DNA separation electrophoresis gels and methods for their use

A novel electrophoresis separation medium comprises polyacrylamide and a dioxane. An electrophoresis separation system comprises a separation channel, the separation channel including the novel separation medium. In a method for electrophoretically separating analytes by introducing analytes into a separation channel, an electric field is applied across the separation channel and the analytes are allowed to electrokinetically migrate within the separation medium. The separation channel comprises the novel electrophoresis separation medium.

BACKGROUND 
The present invention generally relates to gels for separation mediums in 
electrophoretic systems. More particularly, the present invention involves 
improved separation medium gels and methods for their use in capillary 
electrophoresis based DNA separations. 
For decades electrophoretic separation techniques have been the method of 
choice for separating charged molecules and in particular for separating 
proteins. Early electrophoresis applications and many current applications 
involve applying electric fields across separation medium gels which are 
prepared in the form of slabs of varying size and thickness. Samples 
loaded at one end of the slab migrate across the slab under the influence 
of the electric field. When the sample's charged components have different 
electrokinetic mobilities they migrate at different rates and physically 
separate as a result of their differing electrokinetic mobilities. 
Traditionally, slabs which vary in size from several inches on each side 
to several feet are fabricated of a separation gel. Typically these gels 
are crosslinked polyacrylamide or other water swellable gel systems such 
as agarose or cellulose. 
In recent years capillary, electrophoresis (CE) techniques have become the 
electrophoretic separation method of choice for many biological 
researchers. Use of capillary electrophoresis for detection of DNA 
fragments is described by McGregor et al., "Detection of DNA Fragments 
Separated by Capillary Electrophoresis Based on Their Native Fluorescence 
Inside a Sheath Flow," Journal of Chromatography A, 680 (1984), 491-496; 
and Nishiwaka et al., "Separation of Long DNA Fragments by Capillary Gel 
Electrophoresis With Laser-Induced Fluorescence Detection," 
Electrophoresis, 1994, 15, 215-220. CE separations involve injecting 
samples into a buffer filled or gel filled capillary and generating the 
electric field across the capillary in order to cause sample components to 
electrophoretically migrate within the capillary. A variety of 
on-capillary column and off-capillary column detection techniques can be 
used to detect the components including uv, visible, fluorescence and 
electrochemical detection. CE offers many advantages over slab gel 
electrophoresis techniques. CE is available in fully automated systems 
which include automated injectors and data storage and analysis features 
and relatively easy to use detection systems. Additionally, CE separations 
are more rapid than slab gel separations and their separation mediums can 
be replaced after each analysis for a subsequent analytical run. 
Electrophoresis applications have expanded to include a wide range of 
charged analytes and analytes which can be derivatized to incorporate at 
least one charge moiety in order to provide the analyte with an 
electrophoretic mobility. Thus, in addition to proteins, peptides, and 
amino acids, electrophoretic separation methods are useful for separating 
derivatized polysaccharides and oligosaccharides, glycoproteins, nucleic 
acids and oligonucleotides and charged compounds in general. Particularly 
noteworthy is the demand by The Human Genome project and other large scale 
DNA sequencing projects for the capability of separating and identifying 
large numbers of DNA fragments in a single analysis. Because of the huge 
number of bases which must be sequenced in these projects, the success of 
the projects largely depends upon the ability to automate and speed the 
sequencing process. Because electrophoresis is the primary analytical 
method used for DNA sequencing, rapid DNA sequencing requires 
electrophoresis techniques which are not only fast but can resolve many 
bases in a single analysis. 
Slab gel electrophoretic methods using slabs fabricated of crosslinked 
acrylamide can separate and resolve over 600 bases in a single analytical 
run. A major disadvantage associated with the slab approach is that they 
require many hours to perform a single analysis. 
Capillary electrophoresis, on the other hand can perform a single run and 
effectively separate up to about 400 bases in less than an hour. Thus, 
capillary electrophoresis systems incorporating multiple capillaries in a 
single automated system offer the advantage of being able to analyze 
multiple samples in less than an hour. The major limitation associated 
with capillary electrophoresis in DNA sequencing is the separation medium 
itself. Like slab gel electrophoresis, DNA sequencing samples 
traditionally have been analyzed using polyacrylamide separation mediums. 
In order to remove the gel medium from the capillary and replace it with 
fresh medium the DNA separation mediums used in capillaries preferably 
have flow characteristics which allow the medium to flow in and out of 
capillaries. Thus, unlike traditional slab gel separation mediums which 
typically use crosslinked polyacrylamide having high viscosity and 
extensive elasticity properties, capillary electrophoresis separation 
mediums typically include linear polymers or very lightly crosslinked 
polymer gels which are present at lower concentrations than slab gel 
separation mediums and which are capable of being pressure forced to flow 
in and out of capillaries. Many of these separation mediums are based upon 
polyacrylamide and include denaturants such as urea and/or formamide. The 
denaturants improve the DNA fragment separation resolution and DNA 
sequencing read length, but often result in problems connected with their 
precipitation from the medium. Moreover, urea containing gels have 
viscosities which are sufficiently high to cause problems in replaceable 
gel systems and gels incorporating formamide are not stable in aqueous 
mediums and thus are associated with short shelf lives. 
While these denaturing containing polyacrylamide systems work reasonably 
well there is an ongoing need for separation mediums which provide longer 
read length and improved resolution. There is also an ongoing need to 
provide CE separation mediums which incorporate denaturants at 
sufficiently high concentration without their precipitation from the 
separation medium. There is further a need to provide CE separation 
mediums having extended shelf lives and sufficiently low viscosity to 
allow the separation mediums to easily flow into and out of capillaries. 
It is accordingly an object of the present invention to provide separation 
mediums suitable for use in capillary electrophoresis systems having read 
lengths of up to over 500 bases. It is also an object of the present 
invention to provide separation mediums having suitable denaturants which 
will not precipitate from the medium system, having extended shelf lives 
and can be used in replaceable gel systems. 
SUMMARY 
This invention includes an electrophoresis gel separation medium comprising 
polyacrylamide and a dioxane. 
The invention further includes a electrophoresis gel separation system 
comprising an elongated separation channel, said separation channel 
including a separation medium comprising polyacrylamide and a dioxane. 
The invention still further includes the method for electrophoretically 
separating analytes by introducing analytes into a separation channel, 
applying an electric field across the separation channel and allowing the 
analytes to electrokinetically migrate within the separation medium, the 
improvement wherein the separation channel comprises an electrophoresis 
gel separation medium comprising polyacrylamide and a dioxane. 
The present invention provides separation mediums and separation systems 
which when used in connection with electrophoresis separations result in 
improved analytical resolution and improved DNA sequencing read length. 
Advantageously, the separation mediums of the present invention include 
denaturing compounds which do not cause an increase in the separation 
medium viscosity and thus are suitable for use in replaceable gel 
electrophoresis systems. The decreased viscosities and improved flow 
properties associated with the separation mediums of the present invention 
provides means that the separation mediums can be forced in and out of 
capillaries using less pressure than prior art systems. Furthermore, the 
separation mediums of the present invention are stable in an aqueous 
environment making them suitable in applications in which the separation 
medium is for a length of time prior to its use. 
The present invention is based upon the discovery that dioxane can be 
incorporated in polyacrylamide solutions to provide electrophoretic 
separation mediums having suitable biopolymer denaturing characteristics 
and highly improved DNA resolution and read length characteristics. Thus, 
in accordance with one aspect, the present invention provides separation 
mediums of polyacrylamide and dioxane. In preferred embodiments, the 
separation mediums incorporate one or more additional denaturants and 
further include one or more buffer compounds which typically acts as the 
electrophoresis electrolyte.

DESCRIPTION 
A gel according to this invention includes polyacrylamide and a dioxane in 
an effective resolution enhancing amount, preferably on the order of about 
1 to about 5 grams polyacrylamide and about 5 to about 30 ml of a dioxane, 
per 100 ml of the gel. 
In the most preferred embodiments, the gel includes about 3 grams 
polyacrylamide, about 15 ml dioxane (1,4 dioxane and/or 1,3 dioxane), 
about 3.5 M urea, and about 100 mM tris-borate buffer, with the balance 
being water, to make a total gel volume of 100 ml. 
In accordance with another aspect of the present invention there is further 
provided electrophoresis separation apparatus including a separation 
channel and disposed within the separation channel a separation medium. 
The separation medium includes polyacrylamide and dioxane and 
preferentially further includes one or more biopolymer denaturants and an 
ionic buffer. 
The present invention further includes methods for electrophoretically 
separating analytes by introducing analytes into a separation channel of 
the present invention, applying an electric field across the separation 
channel and allowing the analytes to electrokinetically migrate within the 
separation medium. Preferred embodiments of this aspect include the 
electrophoretic separation of DNA fragments obtained using standard DNA 
sequencing chemistries. 
This invention includes the use of 1,3-dioxane, 1,4-dioxane, as well as 
mixtures of the isomers. 
An apparatus suitable for use of the electrophoresis gel of the present 
invention is shown in the FIGURE. The apparatus comprises a gel syringe 12 
for introducing a sample through a valve 14 into a capillary 16. The 
distal end of the capillary is provided with a window 18 for incoming 
laser light for detection. The laser light is provided by laser light 
source 20, with the laser light going through a filter 22 and a focusing 
lens 24, and into the window through a parabolic light shield 26. Light 
emitted from the capillary passes through emission filters 30 into a 
photomultiplier 32, and signals from the photomultiplier 32 are collected 
by data acquisition means 34. A power supply 42 provides the electrical 
charge across the capillary. Spent sample and gel are withdrawn from the 
capillary through a valve 50. 
The following Examples are illustrative. 
EXAMPLE 1 
Procedure For The Preparation Of Gel 
The materials listed here are for the preparation of 150 mL. of 3T gel 
containing 15% dioxane, 100 mM Tris-Borate EDTA and 3.5 M urea. 
______________________________________ 
Acrylamide(Ultra Pure) 4.5 g 
Urea(Ultra Pure) 10.5 g 
1,4 Dioxane 7.5 mL. 
Tris (Trishydroxymethyl) 
30.25 g 
amino-methane! borate)(Ultra Pure) 
Boric Acid 15.46 g 
EDTA-(Ethylenediamine Tetraacetic acid) 
14.61 g 
Resin* 1.5 g 
(AG 501-XA 20-50 mesh) 
APS(Ammonium Persulfate) 0.1 g 
TEMED(N,N,N.sup.1,N.sup.1 -Tetramethylenediamine) 
100 uL 
Shaker and/or Roller 
______________________________________ 
*The Resin is sold by Bio Rad Laboratories under the designation 142-6425 
The resin serves to remove unwanted ions from the buffer and the gel 
solution. 
Buffer and Preparation 
A. TRIS-BORATE-EDTA (500 mL): 
a) Prepare 0.5 M EDTA stock solution by dissolving 14.61 g of EDTA in 100 
mL (total volume after adjusting the pH) of deionized water. 
b) Adjust the pH to 8.0 with concentrated NaOH. 
c) Prepare Tris-Borate EDTA solution by dissolving 30.25 g of Tris, 15.46 g 
of Boric Acid and 5 mL of 0.5 M EDTA (from step b) in water to a total 
volume of 500 mL. Filter through 0.2 micron filter. This solution is 
stable at room temperature for 60 days. 
B. Gel (50 ml): 
a) Dissolve, very slowly, 10.5 g of urea and 7.5 mL of 1,4 dioxane in 
deionized water to a final volume of 40 mL. Wait until completely 
dissolved. 
b) Add 4.5 g of acrylamide to the solution and dissolve completely. 
c) Add 1.5 g of resin and stir for 15 minutes. Filter the solution through 
0.2 micron filter to remove the Resin which is discarded. 
d) Transfer the solution to a 60 mL serum bottle. 
e) Add 10 mL of the Tris-Borate-EDTA solution from step A(c) to the bottle 
and mix. Cap and seal the bottle. 
f) Purge the solution with Helium for 1 hour. 
g) Pressurize the bottle with Argon at 20 psi for 30 seconds. 
h) Remove the bottle and keep in ice for 15-20 minutes. To prevent partial 
polymerization of the gel, cool all sides of the bottle uniformly. 
C. Gel Polymerization: 
The following steps preferably are completed in less than five minutes for 
proper polymerization. 
a) Dissolve 100 uL of TEMED in 900 uL of deionized water and keep on ice. 
b) Dissolve 0.1 g of APS in 1.0 mL of deionized water and keep on ice. 
c) Add 25 uL of each TEMED and APS solution to the bottle containing the 
acrylamide solution from step B(h). 
d) Mix the solution and keep at 2-8.degree. C. (with no disturbance) for 
20-24 hrs. 
D. Gel Preparation: 
a) Prepare 100 mL of 100 mM Tris-Borate-EDTA, 15% dioxane and 3.5 M Urea 
buffer by dissolving 21.0 g of Urea, 15 mL of 1,4 Dioxane and 20.0 mL of 
Tris-Borate-EDTA in deionized water to a final volume of 100 mL. Filter 
the solution through 0.2 micron filter. 
b) Transfer the buffer to a 250 mL glass bottle. 
c) Add the polymerized gel from step C(d) to the bottle. 
d) Purge the solution with pure oxygen for 15 minutes. 
e) Cap the bottle and shake for 24-30 hrs at room temperature. f) Remove 
the bottle from shaker and keep at 2-8 C. 
E. Gel Composition: 
The electrophoresis separation gel resulting from step D has the following 
composition: 
Polyacrylamide=3.0 grams/100 mL (total gel volume) 
Dioxane (15%)=15 mL/100 mL (total gel volume) 
Urea (3.5 Molar)=21.02 grams/100 mL (total gel volume) 
Tris (100 millimolar)--1.21 grams/100 mL (total gel volume) 
Boric Acid (100 millimolar)--0.62 gram/100 mL (total gel volume) 
TEMED (0.0017%)=0.0017 mL/100 mL (total gel volume) 
APS (0.0017%)=0.0017 gram/100 mL (total gel volume) 
EDTA (1.0 millimolar)=0.292 gram/100 mL (total gel volume) 
Water is used to dissolve the materials to reach final volume of 100 mL gel 
solution. 
EXAMPLE 2 
A first electrophoretic gel which contained 1,4 dioxane was prepared 
according to Example 1 and had the composition set forth in Step E. 
A second gel was prepared which was identical to the first gel except that 
the 1,4-dioxane was omitted. 
Each of the gels was incorporated in a standard electrophoresis capillary 
utilizing the equipment of FIG. 1. Electrophoresis was carried out on a 
sequencer provided with a laser equipped with an optical system which 
causes the beam to impinge on the capillary. The light reflected by the 
capillary is sensed by a detector and the detector output is computer 
processed to generate a tracing. An array of DNA fragments containing over 
500 base pairs was used as the analytes to be separated. The mixture of 
DNA fragments was prepared for sequencing by conventional cleavage 
techniques. The same DNA fragments were used with both gels. 
The sequencing labeling chemistries were a single dye reaction performed on 
a M13 mp18 DNA template and terminated with dideoxythymidine triphosphate. 
The product was purified by ethanol precipitation. Prior to injection the 
sample was heated to 95.degree. C. for 2 minutes and then cooled to room 
temperature. 
A manual hand operated pressure pump was used to introduce the polymer into 
the capillary (45 cm long) in less than one minute. The sieving matrix was 
used as the run buffer for each electrode (cathode and anode). After the 
filling process was complete, a 5 minute pre-run electrophoresis was done 
at 10-15 kilovolts (kV) prior to sample injection to monitor the current. 
Electrophoresis (at 10 kV) was continued after sample injection which was 
introduced by electrokinetic injection at 10 kV for 3-5 seconds. The 
electrophoresis voltage was supplied by a 15 kV power supply and runs were 
performed at 30.degree. C. Capillary sieving buffer was contained in 4 mL 
glass vials placed in a vial holder inside the CE breadboard device. 
The equipment used was a basic single capillary electrophoresis system with 
laser excitation source. The light from a laser source was passed through 
a filter to isolate the desired wavelength. The laser emission was then 
reflected by a mirror positioned at 90.degree. relative to the lens. The 
lens was used to focus the laser light into the capillary. The capillary 
(100 micron i.d. and 735 micron o.d.) was maintained horizontal. Detection 
was accomplished with a photomultiplier tube. Full spectral data were 
acquired. 
Both gels were effective in detecting the separation in the range up to 
about 480 base pairs. However, without dioxane, the peaks in an 
electrophoretic tracing are not clearly resolved at peaks 485 to 487, 505 
& 506. The gel containing the dioxanes clearly resolved these peaks 
reflecting improved resolution. 
These results demonstrate that the present invention provides a higher 
detection limit which permits the detection of a greater number of DNA 
fragments in a single separation, thereby facilitating more rapid analysis 
and sequencing of fragments. 
These and other aspects and advantages associated with the present 
invention will become apparent to those skilled in the art upon an 
understanding of the invention as described in the detailed description of 
the invention taken in combination with the enclosed drawing.