 502/402, 502/439
View Patent Images:
Download PDF 4699717 pdf PDF help
US Patent References:
4029583 Chromatographic supports and methods and apparatus for preparing the same June,
1977 Chang 210/198.2
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Foreign References:
DE2601930 July, 1977
FR2462183 February, 1981
GB2075362 November, 1981
Primary Examiner:
Therkorn, Ernest G.
Attorney, Agent or Firm:
Hayes, Davis & Soloway
Parent Case Data:

This application is a continuation-in-part application of our copending application Ser. No. 560,931 filed
Nov. 25, 1983, filed Mar. 25, 1983, published as WO83/03363 on Oct. 13, 1983, now abandoned.
Claims:
We claim:

1. A process for the chromatographic separation of nucleic acids using a chromatographic carrier
prepared by reacting a porous carrier material that contains cavities with a silanization material, wherein
said porous carrier material has a grain size of from 2 to 100 μm, a cavity size cross section of 1 to 40
times the maximum dimension of the nucleic acids contained in the mixture and a specific surface of from
5 to 800 m2 /g, and said carrier material is reacted with a silanization reagent of the general formula R1
R2 R3 SiR4 I.

wherein R1 is selected from the group consisting of an alkoxy radical having from 1 to 10 C atoms, a
halogen group, and a dialkyl amino group with identical or different alkyl radicals having from 1 to 6 C
atoms, R2 and R3 each are selected from the group consisting of a hydrocarbon radical having from 1 to
10 C atoms, an alkoxy radical having from 1 to 10 C atoms, a halogen atom, an alkyl radical having from
4 to 20 C atoms interrupted by at least one oxy or amino group, and at least one halogen, cyano, nitro,
amino, monoalkylamino, dialkylamino, hydroxy or aryl group, and R4 is selected from the group consisting
of a hydrocarbon chain having from 1 to 20 C atoms and an alkyl radical interrupted by at least one oxy or
amino group, or at least one halogen, cyano, nitro, amino, monalkylamino, dialkylamino, alkoxy, hydroxy,
aryl and/or epoxy group, and then, reacting the resulting carrier material with a reagent of the general
formula X-R-Y II

wherein X is selected from the group consisting of an amino, hydroxy, and an epoxy group and, a halogen
atom, R is selected from the group consisting of a hydrocarbon chain having from 2 to 20 C atoms, and
an alkyl radical interrupted by at least one oxy or amino group, or at least one halogen, cyano, nitro,
amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, aryl and/or epoxy group and Y is selected from the
group consisting of a hydrocarbon radical having from 1 to 10 C atoms and functional groups that form
anion or cation exchangers, wherein said radical can be replaced once or more by amino,
monoalkylamino, dialkylamino, quaternary alkylamino, carboxyl, boric acid, alkyl and aryl sulfonic acid
groups.

2. A process according to claim 1, wherein the carrier material has a cavity size of from 50 to 1000 μm
and a specific surface of at most 200 m2 /g.
3. A process according to claim 1, wherein said carrier material comprises silicon dioxide.

4. A process according to claim 1, wherein the diameter of the cavities amounts to from 1 to 20 times the
maximum dimension of the respective nucleic acid to be isolated or the maximum dimension of the
largest of all nucleic acids contained in the mixture.

5. A process according to claim 1, wherein the cavities comprise semispherical recesses.

6. A process according to claim 1, wherein the cavities are tubular in cross section.

7. A process according to claim 1, wherein R1 comprises an alkoxy radical selected from the group
consisting of --OCH3, --OC2 H5 and --OC3 H7.

8. A process according to claim 1, wherein R1 comprises a chlorine group.

9. A process according to claim 1, wherein R2 and R3 each comprise a hydrocarbon radical selected from
the group consisting of --CH3, --C2 H5 and --C3 H7.

10. A method according to claim 1, wherein R2 and R3 each comprise an alkoxy radical selected from the
group consisting of --OCH3, --OC2 H5 and --OC3 H7.

11. A process according to claim 1, wherein R4 comprises an alkyl radical replaced by an epoxy group
consisting of alkoxy, hydroxy, aryl and/or epoxy group.

12. A process according to claim 11, wherein said epoxy group comprises. ##STR2##.

13. A process according to claim 11, wherein the solution containing nucleic acids to be separated and the
elution substance are kept free from bivalent metal ions by using noble metal, glass and/or plastic for
columns, pipes, valves and pumps of the chromatographic equipment.

Description:

The invention concerns a chromatographic process for separating natural and synthetic nucleic acids
from low to very high molecular weight (molecular weight up to 50×10 6 ) by using surface modified
carrier materials that contain cavities, and in particular to the chromatographic separation of nucleic acids
having a chain length from about 5 to about 5000 nucleotides, natural and synthetic ribonucleic acids
having one or two strands (RNA), like transfer RNA, ribosomal RNA, messenger RNA and viral RNA,
natural desoxyribonucleic acid (DNA) and DNA fragments, particularly plasmid DNA and phagene DNA by
using such carrier materials.

The progress in biochemistry, molecular biology and genetic engineering, and the application thereof to
medicine, pharmacology and agriculture requires the quick separation and purification of discrete nucleic
acids species. Thus, for instance, there often arises in genetic engineering the problem that from a
naturally occurring mixture of 100 and more different nucleic acids of high molecular weight, a single
molecular species must be purified homogeneity. The individual nucleic acids are known to be
characterized by nucleotide sequence, molecular weight, size and shape.

Of special interest are long-chain ribo- and deoxyribo-oligonucleotides, natural ribonucleic acids (RNA),
like transfer RNA and 7S RNA, viral RNA and messenger RNA, deoxyribonucleic acids (DNA), DNA
fragments and plasmid DNA.

Traditional techniques force scientists to choose between resolution and recovery and are time
consuming often hours or days to complete. Separation of high molecular weight nucleic acids is often
done by gel electrophoresis or ultracentrifugation in CsCl density gradients. The high resolution of gel
electrophoresis is very favorable but the recovery of the purified samples is low and the quality is often
diminished because of contaminations with the gel matrix (soluble oligomeric agarose or acrylamide)
yielding an inefficient biological activity in cell transformation and enzymatic reactions, e.g. digestion with
restriction endonucleases, ligation, and reverse transcription. In addition electrophoresis is time
consuming requiring hours or days to complete. Although for the special case of plasmid preparation
banding in CsCl density gradients may be performed, it requires prolonged ultracentrifugation at high
speed and consumes large amounts of CsCl and has the disadvantage of being expensive in respect to
CsCl and service cost for the ultracentrifuge.

Chromatographic processes have proved advantageous for many separation problems in organic
chemistry. High-performance liquid chromatography (HPLC) offers the most advantages in relation to
resolution, short consumption of time and reproducibility. Said process has hithereto been used in the
form of gel permeation chromatography (GPC), ion-exchange chromatography and reversed-phase
chromatography (RP chromatography). However, for the separation of nucleic acids, these processes had
the following disadvantages:

GPC is able only to separate small from very large molecules and resolution decreases above molecular
weights of about 250,000, i.e. about 350 basepairs DNA.

The prior art ion exchangers and reversed-phase chromatography resins could only be used with high
resolution for small molecules such as oligonucleotides, e.g. chain length up to 15 nucleotides (Fritz et al.;
Biochemistry (1978) 17, 1257-1267). In the separation of nucleic acids of high molecular weight such as
long-chain ribo- and deoxyribo-oligonucleotides, natural RNA's, like transfer RNA and 7S RNA, viral RNA
and messenger RNA, DNA, DNA fragments and plasmid DNA, the required resolution into individual
nucleic acid species could not be obtained.

Although hydrophobic-ionic RPC-5 chromatography material such as described by Larson, J. E. et al (The

Journal of Biological Chemistry (1979) 254, 5535-5541) had been successfully used in the separation of
DNA fragments, the flow rates that is, very long durations of chromatography, and the low
chromatographic stability of the RPC-5 material are of great disadvantage. Due to the chemical properties
of the RPC-5 material, bleeding of the liquid ion-exchanger Adogen 464 occurs, contaminating the purified
samples.

With prior art chromatography, it was not possible to separate or isolate into the individual molecular
species complex nucleic acid mixtures, with high resolution and high velocity, or to analyze said mixtures.
It is thus an object of the present invention to provide a chromatographic process, wherewith the above-
mentioned disadvantages are eliminated. Yet another object of the present invention is to provide in a
single run the separation into their components, with very high resolution and high velocities, of
macromolecular mixtures of the most varied kinds, which contain components of very different
dimensions, for example, in the range of 30 Angstroms to 1,000 Angstroms. Still another object of the
invention is to provide separation materials suitable for employment at high flow rates, wide temperature
ranges and with extended durability. A high loading capacity is desirable with the nucleic acid mixtures to
be separated. Contrary to the contraindications of the prior art, the present invention provides a
chromatographic resin, with which it is possible to separate in a chromatographic process complex
nucleic acid mixtures having a very broad spectrum of molecular size at very high resolution.

Because of its simplicity and the stable resin, this process is suitable for routine use in industry and
research.

More particularly, the present invention provides the surface modification of a carrier material of a
chromatographic resin, for the chromatographic separation of nucleic acids, using an inert particle,
preferably silica gel. This silica gel is a high performance liquid chromatography silica gel, and preferably
has a particle size of about 2 to 100 microns. A suitable, commercially available silica gel for this purpose
is 10 micron LiChrosphere SI 4000 sold by E. Merck, Germany. However, the silica gel may be any
chromatographic grade silica gel.

In the initial step, the silica is reacted with a bifunctional silane, e.g. γ-glycido-oxypropyl-trimethoxysilane
in toluene. The reaction is conducted for a time and at a temperature to produce a product comprising γ-
glycido-oxypropyl-trimethoxysilane covalently bound to the silica. An excess of γ-glycido-oxypropyl-
trimethoxysilane to reactive silanol sites on the silica is used, a 5-fold excess being preferred. After
reaction, the product epoxy-silica is filtered through a glass filter funnel yielding a filter cake. The filter
cake is washed with hexane, dioxane and methanol to remove the silane and soluble impurities. Then the
epoxy-silica is dried in vacuo. In the next step, if for example, the surface is modified into an anion-
exchanger, the epoxy-silica is reacted with a N,N-dialkylalcohol, e.g. N,N-diethylethanol in toluene. After
reaction, the resulting anion-exchanger diethylaminosilica gel (DEAE-silica) is removed by suction
through a glass filter funnel and washed with methanol, acetone and ether and dried in vacuo.

For chromatography of nucleic acids the resulting chromatographic resin is dispersed in methanol and fed
under pressure into a stainless steel column (diameter 6 mm, length 125 mm) at a flow-rate of 5 ml/min
with a high-pressure pump with methanol as an eluent. The packed column is washed with water and
connected to a liquid chromatograph (DuPont LC 850) equipped with an UV-detector. The separation is
carried out with a gradient elution of increasing ionic strength in aqueous buffers at pH between 4 to 8.
With this chromatographic resin water soluble nucleic acids can be separated conveniently and rapidly
with high resolution.

The process described according to the invention is the first chromatographic process that has the
following properties:

1. General applicability to the separation of nucleic acids.

2. Short chromatographic periods and high reproducibility of the elution profiles by the use of pressure-
stable resins in HPLC equipments.

3. High loading capacity.

4. Chromatographic materials having long-term stability (no "bleeding" of the chromatography columns).

Unlike prior art chromatographic resins, which were exclusively based on the chemical properties of the
carrier materials, the point of departure of the instant invention is the finding that the size and/or the
shape of the cavities of the resin is of quite essential importance for the separation and must be in a
specific relationship to the size of the nucleic acid species to be isolated. It has been found that the size of
the cavities must amount to 1 to 40 times that of the components to be isolated. If the dimensions of the
individual components to be separated differ from each other by more than a factor 40, it is possible to
carry out the separation in several steps using carrier materials having suitably sized cavities. According
to a preferred embodiment of the invention, there is provided a suitable modification of the surface. It has
proved very advantageous here that the groups responsible for the interaction with the substances to be
separated have been anchored to the surface by flexible chain molecules. This effect is obtained, for
instance, by using γ-glycido-oxypropyl-trimethoxysilane. As interaction-producing groups there are
contemplated strongly and weakly basic anion exhangers, strongly and weakly acidic cation exchangers,
groups having hydrophobic interactions, groups having polarization interactions, and groups that combine
several of the aforementioned properties. Since it has been found that in many applications bivalent metal
ions can give rise for considerable hindrances, it has further been proposed according to the invention
that all parts that come in contact with the solvents consist of noble metal, glass or plastic, or have
adequate coatings of noble metal, glass or plastic.

More particularly, in accordance with a preferred embodiment of the invention, there is provided a process
for the chromatographic separation of nucleic acids using a chromatographic carrier prepared by reacting
a carrier material that contains cavities and has a grain size of from 2 to 100 μm, a cavity size of from 10
to 4000 nm and a specific surface of from 5 to 800 m 2 /g with a silanization reagent of the general
formula R 1 R 2 R 3 SiR 4 I.

wherein R 1 corresponds to one alkoxy radical having from 1 to 10 C atoms, preferably --OCH 3 , --OC 2
H 5 or --OC 3 H 7 , or one halogen atom, preferably --Cl, or one dialkyl amino group with identical or
different alkyl radicals having from 1 to 6 C atoms, R 2 and R 3 correspond to a hydrocarbon radical
having from 1 to 10 C atoms, preferably --CH 3 , --C 2 H 5 or C 3 H 7 , or an alkoxy radical having from 1
to 10 C atoms, preferably --OCH 3 , --OC 2 H 5 or --OC 3 H 7 , or one halogen atom or one alkyl radical
having from 4 to 20 C atoms interrupted by at least one oxy or amino group, wherein said radical can also
be replaced once or more by halogen, cyano, nitro, amino, monoalkylamino, dialkylamino, hydroxy or aryl,
and R 4 corresponds to a hydrocarbon chain having from 1 to 20 C atoms or an alkyl radical interrupted
by at least one oxy or amino group, wherein said radical can also be replaced once or more by halogen,
cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, aryl and/or epoxy, preferably
##STR1## and then, to form the final carrier, reacting the carrier containing said cavities with a reagent of
the general formula X-R-Y II

wherein X is an amino, hydroxy, epoxy group or one halogen atom, R is a hydrocarbon chain having from
2 to 20 C atoms, or an alkyl radical interrupted by at least one oxy or amino group, wherein said radical
can also be replaced once or more by halogen, cyano, nitro, amino, monoalkylamino, dialkylamino,
alkoxy, hydroxy, aryl and/or epoxy and Y is a hydrocarbon radical having from 1 to 10 C atoms and
functional groups that form anion or cation exchangers, wherein said radical can be replaced once or
more by amino, monoalkylamino, dialkylamino, quaternary alkylamino, carboxyl, boric acid, alkyl and aryl
sulfonic acid groups, wherein the diameter of the cavities amounts to from 1 to 40 times, more preferably
1 to 20 times the maximum dimension of the respective nucleic acid to be isolated or the maximum
dimension of the largest of all nucleic acids contained in the mixture.

In a particularly preferred embodiment of the invention, the carrier material used has a cavity size of from
50 to 1000 nm and a specific surface of at most 200 m 2 /g. Preferably silicon dioxide is used as carrier
material. In one preferred embodiment of the invention, the cavities are formed by semispherical
recesses; in another preferred embodiment the cavities are tubular. Preferably the solution containing
nucleic acids to be separated and the elution substance are kept free from bivalent metal ions by using
noble metal, glass and/or plastic for columns, pipes, valves and pumps of the chromatographic
equipment.

BRIEF DESCRIPTION OF THE DRAWING

The invention is explained in more detail with reference to the figures, which show:

FIGS. 1A, 1B, and 1C show segments and cross sections through carrier materials of different cavity
sizes; and

FIGS. 2, 3, 4, 5 and 6 are graphic illustrations of separations profiles obtained in accordance with the
present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is explained in more detail in the following examples:

EXAMPLE 1

A weak anion exchanger according to the present invention was synthesized by the following process:

Commercial 50 g LiChrosphere SI 4000 silica gel particles (E. Merck, Darmstadt, Germany) with a partical
size of 10 μm and a poresize of 4000 Angstroms is activated in a 1000 ml three-necked flask at a
pressure of <1 mbar for 24 hours at the temperature of 200° C. After cooling, it was aerated with dry
nitrogen and suspended in 100 ml dry γ-glycidooxypropyl-trimethoxysilane in 500 ml dry toluene and 1 ml
tributylamine. The reaction took place for 10 hours reflux under a nitrogen atmosphere and with
continuous stirring at 400 rpm. After the reaction, the excess γ-glycidooxypropyl-trimethoxysilane and
toluene were removed by suction and the product epoxy-silica was washed four times with 400 ml dry
hexane and two times 400 ml dry ether and dried in vacuo. With a four-necked flask including an inner
thermometer, return-flow cooler, stirrer and nitrogen inlet pipe, the epoxy-silica gel was reacted with 100
ml dry N,N-diethyl-aminoethanol in 400 ml toluene. The reaction was catalyzed by addition of 1 ml BF 3
/ether and boiled for 12 hours under reflux. After reaction, the final chromatographic resin dimethylamino-
silica gel (DEAE-Silica) was removed by suction and washed two times with 400 ml dioxane, 400 ml
methanol and 200 ml ether, and dried at 50° C. in vacuo. The yield amounted to 51.5 g.

For column packing 3 g of the resulting chromatographic resin was dispersed in 50 ml methanol and was
fed under pressure into a stainless steel column (diameter 6 mm, length 125 mm) connected to a 50 ml
packing reservoir at a flow-rate of 5 ml/min with a high-pressure pump with methanol as an eluent. The
packed column was disconnected from the packing reservoir, washed with methanol and water and
connected to a liquid chromatograph (DuPont LC 850) equipped with a UV-detector. The separation is
carried out with a gradient elution of increasing KCl concentration in 5 M urea, 30 mM potassium-
phosphate buffer, pH 6.5

EXAMPLE 2--EFFECT OF PORE SIZE

The effect of the cavity size of the chromatographic resin on the interaction with the macromolecule is
explained with reference to FIG. 1. A macromolecule (1) cannot sufficiently penetrate in the too small
cavity (2) of the carrier material (3) in order to enter into an optimal interaction. On the other hand, the
cavity (4) of more favorable dimensions permits very intensive interactions. To increase the interactions,
the interaction producing groups are anchored on the cavity surface by flexible chain molecules (5). If on
the contrary the cavity (6) is too large, then a reduction in interaction is again to be expected.

In FIG. 2 are illustrated separation examples of long-chain nucleic acids. The cavity diameters are - as
indicated in the drawing--100 Angstroms, 300 Angstroms, 500 Angstroms and 4000 Angstroms. As
example for a separation of long-chain nucleic acids, there was selected a natural mixture of transfer RNA
(80 Angstroms size), ribosomal 5S RNA (110 Angstromes size), 7S RNA (300 Angstroms size) and viroid
RNA (450 Angstroms size, a plant pathogene infectious RNA). It can be clearly seen in FIG. 2 that the
largest pore size selected gave the best separation, and it is not to be ruled out that with a cavity size
between 1000 Angstroms and 4000 Angstroms a still better separation would be obtained. In FIG. 4 the
example from FIG. 2 has been further optimized by a shallower gradient elution. A complete separation of
all four components is obtained. The diethylamino silica gels used in the examples had the loading
capacity of 4.8 mg nucleic acid mixture/g (100 Angstroms), 17 mg nucleic acid mixture/g (500 Angstroms)
and 5.6 mg nucleic acid mixture/g (4000 Angstroms).

EXAMPLE 3--SEPARATION OF OLIGORIBONUCLEIC ACIDS

Oligo-ribo-adenylic acids with chain lengths from 3 to 40 nucleotides were chromatographically purified
using the anion-exchange resin obtained in Example 1 (FIG. 3). Synthetic oligonucleotides of defined
length and sequence are required for modern genetic engineering and molecular biology.

Column: 6 mm×125 mm stainless steel, elution with a linear gradient from 0 to 1 M KCl in 200 min, in 5 M
urea, 30 mM potassium-phosphate buffer, pH 5.5, at a flow rate of 1 ml/min, 35 bar, 22° C.

EXAMPLE 4--VIROID RNA

Viroid RNA (PSTV) from total RNa from infected plants was chromatographically purified using the anion-
exchange resin obtained in Example 1 (FIG. 4). The purified nucleic acid was pure to spectroscopic,
hydrodynamic and thermodynamic properties and was fully active in enzymatic experiments.

Column: 6 mm×125 mm stainless steel, elution with a linear gradient from 250 mM to 1000 mM KCl in
200 min, in 5 M urea, 30 mM potassium-phosphate buffer, pH 6.5, flow rate 1.5 ml/min, 45 bar, 22° C.

EXAMPLE 5--DNA-RESTRICTION FRAGMENTS

Chromatography of high molecular weight DNA restriction fragments was carried out using the anion-
exchange resin obtained in Example 1 (FIG. 5). The DNA fragments were obtained by digestion of pBR
322 plasmid DNA with Hinf 1 yielding following sizes: 75, 154, 220, 298, 344, 396, 506, 517 and 1631
basepairs, respectively. It is remarkable that even the 506 and 517 basepair fragments could be
separated from each other.

Column: 6 mm×125 mm stainless steel, elution with a linear gradient from 700 mM to 1200 mM KCl in
100 min, in 5 M urea, 30 mM potassium-phosphate buffer, pH 6.5, flow rate 1 ml/min, 35 bar 22° C.

EXAMPLE 6--PLASMID DNA FROM CRUDE CELL LYSATE

Purification of 50 μg plasmid pBR 322 DNA from crude cell lysate prepared by the lysozyme/EDTA
method (D. B. Clewell and D. R. Hellinsky, proceedings of the National Academy of Sciences, USA
(1969), 62, 1159-1166) was carried out using the anion-exchange resin obtained in Example 1 (FIG. 6).
Column: 6 mm×125 stainless steel, elution with a linear gradient from 300 mM to 1500 mM KCl in 50 min,
in 5 M urea, 30 mM potassium-phosphate buffer, Ph 6.5, flow rate 1.5 ml/min, 45 bar, 22° C.

Various changes may be made in the foregoing process without departing from the spirit and scope of the
present invention.

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NCBI » Bookshelf » Molecular Biology of the Cell » Molecular Genetics » Recombinant DNA Technology
» The Fragmentation, Separation, and Sequencing of DNA Molecules 1

cell
Molecular Biology of the Cell
3rd
Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D Watson
University of California, San Francisco, USA
Department of Zoology, University of Cambridge, Cambridge, England
Imperial Cancer Research Fund Developmental Biology Unit, University of Oxford, England
MRC Laboratory for Molecular Cell Biology and Biology Department, University College London, England
Department of Cell Biology, John Innes Institute, Norwich, England
Cold Spring Harbor Laboratory, USA
Garland Science
New York
0-8153-1619-41994
Copyright © 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D
Watson
1997
books-source-type
Book
books-subject
Molecular Biology
books-subject
Cell Biology
See "Molecular Biology of the Cell, Fourth Edition"
By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.
Chapter 7: The Fragmentation, Separation, and Sequencing of DNA Molecules 1
Introduction

Before the 1970s the goal of isolating a single gene from a large chromosome seemed unattainable.
Unlike a protein, a gene does not exist as a discrete entity in cells, but rather as a small region of a much
larger DNA molecule. Although the DNA molecules in a cell can be randomly broken into small pieces by
mechanical force, a fragment containing a single gene in a mammalian genome would still be only one
among a hundred thousand or more DNA fragments, indistinguishable in their average size. How could
such a gene be purified? Since all DNA molecules consist of an approximately equal mixture of the same
four nucleotides, they cannot be readily separated, as proteins can, on the basis of their different charges
and binding properties. Moreover, even if a purification scheme could be devised, vast amounts of DNA
would be needed to yield enough of any particular gene to be useful for further experiments.

The solution to all of these problems began to emerge with the discovery of restriction nucleases. These
enzymes, which can be purified from bacteria, cut the DNA double helix at specific sites defined by the
local nucleotide sequence, producing double-stranded DNA fragments of strictly defined sizes. Different
species of bacteria make restriction nucleases with different sequence specificities, and it is relatively
simple to find a restriction nuclease that will create a DNA fragment that includes a particular gene. The
size of the DNA fragment can then be used as a basis for partially purifying the gene from a mixture. Most
important, the DNA fragment usually serves as the starting material for the production of the highly
purified gene in unlimited amounts by DNA cloning.

We begin this section by discussing how restriction nucleases are used to produce specific DNA
fragments and how these (and other) DNA molecules are separated according to their size. We then
explain how, after the purification and amplification of a DNA fragment by DNA cloning, this DNA can be
sequenced to determine the order of its nucleotides.
Restriction Nucleases Hydrolyze DNA Molecules at Specific Nucleotide Sequences 2

Many bacteria make restriction nucleases, which protect the bacterial cell from viruses by degrading the
viral DNA. Each such nuclease recognizes a specific sequence of four to eight nucleotides in DNA. These
sequences, where they occur in the genome of the bacterium itself, are protected from cleavage by
methylation at an A or a C residue; where the sequences occur in foreign DNA, they are generally not
methylated and so are cleaved by the restriction nucleases. Large numbers of restriction nucleases have
been purified from various species of bacteria; more than 100, most of which recognize different
nucleotide sequences, are now available commercially.

Figure 7-1
Figure 7-1

Figure 7-1
The DNA nucleotide sequences recognized by four widely (more...)
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f1.jpg.

Figure 7-1
.

The DNA nucleotide sequences recognized by four widely used restriction nucleases

As in the examples shown, such sequences are often six base pairs long and "palindromic" (that is, the
nucleotide sequence is the same if the helix is turned by 180 degrees around the center of the short
region of helix that is recognized). The enzymes cut the two strands of DNA at or near the recognition
sequence. For some enzymes, such as Hpa I, the cleavage leaves blunt ends; for others, such as Eco RI,
Hind III, and Pst I, the cleavage is staggered and creates cohesive ends. Restriction nucleases are
obtained from various species of bacteria: Hpa I is from Hemophilus parainfluenzae, Eco RI is from
Escherichia coli, Hind III is from Hemophilus influenzae, and Pst I is from Providencia stuartii.
Figure 7-2
Figure 7-2

Figure 7-2
Many kinds of restriction nucleases produce DNA fragments (more...)
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f2.jpg.

Figure 7-2
.

Many kinds of restriction nucleases produce DNA fragments with cohesive ends

DNA fragments with the same cohesive ends can readily join by complementary base-pairing between
their cohesive ends as illustrated. The two DNA fragments that join in this example were both produced
by the Eco RI restriction nuclease, whereas the three other fragments were produced by different
restriction nucleases that generate different cohesive ends (see Figure7-1).
Some restriction nucleases produce staggered cuts, which leave short single-stranded tails at the two
ends of each fragment (Figure7-1). Ends of this type are known as cohesive ends, as each tail can form
complementary base pairs with the tail at any other end produced by the same enzyme (Figure7-2). The
cohesive ends generated by restriction enzymes allow any two DNA fragments to be easily joined
together, as long as the fragments were generated with the same restriction nuclease (or with another
nuclease that produces the same cohesive ends). DNA molecules produced by splicing together two or
more DNA fragments in this way are called recombinant DNA molecules; they have made possible many
new types of cell biological studies.
Restriction Maps Show the Distribution of Short Marker Nucleotide Sequences Along a Chromosome 3

Figure 7-3
Figure 7-3

Figure 7-3
Restriction mapping
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f3.jpg.

Figure 7-3
.

Restriction mapping

A simple example illustrating how the positions of cutting sites for different restriction nucleases (known
as restriction sites) are determined relative to one another on double-helical DNA molecules to create a
restriction map. (kb = kilobases, an abbreviation designating either 1000 nucleotides or 1000 nucleotide
pairs.)
Figure 7-4
Figure 7-4

Figure 7-4
Restriction maps of human and various primate DNAs (more...)
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f4.jpg.

Figure 7-4
.

Restriction maps of human and various primate DNAs in a cluster of genes coding for hemoglobin

The two red squaresin each map indicate the positions of the DNA corresponding to the two α-globin
genes. Each letter stands for a site cut by a different restriction nuclease. As in Figure 7-3, the location of
each cut was determined by comparing the sizes of the DNA fragments generated by treating the DNAs
with the various restriction nucleases, individually and in combinations. Note that the chimpanzee, which
is most closely related to humans, has the most similar restriction map, whereas the gibbon is more
distantly related and has the most diverged mapincluding three DNA insertions. (Courtesy of Elizabeth
Zimmer and Allan Wilson.)
A particular restriction nuclease will cut any double-helical DNA molecule extracted from a cell into a
series of specific DNA fragments (known as restriction fragments). By comparing the sizes of the DNA
fragments produced from a particular genetic region after treatment with a combination of different
restriction nucleases, a restriction map of that region can be constructed showing the location of each
cutting (restriction) site in relation to neighboring restriction sites (Figure 7-3). The different short DNA
sequences recognized by different restriction nucleases serve as convenient markers, and the restriction
map reflects their arrangement in the region. This allows one to compare the same region of DNA in
different individuals (by comparing their restriction maps) without having to determine the nucleotide
sequences in detail. By comparing the restriction maps illustrated in Figure 7-4, for example, we know
that the chromosomal regions that code for hemoglobin chains in humans and various other primates
have remained largely unchanged during the 5 to 10 million years since these species first diverged.
Restriction maps are also used in DNA cloning and DNA engineering, where they make it possible to
locate a gene of interest on a particular restriction fragment and thus facilitate its isolation.
Gel Electrophoresis Separates DNA Molecules of Different Sizes 4

Figure 7-5
Figure 7-5

Figure 7-5
Gel electrophoresis techniques for separating DNA molecules (more...)
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f5.jpg.

Figure 7-5
.

Gel electrophoresis techniques for separating DNA molecules by size

In the three examples shown, electrophoresis is from top to bottom, so that the largest - and so slowest-
moving - DNA molecules are near the top of the gel. In (A) a polyacrylamide gel with small pores is used
to fractionate single-stranded DNA. In the size range 10 to 500 nucleotides, DNA molecules that differ in
size by only a single nucleotide can be separated from each other. In the example the four lanes
represent sets of DNA molecules synthesized in the course of a DNA-sequencing procedure. The DNA to
be sequenced has been artificially replicated from a fixed start site up to a variable stopping point,
producing a set of partial replicas of differing lengths. (Figure 7-8 explains how such sets of partial
replicas are synthesized.) Lane 1 shows all the partial replicas that terminate in a G; lane 2, all those that
terminate in an A; lane 3, all those that terminate in a T; and lane 4, all those that terminate in a C. Since
the DNA molecules used in these reactions are radiolabeled, their positions can be determined by
autoradiography, as shown. In (B) an agarose gel with medium-sized pores is used to separate double-
stranded DNA molecules. This method is most useful in the size range 300 to 10,000 nucleotide pairs.
These DNA mole-cules are restriction fragments produced from the genome of a bacterial virus, and they
have been detected by their fluorescence when stained with the dye ethidium bromide. In (C) the
technique of pulsed-field agarose gel electrophoresis has been used to separate 16 different yeast
(Saccharomyces cerevisiae) chromosomes that range in size from 220,000 to 2.5 million nucleotide pairs.
The DNA was stained as in (B). DNA molecules as large as 107 nucleotide pairs can be separated in this
way. (A, courtesy of Leander Lauffer and Peter Walter; B, courtesy of Ken Kreuzer; C, from D. Vollrath
and R.W. Davis, Nucleic Acids Res. 15:7876, 1987, by permission of Oxford University Press.)
In the early 1970s it was found that the length and purity of DNA molecules could be accurately
determined by the same types of gel electrophoresis methods that had proved so useful in the analysis of
proteins. The procedure is actually simpler than for proteins: because each nucleotide in a nucleic acid
molecule already carries a single negative charge, there is no need to add the negatively charged
detergent SDS that is required to make protein molecules move uniformly toward the positive electrode.
For DNA fragments less than 500 nucleotides long, specially designed polyacrylamide gels allow
molecules that differ in length by as little as a single nucleotide to be separated from each other (Figure7-
5 A). The pores in polyacrylamide gels, however, are too small to permit very large DNA molecules to
pass; to separate these by size, the much more porous gels formed by dilute solutions of agarose (a
polysaccharide isolated from seaweed) are used (Figure 7-5B). These DNA separation methods are
widely used for both analytical and preparative purposes.

A variation of agarose gel electrophoresis, called pulsed-field gel electrophoresis, makes it possible to
separate even extremely long DNA molecules. Ordinary gel electrophoresis fails to separate such
molecules because the steady electric field stretches them out so that they travel end-first through the gel
in snakelike configurations at a rate that is independent of their length. In pulsed-field gel electrophoresis,
by contrast, the direction of the electric field is changed periodically, which forces the molecules to
reorient before continuing to move snakelike through the gel. This reorientation takes much more time for
larger molecules, so that progressively longer molecules move more and more slowly. As a consequence,
even entire bacterial or yeast chromosomes separate into discrete bands in pulsed-field gels and so can
be sorted and identified on the basis of their size (Figure 7-5C). Although a typical mammalian
chromosome of 108 base pairs is too large to be sorted even in this way, large subregions of these
chromosomes are readily separated and identified if the chromosomal DNA is first cut with a restriction
nuclease selected to recognize sequences that occur only extremely rarely (once every 106 to 107
nucleotide pairs, for example).

The DNA bands on agarose or polyacrylamide gels are invisible unless the DNA is labeled or stained in
some way. One sensitive method of staining DNA is to expose it to the dye ethidium bromide, which
fluoresces under ultraviolet light when it is bound to DNA (see Figure 7-5B and C). An even more
sensitive detection method involves incorporating a radioisotope into the DNA molecules before
electrophoresis; 32P is often used as it can be incorporated into DNA phosphates and emits an energetic
β-particle that is easily detected by autoradiography (Figure 7-5A).
Purified DNA Molecules Can Be Specifically Labeled with Radioisotopes or Chemical Markers in Vitro5

Figure 7-6
Figure 7-6

Figure 7-6
Two procedures are used routinely for making DNA molecules (more...)
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f6.jpg.

Figure 7-6
.

Two procedures are used routinely for making DNA molecules radioactive
(A) A purified DNA polymerase enzyme labels all the nucleotides in a DNA molecule and can thereby
produce highly radioactive DNA probes. (B) Polynucleotide kinase labels only the 5' ends of DNA strands;
therefore, when labeling is followed by restriction nuclease cleavage, as shown, DNA molecules
containing a single 5'-end-labeled strand can be readily obtained. The method in (A) is also used to
produce nonradioactive DNA molecules that carry a specific chemical marker that can be detected with an
appropriate antibody (see Figure 7-18).
Two procedures are widely used to add distinct labels to isolated DNA molecules. In the first the DNA is
copied by an E. coli enzyme, DNA polymerase I, in the presence of nucleotides that are either radioactive
(usually labeled with 32P) or chemically tagged (Figure 7-6A). In this way "DNA probes" containing many
labeled nucleotides can be produced for nucleic acid hybridization reactions (see below). The second
procedure uses the bacteriophage enzyme polynucleotide kinase to transfer a single 32P-labeled
phosphate from ATP to the 5' end of each DNA chain (Figure 7-6B). Because only one 32P atom is
incorporated by the kinase into each DNA strand, the DNA molecules labeled in this way are often not
radioactive enough to be used as DNA probes; because they are labeled at only one end, however, they
are invaluable for DNA sequencing and DNA footprinting, as we see next.
Isolated DNA Fragments Can Be Rapidly Sequenced 6

Figure 7-7
Figure 7-7

Figure 7-7
The chemical method for sequencing DNA
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f7.jpg.

Figure 7-7
.

The chemical method for sequencing DNA

(A) The procedure starts with a set of identical end-labeled double-stranded DNA molecules produced by
the method outlined in Figure 7-6B. In the first step the strands of the double helix are dissociated and
exposed to mild treatment with a chemical that destroys one of the four bases (in this case, A residues) in
the DNA. Because the treatment is mild, usually only one of the A residues in each molecule is destroyed
at random. This generates a family of DNA fragments of different lengths, reflecting the different sites at
which A residues occur in the original DNA. These fragments are separated on a gel and detected by
autoradiography: only the fragments possessing a 5'-terminal 32P-phosphate group show up on the gel,
and their sizes reveal the distances from the labeled end at which A residues occur. (B) To determine the
full sequence, similar procedures are carried out simultaneously on four separate samples of the same 5'-
end-labeled DNA molecule using chemicals that cleave DNA preferentially at T for the first sample, C for
the second, G for the third, and A for the fourth. The resulting fragments are separated in parallel lanes of
a gel like that shown in Figure7-5A, giving a pattern of radioactive DNA bands from which the DNA
sequence is read. The nucleotide closest to the 5' end of the sequence is determined by looking across
the gel at level 1 (at the bottom of the gel) and seeing in which lane a band appears (T). The same
procedure is repeated for level 2, then level 3, and so on, to obtain the sequence. The method has been
idealized here; the actual chemical treatments are less specific than shown.
Figure 7-8
Figure 7-8

Figure 7-8
The enzymatic method for sequencing DNA
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f8.jpg.

Figure 7-8
.

The enzymatic method for sequencing DNA

(A) The DNA to be sequenced is used as template for the in vitro synthesis, by DNA polymerase, of a set
of partial replicas, all beginning at the same place, but terminating at different points along the DNA chain.
The key to this method is the use of dideoxyribonucleoside triphosphates in which the deoxyribose 3'-OH
group present in normal nucleotides is missing; when such a modified nucleotide is incorporated into a
DNA chain, it blocks the addition of the next nucleotide. In the example illustrated, dideoxy ATP (ddATP)
competes with an excess of deoxy ATP (dATP), so that each newly synthesized DNA strand made in a
test tube by DNA polymerase will stop at a randomly selected A in the sequence. This reaction therefore
generates a ladder of DNA fragments similar to that shown previously for the chemical method (Figure 7-
7); these fragments are detected by a label (chemical or radioactive) that is either incorporated into the
oligonucleotide primer (orange), or into one of the deoxyribonucleoside triphosphates (green) used to
extend the DNA chain. (B) To determine the full sequence, four different chain-terminating nucleoside
triphosphates (red) are used in separate DNA synthesis reactions on the same primed single-stranded
DNA template. When the products of these four reactions are analyzed by electrophoresis in four parallel
lanes of a polyacrylamide gel, the DNA sequence can be derived in the manner illustrated for the
chemical method in Figure7-7B.
In the late 1970s methods were developed that allow the nucleotide sequence of any purified DNA
fragment to be determined simply and quickly. They have made it possible to determine the complete
DNA sequences of thousands of genes, including those coding for such well-known proteins as insulin,
hemoglobin, interferon, and cytochrome c. The volume of DNA sequence information is already so large
(many tens of millions of nucleotides) that computers must be used to store and analyze it. Several
continuous stretches of DNA sequence have been determined that each contain more than 105
nucleotide pairs; these include the entire genomes of the Epstein-Barr virus (which infects humans and
causes infectious mononucleosis) and of a plant chloroplast, as well as an entire chromosome of yeast.
Two powerful DNA sequencing methods were originally developed: the principle underlying the chemical
method is illustrated in Figure 7-7, and the enzymatic method is explained in Figure7-8. The latter
method, which is based on in vitro DNA synthesis carried out in the presence of chain-terminating
nucleoside triphosphates, has now become the standard procedure for sequencing DNA.

Figure 7-9
Figure 7-9

Figure 7-9
Finding the regions in a DNA sequence that encode a (more...)
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f9.jpg.

Figure 7-9
.

Finding the regions in a DNA sequence that encode a protein

(A) Any region of the DNA sequence can, in principle, code for six different amino acid sequences,
because any one of three different reading frames can be used to interpret the nucleotide sequence on
each strand. Note that a nucleotide sequence is always read in the 5'-to-3' chain direction and encodes a
polypeptide from the amino (N) to the carboxyl (C) terminus. For a random nucleo-tide sequence, a stop
signal for protein synthesis will be encountered, on average, about once every 21 amino acids (once
every 63 nucleo-tides). In this sample sequence of 48 base pairs, each such signal (stop codon) is
colored green, and only reading frame 2 lacks a stop signal. (B) Search of a 1700 base-pair DNA
sequence for a possible protein-encoding sequence. The information is displayed as in (A), with each
stop signal for protein synthesis denoted by a green line.In addition, all of the regions between possible
start and stop signals for protein synthesis (see p. 234) are displayed as red bars.Only reading frame 1
actually encodes a protein, which is 475 amino acid residues long.
DNA sequencing methods are so rapid and reliable that the easiest and most accurate way to determine
the amino acid sequence of a protein is to determine the nucleotide sequence of its gene: a cDNA clone
is made from the appropriate mRNA (see below), its nucleotide sequence is determined, and the genetic
code is then used as a dictionary to convert this sequence back to an amino acid sequence. Although in
principle there are six different reading frames in which a DNA sequence can be translated into protein
(three on each strand), the correct one is generally recognizable as the only one lacking frequent stop
codons (Figure 7-9). A limited amount of amino acid sequence can be determined from the purified
protein to confirm the sequence determined from the DNA.

As techniques for DNA sequencing have improved, scientists have begun to envisage the possibility of
determining the entire DNA sequence of a human genome of 3 x 109 nucleotides. Because this sequence
specifies all of the possible RNA and protein molecules that are used to construct the body, knowledge of
it will provide us with a "dictionary of the human being," which will greatly expedite future studies of
human cells and tissues. DNA sequencing on such a scale will require the development of highly
automated methods that will lower the current cost of DNA sequencing by at least fivefold.
DNA Footprinting Reveals the Sites Where Proteins Bind on a DNA Molecule 7

Figure 7-10
Figure 7-10

Figure 7-10
The DNA footprinting technique
An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of
referred object is ch7f10.jpg.

Figure 7-10
.

The DNA footprinting technique

(A) This technique requires a DNA molecule that has been radioactively labeled at one end (see Figure7-
6B). The protein shown binds tightly to a specific DNA sequence that is seven nucleotides long, thereby
protecting these seven nucleotides from the cleaving agent. If the same reaction were carried out without
the DNA-binding protein, a complete ladder of bands would be seen on the gel (not shown). (B) An actual
footprint used to determine the binding site for a human protein that stimulates the transcription of specific
eucaryotic genes. These results locate the binding site about 60 nucleotides upstream from the start site
for RNA synthesis. The cleaving agent was a small, iron-containing organic molecule that normally cuts at
every phosphodiester bond with nearly equal frequency. (B, courtesy of Michele Sawadogo and Robert
Roeder.)
A modification of the chemical method for DNA sequencing can be used to determine the nucleotide
sequences recognized by DNA-binding proteins. Some of these proteins play a central part in determining
which genes are active in a particular cell by binding to regulatory DNA sequences, which are usually
located outside the coding regions of a gene. In analyzing how such a protein functions, it is important to
identify the specific sequences to which it binds. A method used for this purpose is called DNA
footprinting. First, a pure DNA fragment that is labeled at one end with 32P is isolated (see Figure7-6B);
this molecule is then cleaved with a nuclease or a chemical that makes random single-stranded cuts in
the DNA. After the DNA molecule is denatured to separate its two strands, the resultant subfragments
from the labeled strand are separated on a gel and detected by autoradiography. The pattern of bands
from DNA cut in the presence of a DNA-binding protein is compared with that from DNA cut in its
absence. When the protein is present, it covers the nucleotides at its binding site and protects their
phosphodiester bonds from cleavage. As a result, the labeled fragments that terminate in the binding site
will be missing, leaving a gap in the gel pattern called a "footprint" (Figure7-10A). The footprint of a DNA-
binding protein that activates the transcription of a eucaryotic gene is shown in Figure7-10B.
Summary

Recombinant DNA technology has revolutionized the study of the cell. The development of this
technology was neither planned nor anticipated. Instead, steady advances in the ability of researchers to
manipulate DNA molecules were made on many different fronts until the combination of techniques
became powerful enough to allow researchers to pick out any gene at will and, after an amplification step,
to determine the exact molecular structure of both the gene and its products. Crucial elements in this
technology are the ability to fragment chromosomes into DNA molecules with specific ends and the
means to separate and sequence the resulting DNA fragments. The fragmentation step is carried out by
proteins that are normally produced by bacteria in order to destroy invading DNA molecules. These
proteins, called restriction nucleases, are purified and used to cut the DNA double helix at a specific short
nucleotide sequence. The resulting DNA fragments can be separated from one another according to their
size by using electrophoresis to move the mixture of fragments through the pores of a gel. Under certain
conditions, even DNA molecules that differ in length by only a single nucleotide can be detected as
separate bands. DNA sequencing takes advantage of this powerful separation method: after
electrophoresis, the sequence of nucleotides in a DNA molecule is determined from the pattern of labeled
DNA bands observed when a fixed end of one of its DNA strands has been labeled and the other end
terminates at a randomly selected nucleotide of a single type (A, G, C, or T).
Copyright © 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D
Watson
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Table of Contents

Browse on

* In this page
* Molecular Biology of the Cell
* Introduction
* Restriction Nucleases Hydrolyze DNA Molecules at Specific Nucleotide Sequences 2
* Restriction Maps Show the Distribution of Short Marker Nucleotide Sequences Along a Chromosome
3
* Gel Electrophoresis Separates DNA Molecules of Different Sizes 4
* Purified DNA Molecules Can Be Specifically Labeled with Radioisotopes or Chemical Markers in
Vitro5
* Isolated DNA Fragments Can Be Rapidly Sequenced 6
* DNA Footprinting Reveals the Sites Where Proteins Bind on a DNA Molecule 7
* Summary

Where Electrophoresis Gels

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DNA electrophoresis’ is used to separate DNA fragments by size. The types of gels most commonly used
for DNA electrophoresis are agarose (for relatively large DNA molecules) and polyacrylamide (for high
resolution of short DNA fragments). The former is also sometimes referred to as submarine
electrophoresis because the gels are submerged in buffer. The buffers used for the separation are Tris-
Borate-EDTA or Tris-Acetate-EDTA and the DNA migrates relative to its size: smaller fragments moving
faster through the gel matrix and larger fragments moving slower. Following the separation, the gels can
be stained or transferred (Southern blotting). DNA separations are usually detected by dyes that bind to
the molecules and are typically viewed under UV illumination. Nucleic acids can be transferred from the
gel matrix to membrane sheets by capillary or electrical transfer for probing.

DNA fragments in the range of 100 bp to 20 kb are typically separated in agarose gels using submarine
style electrophoresis units. Hoefer offers 8 sizes of submarine units for quick results or high throughput.

Smaller fragments, <100 bp, are typically separated on polyacrylamide gels in a vertical format. We
recommend the Hoefer MiniVE or the Mighty Small II units for small fragments up to 1 kb.

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Nucleic acid separation method
United States Patent 5096818
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An improved method for isolating and purifying nucleic acid from cell culture media contemplating adding
to resuspended cell solutions a lysing solution and neutralizing/deproteinating agent without mechanical
mixing, followed by centrifugation to partially pellet cellular debris, mixing the solution to complete the
reaction, and additional centrifugation to fully pellet the debris.
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US Patent References:
Method of isolating and purifying nucleic acids from biological samples
Seligson et al. - June, 1990 - 4935342

Nucleic acid isolation process

Longmire et al. - May, 1990 - 4921952

Nucleic acid capture method

Gebeyehu et al. - May, 1990 - 4921805

Process for rapid isolation of high molecular weight DNA

Hewitt - February, 1990 - 4900677

Double-stranded DNA having sequences complementary to a single-stranded DNA and derived from a
bean golden mosaic virus
Morinaga et al. - August, 1989 - 4855237 begin_of_the_skype_highlighting 1989 - 4855237
end_of_the_skype_highlighting

Inventors:
Debonville, David A. (Beverly, MA)
Application Number:
07/534025
Publication Date:
03/17/1992
Filing Date:
06/04/1990
Export Citation:
Click for automatic bibliography generation
Assignee:
AutoGen Instruments, Inc. (Beverly, MA)
Primary Class:
435/270
Other Classes:
536/25.420, 435/91.100, 435/259, 536/25.410, 435/91.400
International Classes:
C12N1/06; C12N1/08; C12N15/10
Field of Search:
435/91, 435/172.1, 435/172.3, 435/259, 935/19-21, 536/27
View Patent Images:
Download PDF 5096818 pdf PDF help
US Patent References:
4843012 Novel composition for nucleic acid purification June, 1989 DeBonville et al.
435/259
4833239 Method for the isolation and purification of DNA molecules May, 1989
DeBonville et al. 536/27
4755464 Preparation of plasmid DNA and products thereof July, 1988 MacPhee et al.
935/19
4752574 Chimeric cloning vectors for use in streptomyces and E. Coli June, 1988
Hershberger et al. 435/172.3
4649119 Cloning systems for corynebacterium March, 1987 Sinskey et al. 435/172.3
4283489 Purification of nucleotide sequences suitable for expression in bacteria August, 1981
Goodman et al. 935/21
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Foreign References:
DE0276692 March, 1990 435/259
Primary Examiner:
Lilling, Herbert J.
Attorney, Agent or Firm:
Iandiorio & Dingman
Claims:
What is claimed is:

1. An improved method for isolating and purifying nucleic acid from cell culture media of the type in which:

(a) cells in the culture are concentrated apart from major contaminants in the media and resuspended in
buffer in a tube;

(b) the cells are lysed in the presence of a lysing solution;

(c) the resulting solution is neutralized and deproteinated in the presence of a neutralizing and
deproteinating agent; and

(d) cellular debris is eliminated;

wherein the improvement comprises:

(e) adding to the resuspended cells the lysing solution and the neutralizing/deproteinating agent without
mechanical mixing of the tube contents by repeated pipetting, bubble blowing or shaking the tube, to at
least partially accomplish the lysing, neutralization and deproteination;

(f) centrifuging the tube to partially pellet the cellular debris (g) mechanically mixing the solution after
partial pelleting; and

(h) centrifuging a second time to fully pellet and eliminate cellular debris.

2. The method of claim 1 in which said mechanical mixing includes repeated pipetting of the solution.

3. The method of claim 1 in which said mechanical mixing includes blowing bubbles into the solution.

4. The method of claim 1 in which said mechanical mixing includes vortexing the solution.

5. An improved method for isolating and purifying nucleic acid from cell culture media of the type in which:

(a) cells in the culture are concentrated apart from major contaminants in the media and resuspended in a
buffer in a tube;

(b) the cells are lysed in the presence of a lysing solution; and

(c) the resulting solution is deproteinated in the presence of a deproteinating agent;

wherein the improvement comprises:

(d) adding the lysing solution and the deproteinating agent to the resuspended cells without mechanical
mixing of the tube contents by repeated pipetting, bubble blowing or shaking the tube, to at least partially
accomplish the lysing neutralization and deproteination;

(e) centrifuging the tube to partially pellet the cellular debris (f) mechanically mixing the solution after
partial pelleting; and

(g) centrifuging a second time to fully pellet and eliminate cellular debris.

6. The method of claim 5 in which said mechanical mixing includes repeated pipetting of the solution.

7. The method of claim 5 in which said mechanical mixing includes blowing bubbles into the solution.

8. The method of claim 5 in which said mechanical mixing includes vortexing the solution.

Description:

FIELD OF INVENTION

This invention relates to an improved nucleic acid separation technique which is particularly suited for
automated separation procedures.

BACKGROUND OF INVENTION

The isolation and analysis of nucleic acids from various sources is a commonly performed procedure in
genetic and recombinant DNA research. As the primary genetic elements, nucleic acids will exist in
various forms depending on the biological source: Mammalian sources (such as blood) contain large,
double-stranded, filamentous DNA (20-500 million bases); viruses (HIV or Epstein-Barr) can contain
single- or double-stranded DNA or RNA, filamentous or closed-circular in structure (10-200 kilobases);
bacteria (particularly variants of E. coli K-12) contain a single chromosome (4 million bases) and extra
chromosomal elements, either plasmids or cosmids (2-50 kilobases), in E. coli these elements are all
double-stranded, closed-circular DNA molecules. The procedures and chemistries commonly employed T.
Maniatis et al.: Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory (1989).

The chemistries typically involve breaking open the cells or viral particles, extracting cellular and protein
contaminants, purifying and concentrating the nucleic acids and then resuspending it in small volume
prior to use. Purification of plasmids or cosmids requires the additional step of separation from the
bacterial chromosome prior to their use. This technique, originally described by H. C. Birnboim and J.
Doly "A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA", Nucleic Acids
Research 7:1513-1523 (1979), accomplishes this separation based on the plasmid or cosmid's capacity
to resist denaturation (i.e. separation of the complementary DNA strands into their single-stranded
components). Since the size of the DNA molecule influences its resistance to denaturation, and
subsequent co-purification with plasmids and cosmids, it is important that the bacterial chromosome not
be broken into smaller fragments during the course of the procedure. For this reason, manual protocols
emphasize gentle mixing. In general, any DNA isolation procedure that will reduce the amount of shearing
of chromosomal DNA is desirable.

A major contributor to the breakup of large bacterial chromosomes are the shear forces generated during
the preparational procedure. When the procedures are performed manually, the undesirable high shear
forces are avoided by accomplishing mixing by slowly inverting the test tubes. However, in automated
separation procedures employing automatic equipment to perform some or all of the separation steps,
such slow, gentle mixing by capping and inverting the test tubes is difficult and costly to accomplish.

In automated DNA separation techniques, the tube contents are typically mixed by repeated pipetting of
the solution, blowing bubbles into the solution, or shaking the tubes to simulate vortexing. Each of these
mixing procedures generates significant shear forces as compared to the slow-inversion manual
technique. Accordingly, the automated DNA separation procedures have been characterized by low yields
of plasmid or cosmid DNA of an inferior quality, at least partially due to contamination by chromosomal
fragments. In addition, the automated separation procedures typically take more time than the manual
methods because the mixing steps are very slow and inefficient.

SUMMARY OF INVENTION

It is therefore an object of this invention to provide an improved DNA separation procedure which
increases the yield and quality of plasmid and/or cosmid DNA.

It is a further object of this invention to provide such a procedure which employs the mixing processes of
automated DNA separation techniques.

It is a further object of this invention to provide such a procedure which when applied to automated
separation techniques provides high quality results.

This invention results from the realization that automated DNA separation techniques can be dramatically
improved by adding the lysing/denaturing and the deproteinating and/or neutralizing agent to the
resuspended cells without mixing, followed by centrifuging the solution to partially pellet the cellular
debris, and then mixing the solution to complete the lysing and deproteinization, followed by a second
centrifugation that completely pellets the cellular debris.

DISCLOSURE OF PREFERRED EMBODIMENT

Other objects, features and advantages will occur to those skilled in the art from the following description
of a preferred embodiment.

This invention may be accomplished in an improved method for isolating and purifying nucleic acid from
cell culture media in which the lysing/denaturing and the deproteinating and neutralizing agent or agents
are added to the resuspended cells without mechanical mixing, the resultant solution is centrifuged to
partially pellet the cellular debris, and then mixed to complete the lysis/denaturization and
neutralization/deproteinization before the cellular debris is finally and fully eliminated by centrifugation. As
a result, the high-shear force mixing procedures available in automated DNA preparation devices may be
employed to accomplish separations at least as good as those accomplished in the more laborious
manual separation techniques.

In standard plasmid/cosmid isolation protocols, performed manually or automatically, the cells are pelleted
by centrifugation and the nutrient broth is siphoned off and discarded. The pelleted cells are then
resuspended in an isotonic buffer by vigorous mixing. A lysing/denaturing reagent is then added to the
resuspended cells, and the tube is mixed. In the manual techniques, the low-shear force, gentle mixing
required to prevent the chromosomal DNA from fragmenting is accomplished by capping and gently
inverting the tube one or more times to completely combine the different reagents. Incubating and mixing
are typically performed at reduced temperatures (i.e. on ice) to further reduce breakup of chromosomal
DNA. Mixing is complete when a very viscous solution is observed. In the automated procedures, such
gentle mixing is typically not available; substituted therefor is mixing accomplished by: repeated pipetting
of the solution, blowing bubbles into the solution, or shaking the tubes to simulate vortexing. However,
each of these procedures provides high shear forces which tend to break the chromosomal DNA into
small fragments, which are then co-purified with the plasmid or cosmid DNA, resulting in lower yields and
lower quantity separations.

After the mixing, the solution is typically incubated on ice for several minutes, and then a neutralizing
reagent is added, followed by further mixing as described above. The tube is then again incubated on ice
for several minutes, followed by a centrifugation to completely pellet the cellular debris. The lysate is then
transferred to a clean tube where the nucleic acid is precipitated with an alcohol solution, followed by a
removal of the supernatant, washing of the nucleic acid, and resuspension in a buffer.

Thus, both the standard manual and automatic protocols provide two mixing steps, one after the addition
of the lysing/denaturing reagent, and one more after addition of the neutralizing reagent. Accordingly, the
chromosomal DNA is twice subjected to the mixing shear forces before the solution is centrifuged to pellet
cellular debris, and there is a great chance of fragmentation of at least some of the chromosomal DNA.

In contrast, in the improved method of this invention, the solution containing the chromosomal DNA is
subject only to a single mixing step before the debris is fully pelleted by centrifugation. In addition, this
mixing step is accomplished only after the tube has been centrifuged a first time to partially pellet the
cellular debris, thereby removing at least some of the bacterial chromosomal DNA with the partially
precipitated cellular debris, reducing susceptibility of the chromosomal DNA to the excessive shear forces
generated by the automated procedure mixing procedures--repeated pipetting, bubble blowing, or
vortexing.

EXAMPLE I

Plasmid/Cosmid Isolation Protocol

The separation method of this invention has been used in isolating plasmid/cosmid DNA as follows:

The E. coli cells were pelleted by centrifugation at 4,000 rpm for two minutes. The nutrient broth was then
removed from the tubes and discarded to waste by pipetting. An isotonic buffer was then added to the
pelleted cells and the cells resuspended in the buffer by pipet mixing the tube contents--in other words, by
repeated pipetting of the contents.

A lysing/denaturing reagent was then added to the resuspended cell sample. The mixing action on
reagent addition partially mixes the reagent with the cell sample to partially react the chemical
components; the tube is not mechanically mixed at this time. There is no incubation on ice following the
addition of the lysing/denaturing reagent.

The neutralizing and deproteinating reagent was then added to the tube. Again, the turbulence generated
by addition of the reagent was the only tube mixing provided. This partial mixing results in a partial
reaction of the chemical components. As before, there was no incubation step following the addition of the
neutralizing reagent.

The tube was then centrifuged at 9,000 rpm for two minutes to partially pellet the cellular debris. The tube
contents were mixed by repeated pipetting. The tube was then recentrifuged again at 9,000 rpm for two
minutes to fully pellet the cellular debris. The procedure then proceeded with the transfer of the lysate to a
clean tube, followed by the nucleic acid precipitation, washing and resuspension as described above. The
result was plasmid/cosmid yield and quality at least as good as that of the manual procedure employing
the two gentle mixing steps employing process steps suited for use in automated separation devices.

EXAMPLE II

Genomic DNA Isolation of Blood

High molecular weight DNA is typically manually separated from tissue sources by adding an
enzymatic/lysing reagent to cells resuspended in an isotonic buffer, and gently mixing the contents,
followed by incubation from two to sixteen hours at 37° C. to 55° C. Protein extracting reagent is then
added to the tube and the tube is again mixed gently by inversion, followed by centrifugation to fully pellet
the cellular debris, and then transfer of the lysate, followed by a repeat of the protein extraction and
centrifugation steps, a second lysate transfer, and then the standard nucleic acid precipitation and
resuspension.

The method of this invention has been applied to isolating genomic DNA from blood samples by adding
the lysing reagent to disrupt the resuspended cells without mechanically mixing the tube contents; the
only mixing is provided by the force of reagent addition into the tube. The two to sixteen hour incubation
step was also not necessary in the procedure, as no enzymatic component was used.

A neutralizing and deproteinating reagent was then added to the tube. Again, the tube contents were not
mixed--the force of reagent addition was the only mixing provided. After addition of the reagent, the tube
was spun at 9,000 rpm for two minutes to partially pellet the cellular debris, followed by a high-shear force
mixing of the tube contents by repeated pipetting. It is believed that the partial pelleting before mixing
reduces shear forces on the DNA caused by the presence of partially reacted cellular debris, thus
providing superior results even with the high-shear force mixing. After the mixing step, the tube was again
spun at 9,000 rpm for two minutes to fully pellet the cellular debris, followed by a transfer of the lysate and
precipitation and suspension of the nucleic acids as before.

Accordingly, when applied to isolation of genomic DNA from tissue samples, the method of this invention
replaces the three mixing steps of the manual method--once after addition of the lysing reagent and once
after each of the two additions of the protein extracting reagent--with a single mixing step provided after
the cellular debris is partially pelleted. In addition, the repeated performance of the protein extraction step
of the manual procedures is obviated by the addition of the lysing reagent followed by the neutralizing
reagent and partial pelleting of the cellular debris before mixing of the tube contents.

The procedures of this invention thus accomplish substantially the same results as the manual isolation
techniques using automated equipment, thereby saving substantial technician's time. In addition, the
method of this invention allows the separations to be performed on an automated machine with quality
levels as good as those provided in the manual methods without the variability of results inherent in the
manual separation techniques.

Other embodiments will occur to those skilled in the art and are within the following claims:

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