1. Field of the Invention
The present invention relates to a method and a device to assist the analysis and processing or gel slice cutting of a biomolecule resolving gel after gel electrophoresis and more specifically to a template device and a method for assisting the tracing, marking, measuring, analyzing, referencing and cutting of slices of the electrophoretic gels.
2. Description of the Prior Art
Gel electrophoresis is one of the most important techniques in life science methods. It is used extensively in the field of biotechnology to separate target biological macromolecules (biomolecules), such as DNA, RNA, or protein form a mixture of biomolecules. This analytical tool is equally important for research purpose as well as quality assurance testing of biomolecules in industry. This procedure, which is done for identification and quantification of biomolecules, separates them into bands, which are then typically stained with a fluorescent dye with an appropriate Stokes shift (that is the discrimination between excitation and emission wavelengths) so that when the gel is irradiated with light at an excitation wavelength the dye emits light at a different wavelength, and the resulting emissions are detected and quantified. Irradiation is commonly achieved by transillumination, i.e., irradiation of the gel at the side opposite the side where detection is performed. A commonly used dye for DNA fragments is ethidium bromide, which is most efficiently excited at wavelengths in the ultraviolet range.
After performing gel electrophoresis, further stages of performance can be divided into “analysis” and “processing” or “gel slice cutting”. For the purpose of definition, “analysis” hereby means viewing of biomolecules in a stained gel on transilluminator for all observable characteristics, for example, band integrity, quality of band separation, expected concentration of biomolecules by comparison with known standard concentration markers, and size of biomolecules by comparison with known standard size markers, analyzing a particular part of gel for size selection or purification of biomolecules and taking the picture of the gel etcetera, on the basis of which decision can be made for “processing” or “gel slice cutting”. For the purpose of definition, “processing” or “gel slice cutting” hereby means removing the desirable part of the gel after analysis with the help of suitable methods available in the art. The removed gel slice can be further used for isolation of biomolecules from the gel slice for purification (gel elution or gel extraction), performing gel electrophoresis again etcetera or any other procedure depending upon the experimental requirement.
Usually biomolecules are observed or analyzed within the stained gel with the help of ultraviolet (UV) irradiation. Conventional method/arrangement of gel analysis includes use of gel on top of UV tans-illuminator with or without gel documentation apparatus. Gel documentation apparatus (GDA) covers the UV emitting area from all sides thus confining the UV radiations within a chamber of the apparatus and this in turn saves a user from UV exposure, however, analysis without GDA is prone to exposure of potentially dangerous UV radiations even with the protective goggles or face shields, especially skin surfaces including throat, hands and arms are at risk. Further, UV radiations also reflect off nearby surfaces thus reaching the user.
After analysis, it is often desirable to remove target biomolecules from a gel polymer (processing or gel slice cutting). Gel slice cutting is generally used for size selection as well as purification of biomolecules. This gel slice cutting is performed directly on the UV trans-illuminator which usually takes more time than analysis. Further, for procedures involving a large number of gel slice cuttings takes longer time from tens of minutes to an hour or even more to cut gel slices. During this time, the user is exposed to harmful UV radiations. Furthermore, prolonged UV exposure may induce photo induced reaction in macromolecule inside the gel matrix that may affect experimental outcome. The use of UV light is therefore of concern not only in the detection of biomolecules but also for a laboratory worker exposed to harmful excitation light.
U.S. Pat. No. 5,327,195 issued to Timothy G. J. Ehr and U.S. Pat. No. 7,030,392 issued to Alex Waluszko describes the basic working principal of transilluminator. Various eye protective means including goggles with ultraviolet rays blocking properties can be employed with these transilluminators as described in U.S. Pat. No. 4,758,079 issued to Wanda Bledose and U.S. Pat. No. 5,016,292 issued to Mark Rademacher.
Smoot et al., in issued U.S. Pat. No. 4,657,655 teaches foto/phoresis apparatus for electrophoretically separating, visualizing and photographing DNA in agarose gels and Ramm et al., in issued patent number WO 98/07022 described a digital imaging system for assays in well plates, gels and blots, both describing the basic principal and working of gel documentation system. Gel documentation systems often come with gel slice cutting means with varying degree of UV protection during the gel cutting process. Slider imager instrument from Maestrogen Inc. of Las Vegas, Nev., USA and other similar instruments provide some degree of protection from UV radiation. However, GelDoc-It® Imaging System from UVP, LLC of Upland, Calif., USA and gel observation and gel cut-out hood named Gel LaBox from MeCan Imaging Inc. of Fujimino-City, Saitama, Japan provides a significant amount of protection from UV radiations, however, these instruments still use UV rays during all the analysis and processing time. Further, the arm and hands of the technician remain restricted to a certain angle during gel slice cutting that may lead to difficult handling of the process. For gel cutting, various means are available in the art besides the traditional method using surgical blade, including U.S. Pat. No. 3,949,471 issued to Leo P. Cawley, U.S. Pat. No. 6,565,728 issued to Brnako Kozulic, U.S. Pat. No. 7,413,908 issued to Caldwell et al.
The present invention comprises a method and an apparatus to assist in gel analysis and processing where UV irradiation is only required for analysis, however processing or gel slice cutting can be performed without the use of harmful UV radiations. As will be better understood from the discussion that follows, the present invention overcomes many of the drawbacks of the prior art devices.
In accordance with the present invention, a method and apparatus to assist gel analysis and processing following the electrophoretic separation is described. In a preferred embodiment, the apparatus of the present invention consists of template sheet(s) and a template sheet holding assembly. The template sheet(s) are of transparent material and consist of horizontal and vertical lines that are visible in both visible as well as UV light. These template sheet(s) are used inside the template sheet holding assembly. The template sheet holding assembly along with template sheet(s) is placed on the UV transilluminator and the gel is placed on top of the template sheet holding assembly. Each individual template sheet can be moved inward and outward in its respective slot in the template sheet holding assembly to adjust them according to the gel.
In accordance with the method described in the present invention, the gel is adjusted according to the pattern of the first template sheet containing plurality of pair of vertical lines, each pair being representative of the respective lane of the gel. At this time looking through the gel will give a view such that each lane of the gel has been vertically underlined or represented with vertical lines on both sides. Now, at this point of time UV irradiation can be turned on to view the actual positions of the bands in each lane of the gel. The lane representing vertical lines will also glow in UV light along with the bands. By performing a quick analysis, the band of choice can be selected for processing or gel slice cutting. Once decision is made for the band of choice, it can also be underlined by adjusting the second template sheet containing a single horizontal line to the upper boundary of the selected band of choice. The lower boundary of the band of choice can be underlined by adjusting the third template sheet also containing a single line to the lower boundary of the selected band of choice. This procedure will take few seconds to less than a minute after which UV irradiation can be turned off. By turning UV irradiation off, the bands in the gel will disappear; however, the adjusted pattern of horizontal and vertical lines will be still visible. The quadrilateral shapes formed by the intersection of horizontal and vertical lines in the lanes represent the bands of choice that can be cut accurately with the help of blade or any other suitable and convenient method available in the art.
In another embodiment of the present invention, the pattern of plurality of pair of vertical lines (that will represent lanes) and first horizontal line (that will represent upper boundary of band of interest) can be made on one template sheet, where as the second horizontal line (that will represent lower boundary of band of interest) can be made on second template sheet.
In still another embodiment of the present invention, the pattern and number of horizontal and vertical lines can be made on one or more than one template sheets according to the requirement of analysis, e.g., analysis and comparison of ladders etc. A scale can also be made on a certain template sheet where band migration distances with reference to time needs to be calculated.
Accordingly, it is a primary objective of the present invention to provide a reliable template device and a method for assisting gel analysis and processing or gel slice cutting.
Still another primary objective of the present invention is to provide a device and a method for gel processing or gel slice cutting with minimal to no use of harmful UV radiations where UV irradiation is only used for gel analysis whereas gel processing or gel slice cutting can be performed without the use of harmful UV radiations.
Still one another primary objective of the present invention is to provide a device and method which is easy to hands and arms during gel slice cutting while also protect from harmful radiation.
Still, another objective of the present invention is to provide referencing vertical lines to mark each lane of the gel during gel analysis and processing.
Still, an additional objective of the present invention is to provide referencing horizontal lines at right angle to the vertical lines for comparison of bands that are in line with each other during gel analysis and processing.
Yet another objective of the present invention is to provide referencing horizontal lines at right angle to the vertical lines for comparison of ladders bands that are in line with each other on both ends of the gel to identify the equal or unequal migration rates on both ends of the gel.
Yet an additional objective of the present invention is to provide a device and a method to select or mark the area of the gel for processing or gel slice cutting for further processing.
Various other objectives, advantages, and features of the invention will become apparent to those skilled in the art from the following discussion, taken in conjunction with the accompanying drawing in which:
Referring now to the invention in more detail,
a (i) describe a pattern 22 marked on a substantially square and flexible transparent plastic sheet or other suitable transparent material comprising a set of vertical lines 13 and a set of horizontal lines 17. The set of vertical lines 13 comprised of vertical lines 2, 4, 6, 8, 10 and 12 and the set of horizontal lines 17 comprised of horizontal lines 14 and 16. In the set of vertical lines 13, each pair of vertical lines (2 and 4, 6 and 8, and 10 and 12) represents the respective lane 1, 2 and 3 marked in between the respective vertical lines in circles. The horizontal lines 14 and 16 intersect vertical lines 2, 4, 6, 8, 10 and 12 and form quadrilateral shapes 18a, 20a, 18b, 20b, and 18c. The horizontal and vertical lines can be visually observed in visible and ultraviolet (UV) light spectrum regions. The pattern 22 can be marked on one transparent sheet or different parts of the pattern can be marked on different transparent sheets which when combined gives the same pattern as illustrated in
b (i) describe a gel matrix 30 having rectangular wells 32a, 32b and 32c, where the length of a rectangular well provides the width of the respective lanes 32d, 32e and 32f in which a macromolecule can be resolved. For exemplary purpose, during the electrophoresis, in lane 32d, a size marker is resolved comprising bands 34, 36, 38, 40, 42, and 44, whereas, lane 32e and 32f resolved the wider size range smears 46 and 48 respectively for two different samples.
The pattern 22 when placed under the gel matrix 30 provides tracing/reference lines template for the analysis of the gel matrix. Each pair of vertical lines including 2 and 4, 6 and 8, and 10 and 12 provides boundary line/reference line/tracing line for lanes 32d, 32e, and 32f respectively. Furthermore, horizontal lines 14 and 16 can mark the area of interest in each lane simultaneously.
For example, in order to analyze the smears 46 and 48 in the lanes 32e and 32f in comparison to size marker in lane 32d from size marker band 36 to band 38, the horizontal lines can be arranged so that line 14 defines the lower boundary of band 36 in lane 32d through lanes 32e and 32f and likewise, the horizontal line 16 can be arranged so that it defines the upper boundary of band 38 in lane 32d through lanes 32e and 32f.
The finalized pattern 22 through gel 30 provides reference line around the lanes (vertical lines) and area of interest between band 36 and 38 (horizontal lines). The intersecting quadrilateral shapes 18a, 18b, and 18c represents size range between band 36 and 38, the size range 50 required to be studied in lane 32e, and the size range 52 required to be studied in lane 32f, respectively. Whereas, the quadrilateral shapes 20a and 20b are between the lanes 32d and 32e and lanes 32e and 32f.
c (i) represent the analysis which is preserved even in the absence of UV light and visible through the gel due to the application of principle of the present invention. The quadrilateral shapes 50 and 52 can further be processed to gel cutting for obtaining the required size out of the wide size smears 46 and 48 in the absence of UV light.
a (ii) provides another pattern 28 comprising of a set of vertical lines 13 and a set of horizontal lines 26 for the analysis against multiple bands of the size marker as described in
The template sheet of the present invention is a sheet of transparent flexible plastic material of substantially square shape comprising a pattern of at least a pair or plurality of pairs of mutually parallel vertical lines and a pattern of at least one or more mutually parallel horizontal lines, wherein the intersection of at least one pair (or plurality of pairs) of vertical lines and at least two horizontal lines make quadrilateral shapes. For exemplary purpose, a template sheet 55a comprises of three pairs of vertical lines, template sheet 55b comprises a single horizontal line and template sheet 55c also comprises a single horizontal line but at different location on the sheet than template sheet 55b. The combination of template sheets 55a, 55b and 55c give rise to a pattern similar to pattern 22 as described in FIG. la (i). Another template sheet 55e comprises a pattern of three pairs of vertical lines and six horizontal lines similar to pattern 28 as described in
The template holding assembly 70 of the present invention as illustrated in
c describes the achievement of a particular pattern for gel analysis using three different template sheets to achieve a final combined pattern of the said three template sheets in accordance with the principle of the present invention. In
d describes the achievement of a particular pattern for gel analysis using a single template sheet comprising a pattern of horizontal and vertical lines 55e inserted in slot 72a of the template holding assembly 70 as illustrated in
a describes the principle setting of the method of the present invention. The template sheets 55a, 55b and 55c comprising different patterns on them are placed in between a gel matrix 54 and the light emitting area 57 of the UV transilluminator 56 with the switch 58 in OFF position. The observer/analysis means can view the pattern through the gel matrix as 74 in the absence of UV light.
b describes the analysis setting of the present invention with the template holding assembly 70, which can hold patterned transparent template sheets 55a, 55b and 55c inside of it through the shelf slots 72a, 72b and 72c of the front panel/entry section 72, respectively. The template holding assembly 70 can be employed as a fixed part of UV tranilluminator 56 or be used as a removable or separate part like a conventional gel tray. The observer/analysis means can view the pattern through the gel matrix as 76 in the absence of UV light. The template sheets 55b and 55c can be moved a little inward or outward in their respective shelf slots to change the location of respective horizontal lines in the final pattern 55d according to the required location for analysis.
c describes the analysis setting similar as
a describes a comb 100 used for making wells in the gel matrix 106 of
b describes a comb 102 used for making wells in the gel matrix 108 of
c describes a comb 104 used for making wells in the gel matrix 110 of
While the invention has been described in complete detail and pictorially shown in the accompanying drawings, it is not to be limited to such details, since many changes and modifications may be made to the invention without departing from the spirit and the scope thereof. Hence it is described to cover any and all modifications and forms which may come within the modifications and forms which may come within the language and scope of claims.
This is a non-provisional application claiming benefit of provisional application 61/741,288 filed on Jul. 18, 2012 entitled A METHOD TO ASSIST GEL ANALYSIS AND PROCESSING AND APPARATUS FOR THE SAME.