WO1993023746A1 - Use of multiple short gels for elongated separation pattern - Google Patents

Abstract

Complex samples are accurately separated using gel electrophoresis by simultaneously employing a plurality of lanes (1-8) having different pore sizes. By performing the electrophoresis simultaneously with each of the lanes and monitoring mobility, one defines the portion of each of the lanes to be used in tandem fashion to prepare an elongated gel where each of the bands is defined for each of the sample components. By employing appropriate software and electronic hardware, one can map the bands in a reproducible spectrum.

Description

USE OF MULTIPLE SHORT GELS FOR ELONGATED SEPARATION PATTERN

INTRODUCTION

Technical Field

The field of this invention is sample component separation by gel electrophoresis.-

Background

Gel electrophoresis plays an essential role in biotechnology. The ability to separate molecules by their ability to travel in an electrical field in relation to pore size is used extensively in analyzing mixtures, confirming manipulations of DNA, identifying particular genes, and in forensic analysis. In many situations, gel electrophoresis is far from ideal, particularly with compositions having a broad molecular weight range. With a single pore size, either one will not see separation of the smaller components or there will be no separation of the larger components. Thus, the resolution tends to be poor.

Also, the usual gel electrophoresis has a relatively long gel, generally at least about 10 cm, usually longer, so that the time for completion of the gel can be many hours. There is substantial interest in being able to develop new methodologies for performing gel electrophoresis in a more efficient and expeditious manner.

Relevant Literature

Stellwagen, N.C. "Gel Electrophoresis of DNA" in: Advances in Electrophoresis, Vol. 1, A. Chrambach, B.J. pp. 177-228 (1987) .

Sealey, P.G., et al . in: Gel Electrophoresis of Nucleic Acids : A Practical Approach, Rickwood D., Hames B.D. eds., IRL Press, Oxford, pp. 39-76 (1982) .

Stellwagen, N.C, Biopolymers , 24, 2243-2255 (1985).

Lerman, L.S., et al . , Biopolymers, 21, 2315-2316 (1982).

Lumpkin, O.J., et al . , Biopolymers, 21, 2315-2316 (1982).

Lumpkin, O.J., et al . , Biopolymers, 24, 1573-1593 (1982).

Southern, E., Anal . Biochem., 105, 304-318 (1980).

Schleif, R.F., et al., Practical Methods in Molecular Biology, Springer Verlag, New York (1981) .

Serwer, P., et al., Biochemistry, 24, 922-927 (1984).

Fisher, M.D., et al., Biochemistry, 10, 1895-1899 (1971).

Mikel, S., et al . , Nucleic Acid Research, 4, 1465-1482 (1977) .

Smith, S.S., et al . . Anal . Biochem. , 128, 138-151 (1983).

Malvy, C, Anal. Biochem. , 143, 158-162 (1984). Sutherland, J.C, et al . , Anal . Biochem. , 139, 390-399 (1984) .

Stellwagen, N.C, Biochemistry, 22, 6186-6193 (1983).

Dingman, C.W. , et al . , Biochemistry, 11, 1242-1250 (1972).

Maxam, A.M., Methods Enzymol . , 65, 499-560 (1980).

Low, C.M.L., et al . , FEBS Lett . , 176, 414-419 (1984).

SUMMARY OF THE INVENTION

Methods and devices are provided for efficient gel electrophoresis separation of complex mixtures. The method employs a gel plate having a plurality of lanes, where the lanes have a predetermined incremental difference in pore size. By monitoring migration in each of the lanes, beginning with the smallest pore lane, one can calculate the "cut-off"-point using mobility data from the higher gel concentration leading zone to calculate the portion of the next gel to be used for an elongated gel without separation pattern overlap. Instrument software can be employed for the mobility data calculation using a Ferguson plot evaluation. One can then obtain a separation pattern, as if each of the relevant gel portions were overlapped in tandem.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a perspective view of a gel plate;

Fig. 2 is a diagrammatic view of gel lanes and bands; and

Fig. 3 shows an elongated "synthetic gel" construction with the bands indicated, as well as the spectral analysis of the bands. DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and devices are provided for performing gel electrophoresis in more efficient and rapid manners. Specifically, relatively short gels are used with a plurality of lanes, where each of the lanes differ incrementally as to pore size, so as to vary mobility of the sample components. Sample is applied to each of the lanes and the lanes electrophoresed simultaneously employing real¬ time detection of zone migration.

Using mobility data from the higher* gel concentration leading zone, the portion of the next gel to be used for an elongated gel construct without separation pattern overlap can be determined. This can be done with software employing a mobility data calculation using a Ferguson plot evaluation. The software can then align each of the lanes in tandem fashion to provide an elongated "synthetic gel pattern" , which provides for a gel band pattern extending over 3 or more logs of molecular weight difference in the components of the gel.

Any of the common electrophoretic gels may be employed, such as agarose, polyacrylamide, gelatin, etc. For the most part, the agarose will have a concentration between about 0.2-2% T (T=total amount of gelling agent present in the gel) , while acrylamide will generally have about 3-15% T, highly crosslinked to maintain wide pore sizes (generally 25-60% C-BIS) , more usually concentrations ranging from 4-7.5% T. Descriptions of forming gels for gel electrophoresis may be found in The Practice of Quantitative Gel Electrophoresis, Chra bach, VCH Publishers.

The incremental difference between the different gels depends on the gel medium which is used. For polyacrylamide gels, it will generally be at least about 0.25% and not more than about 1%, more usually not more than about 0.5%. For agarose gels, the incremental difference will be at least about 0.1% and not more than about 0.25%, more usually not more than about^" 0.1%. Desirably, one may wish to occupy the entire useful range which is available for the particular thickening agent. The significant factor is that the portion of the gel which is used for the "synthetic gel" must not be so large that the distance between two adjacent cut-off points for two adjacent gels is more than about 75% of the length of the gel. Usually, the distance will be less than about 50% the length of the gel, and will usually be selected to provide at least about 5% the length of the gel.

Usually, there will be at least 4 lanes, more usually at least 6, preferably 8, and if desired 10-12 or more lanes may be employed. The lanes may be of varying widths, including capillary size, generally being from about 0.2-1 mm as a capillary, otherwise generally ranging from about 0.2-1 cm in width. The gel thickness will vary, generally being from about 0.5 mm to 3.0 mm.

The length of the gel used in the subject invention will generally be at least about 2 cm, more usually at least about 5 cm, and not more than about 15 cm, usually not more than about 12 cm.

Conventional gel plates may be employed or gel plates as described in application Serial No. 522,325, filed May 14, 1990, now U.S. Patent No. 5,104,512. The plates will normally have separation walls between the lanes, so that each lane may be individually prepared. The level of porosity can be achieved by using varying amounts of the thickening agent in the different lanes, using varying amounts of crosslinking agents, or using varying amounts of curing agents, such as free radicals, ultraviolet radiation, or the like, or combinations thereof. The thickening agent may be the same in each of the lanes which are employed. The incremental amount of gelling agent or curing will be based upon the number of lanes which are available, the nature of the sample, the length of each lane, and the molecular weight range of the sample components and the optimum resolution. For example, longer gel lengths and increasing number of lanes will be desirable with an increasingly complex mixture having components ranging over a large molecular weight range.

The gel will normally have a sample well or a site for sample application, where the sample may be uniformly applied. A stacking gel may be present, which will normally be not more than about 20% of the length of the gel, usually not more than about 10% of the gel, so as to allow for all of the components of the sample reaching the separation gel at about the same time.

In carrying out the electrophoresis, various buffers may find application. These buffers are well known in the literature and need not be described here.

The electrophoresis is applicable to any type of sample which may be electrophoresed, including nucleic acids, proteins, sugars, lipids. combinations thereof, and the like. In addition, aggregations of various compounds may be electrophoresed, including such particles as viruses, organelles, and the like.

After the sample has been loaded onto the gels in the various lanes, the electrophoresis may now be initiated. Zone migration is monitored. Monitoring can be achieved using a variety of dyes or markers which allow for detection of the presence of a band of a sample component. The electrophoresis will usually be terminated when a band in the lane having the lowest porosity approaches the end of the lane. One may monitor all the lanes and terminate when a satisfactory separation in each of the lanes has been achieved. By monitoring the zone migration, one may calculate the "cut-off"-point from lane to lane, using mobility data from the higher gel concentration leading zone to calculate the proportion of the next gel to be used for the elongated gel 5 without separation pattern overlap. This can be done by instrument software, where the calculation involves:

(1) Calculation of mobility data for the leading zone (most anodic migrating sample component) from migration distance, runtime, voltage gradient and gel concentration.

10 (2) Search for the corresponding zone position in the neighboring lane with higher gel concentration (in migration direction located right to the other lane) using the mobility data obtained from 1 and recalculating the corresponding mobility at that gel concentration.

15 (3) Calculation of the "cut-off"-point in this lane to avoid zone overlapping.

(4) Addition of data points from the second lane starting from the "cut-off"-point to the end zone in this lane, after the last data point from the neighboring left lane (see

20 Fig. 2) .

(5) Proceed to the next lane, repeating calculation starting at 1, until all eight lanes are evaluated and the complete long gel is synthesized from the data of the corresponding gel fragments.

25 The electrophoresis is complete when the sample in the largest pore-sized gel shows a band approaching the end of the gel.

The elongated separation pattern ("synthetic gel") is then constructed, which can be done by appropriate software. The

30 synthetic gel is prepared by adding onto the separation pattern achieved in lane 1 the smallest pore size gel, all other lane segments from their "cut-off"-point to the lane terminus, to the cut-off point of the next respective gel and so on, in order of decreasing gel concentration (larger pore size) . A synthetic gel pattern is then obtained, where one now has the e fect of having run a very long gel with separation of molecules which can range over 3 log orders or more difference in molecular weight. The bands can be readily graphed so as to provide a spectrum indicating the separation distance and intensity of the various bands.

The subject invention provides numerous advantages. It allows for a significantly shorter gel length and concomitantly shorter run times. Optimum resolution for each particular molecular range is achieved, since only that part of the gel is used that allows for the best separation with least diffusion (optimum pore sized gel) . Because of the shorter length, and matching pore size to molecular weight, less zone distortion is obtained.

The subject invention may be employed with the device of U.S. Patent No. 5,104,512, which allows for continuous monitoring of the migration of the sample components. However, this device is not required and any other device which allows for monitoring of the movement of the components, where the information may be fed to a computer for analysis may also be employed.

The following example is offered by way of illustration and not by way of limitation.

EXPERIMENTAL

DNA restriction fragments with enlarged size ranges were analyzed. The mixture was obtained by mixing the following marker/size standard products: Pharmacia-LKB Lamda-DNA PFGE No. 27-4530 (48,000-1,212,500 bp) , Pharmacia-LKB Lamda-DNA- HindIIl/PHI-X-174 DNA-Haelll Digest No. 27-4060 (72-23130 bp) . The DNA-mixture was pre-labelled with Ethidium-bromide (1 μg/μl) prior to run to allow for Real- Time fluorescence detection during the separation process. The gel range was 0.5% T-l.9% T (w/v) GTG-agarose, with 0.2% increment from lane to lane. The buffer system was TAE (Tris/acetate/EDTA) . The size range for separation was 80 bp to 2 Mbp, covering a size differential of about 2 x 10⁴ units.

The gel length was 120 mm/lane (600 data points) while the synthetic gel length was 640 mm (3200 data points) . (The data points are the scan resolution, 5 data points/mm equal 0.2 mm resolution.) The conditions for the separation were selected as follows: temperature—15°C; power supply mode—constant voltage across the gel; photometer mode—fluorescence emission; voltage limit—500 volts; current limit—30.0 A; fieldstrength—10.0 V/cm across the gel; sample application position—0 mm from start; sample entry condition—5 V/cm, 300 sec; number of scans—8; scan interval—15 min. (=2 hours runtime) .

In Fig. 2 is given a diagrammatic view of what each of the bands in the lanes looked like with the diagonal lines between each lane indicating the cut-off point, which indicates the portion of the gel above the bottom of the cut-off point, which is used in the elongated gel, with the exception of lane 1 which is used in its entirety.

Fig. 3 shows the elongated gel and a spectrum of the peaks associated with each of the bands, indicating the intensity of the band.

Fig. 1 shows a perspective view of a gel plate having 8 lanes. The plate 10 has a plurality of lanes separated by walls 12 with gel 14 in each lane. The gels have sample well 16 for receiving the sample. Alternatively, the sample may be applied by an automatic device on the gel at 0-20 mm from the start position. Each of the wells differs from its adjacent well by having a different polymer concentration to provide for different pore sizes in a predetermined order. The ends 18 and 20 of the gels in the lanes are open to be able to contact buffer solution for carrying out the electrophoresis.

It is evident from the above description, that the subject invention provides for numerous advantages. In a single electrophoresis, one may separate a complex mixture of components of varying molecular weight and mobility in an accurate manner. Using a single gel plate, where the electrophoresis is carried out under the same conditions, separations are achieved where a spectrum of the bands can be obtained which defines each of the components in the mixture. Thus, reproducible results are achieved in a rapid and efficient manner. Lower diffusion of bands is obtained for more accurate readable results. Better separation can be achieved, since for each molecular weight subset of components, the gel is optimized for that subset.

All publications and patent applications cited in this specification are herein incorporated b reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for separating a mixture of like molecules of molecular weights differing over a wide range using gel electrophoresis, said method comprising: applying said mixture to a plurality of gels in different lanes, where each of the gels has a different porosity to provide a graded order of porosities among the lanes; separating said mixture of molecules in each of said lanes under electrophoretic conditions; determining the cut-off point of each lane to define the region to be used in constructing a synthetic gel comprising bands of molecules separated by differences in electrophoretic mobility extending over the molecular weight range of interest, wherein each lane provides separation of a different group of molecules; and combining each of said regions in tandem in order of decreasing porosity to construct said synthetic gel.

2. A method according to Claim 1, wherein said molecules are proteins.

3. A method according to Claim 1, wherein said molecules are nucleic acids.

4. A method according to Claim 1, wherein said graded order has a constant percentage difference in porosity as related to a constant percentage difference in thickening agent.

5. A method according to Claim 1, wherein the number of lanes is at least 4.

6. A method according to Claim 5, wherein the length of the lanes is at least about 2 cm.

7. A method according to Claim 1, including the additional step of monitoring the lane with the lowest porosity and terminating said electrophoresis when a band becomes proximal to the end of said lane.

8. A gel plate comprising at least 4 lanes, wherein said lanes have gels of a graded porosity.

9. A gel plate according to Claim 8, wherein said graded porosity has a uniform difference between succeeding porosities based on the amount of thickening agent used.

10. A gel plate according to Claim 8, wherein said gel is thickened with agarose.

11. A gel plate according to Claim 8, wherein said gel is thickened with acrylami.de.

12. A synthetic gel comprising a series of bands related to molecules in order of decreasing mobility produced by the method according to Claim 1.