Title of Invention

"AN IMPROVED SINGLE STRIP COMB FOR ELECTROPHORESIS"

Abstract "An improved single strip comb for electrophoresis" This invention relates to an improved single strip comb for electrophoresis comprises of a comb block (1), a comb beam (10) and a set of comb clamps (21), the comb block (1) is screwed on to comb beam (10) with the help of set of block/bearr screws (28) passing through block holes (8) and beam holes (17), the comb beam (10) is screwed onto the comb clamps (21) having one comb clamp on each side/face (13 and 14) of the comb beam (10) with the holes (28) and beam holes (19).
Full Text This invention relates to an improved single strip comb for electrophoresis.
PRIOR ART OF THE INVENTION;
Yeast is a cimple, unicellular eukaryote with small nuclear deoxyribonucleic acid, referred herein after as DNA, contents and relatively better characterised genome. It has 16 chromosomes in a haploid cell and sizes of chromosomes range from 240 kb to 2200 kb (kilobases). Because of their small sizes it has been possible to separate chromosomes individually using pulsed-field gel electrophoresis, herein after referred as PFGE, technique and this has encouraged attempts to study DNA lesions and repair in individual chromosomes regulated by 3 different epistasis groups have been identified. Such identified DNA repair pathways known in the art are, by the name of the leader radiation sensitive gene, herein after referred as RAD, of the group., are (i) RAD3 dependent error—free excision repair, (ii) RADS dependent error-prone repair and (iii) RAD52 dependent recombination repair pathway. These repair and fixation pathways act competitively on DNA
lesions and modification in the repair pathways could alter the rates of DNA repair and fixation processes and thereby influence the resultant cellular responses i.e. cell survival., mutagenesis and transformations.
It is well understood by those, who are well conversant with the art that modifications in the repair pathways, as referred herein above, generally means the modification of DNA damage induced by damaging agents such as radiations. The irradiation of the cell culture in general, or yeast in particular, as referred herein this description, by ionising radiations may induce the alterations in the cellular response in general and alter the cell survival and generate mutagenesis in particular.
Study of effects of such modifications of ionising radiations, as referred herein above, induced DNA damage, cell lethality and mutagenesis have been
reported in the art.
To study such effects, the DNA samples are prepared and developed by such known methods. The agarose gels formed/'developed are photographed after staining and d e s t a i n i n g the gels. Such developed photographs/ photonegatives are used to estimate the DNA damage and repair preferably after scanning under the dens i tometer.
The effects of ionizing radiations induced
alterations in cellular responses in general and on cell lethality and mutagenesis in particular, as referred
herein above, have been studied by pulsed field e1ectrophoresis, referred herein as PFE, in general and pulsed field gel e1ectrophoresis, referred herein after as PFGE, in particular, technique. In the pulsed field e 1 ectrophoresed agarose gels, thus obtained, DNA bands, each representing a chromosome, are possilble to be resolved clearly in unirradiated and some times in samples irradiated with low doses of ionizing radiations. However on irradiation, the f 1 ou r e.seencr intensities of bands decreases and the. smear appears, particularly in the low molecular weight range. The decrease in the flourescence intensities and increase in the smearing of bands is ionizing radiation dose dependent. This dependency can be observed in any such
study by observing the disappearance of the bands, which show relatively lower intensities in (.he irradiated samples, on irradiation even by 100 Gy (Gray). The increase in smearing of bands is observed on increasing the dose of ionising radiations.
The smear appeared particularly, in the low molecular weight range, on7 the bands after irradiation, limits the detailed analysis of only clearly resolved bands preferably in the high molecular weight region only. Therefore, instead of quantifying different types
of DNA damage and repair, by such known methods, using
such known techniques and by adopting such known devices
is restricted to quantitation of average DNA damage and repair on the basis of alterations in the intensity ratios, referred as pn, of the individual bands.
The intensity ratio pn , as referred herein above, is calculated by known methods. For understanding, which may be referred as
pn=In/It;
where,
In is the intensity of nth band in a lane, and
1t is the sum of intensities of all bands and the
we 11 in the 1ane .
ch limitations of such known methods using such
known techniques by adopting such known devices, as staled herein above, that is being able to quantify only the average DNA damage and repair rather than being able to quantify the actual or specific DNA damage or repair to the individual chromosomal DNA bands are mainly due to the known e1ectrophoretic combs.
The known electrophoretic combs, when used with the known PFE or PFGE/techniques results in the formation of smear on the chromosomal bands, after it is irradiated as stated herein above, which in turn can not be resolved to separate the DNA fragments from the chromosomal bands.
4
Still another disadvantage of formation of smear by use of known electrophoretic combs with known PFE or PFGE technique is that the formation of such smear does not allow to identify the belongingness of the smear of DNA fragments with individual or particular or specific chromosome.
Still another disadvantage of formation of such smears by use of such known electrophoretic combs with known PFE or PFGE technique is that the formation of such smear does not allow to resolve the mixture of DNA fragments, according to their be 1ongingness to the individual or specific chromosomal band, obtained after irradiation and which in turn would have been separated
after pulsed field gel eIoctrophoresis.
The known e1ectrophoretic combs used to cast wells in the agarose gels as referred herein above, arc mainly used to cast many wells, herein after referred as multiple wells. The multiple wells, casted by such known e1ectrophoretic combs aro closely placed in series and are of much smaller size and much lesser thickness.
The multiple wells that is the number of wells generated by known such electrophoretic combs as
referred herein abqve, may vary from four to fifteen or
even twenty in number and thefr size particularly the length of such wells, generated in the agarose gels by adopting such known electrophoretic combs, may vary from 3.0 mm to 20.0 mm and the width of such wells may vary from 0.5 mm to 2.0 mm.
The main disadvantage of such mutiple wells generated by electrophoretic combs, known in the art, is that wells thus generated in the a.garose gels, aro unsuitable to hold the samples for further developing the assay to study the in-vitro DNA damage to individual or say specific chromosome, irrespective of the cell cycle stage stage, which in turn results in t he disadvatages/ drawbacks of the prior art, as described herein above in the foregoing d e s r r i p t ion, that is
limiting the quantification only to average DNA damage and repair instead of actual or specific DNA damge or repair to individual chromosomal bands.
Therefore, the known e 1 ect roplioret i c combs, as known in the prior art, do not permit t o rec o r d the m o b i 1 i t y of o a. c h i n d i v i d u a 1 c h r o m n s o m e or i I s DNA fragments to estimate the DNA damage and repair.
Still another disadvantage of such known combs is that, there are no means on the comb block to accurately adjust the height of the block with respect to the comb beam, which in turn may result in wells of
non-uniform depth, and hence such wells generated by use of known such combs of prior art are unsuitable for further development of the assay.
Still another disadvantage of such known combs is that there is no provision of height adjustment of comb beam with respect to the comb clamps and hence, the height of the beam and the block with respect to the ground level can not be adjusted after the height of comb block has been adjusted.
This is further disadvantage of such known comb:; that, readjustment of the height of beam or block in known such combs may contaminate the comb, particularly
the comb block, which in turn generates the damaged wells and such wells, once damaged, are unsuitable for the further development of the already developed assay.
NEED OF THE INVENTION
Therefore, there is a need to have an improved electrophoretic comb, which can overcome the disadvantages and limitations, as described herein above, of the known such electrophoretic combs.
OBJECTS OF THE INVENTION
An object of the present invention is to propose an improved single strip comb for electrophoresis which obviates the disadvantages and limitations of the prior art.
Another object of the invention is to propose an improved single strip comb for electrophoresis which can permit the detailed analysis even of unresolved bands comprising of chromosomes and chromosomal DNA fragments.
Still another object of this invention is to propose an improved electrophoretic comb which can permit the analysis of and quantification of the actual or specific. DNA damage and repair to the individual chromoBomes„
Yet another object of the present. invention is to propose an improved single strip comb for electrophoresis which can permit to resolve the smear to separate the DNA fragments, particularly which can permit to identify the belongingness of the smear of DNA fragments to the individual or particularly or specific chromosomal DNA band, more particularly, which can permit to recolve the mixture of DNA fragments, according to their belongingness to the individual or specific chromosome obtained after irradiating the unirradiated agarose gel strip, which in turn has been already obtained by using he known such electrophoretic combs with known such PFE or PFGE technique.
Still another object of this invention is to propose an improved device which can generate a single well of much large size, which in turn is suitable to hold the sample for further developing the assay, more particularly the chromosomal profile, which has already been developed once by using the known electrophoretic comb, having capability of generating multiple wells, and each such well of mutiple well formed is of small length and width as described herein above.
Still further object of this invention is to propose an improved device which can permit to record the mobility of each individual chromosome or its fragments already separated by using known electrophoretic comb with known PFE or PF6E technique, to estimate the ionising radiation induced DNA damage and repair.
The present invention is to propose an
improved single strip comb for electrophoresis which can
facilitate the resolution of smear, which in turn is the
mixture of DNA gragments of each individual or
particu1ar chromosome.
BRIEF DESCRIPTION OF THE INVENTION
Accordingly, the present invention discloses an improved single strip comb for electrophoresis and the method of use thereof, particularly an improved single strip comb for gel electrophoresis and the method of use thereof, more particularly an improved single strip comb for direct current horizontal gel electrophoresis and the method of use thereof, primarily
comprising of a block, herein referred as comb block and of block holding means herein referred as beam, particularly comb beam and of a set of beam holding means, herein after referred as clamps, particularly comb clamps.
The comb block, comb beam and comb clamps are characterised by structures having six faces.
The comb block is rectangular in shape and is vertically screwed on to the comb beam, through two or more longitudinal holes, herein after referred as block holes, preferably two. The faces measuring the height of the comb block are provided with horizontal grooves.
The comb beam is longitudinally extending, rectangular or square in shape, provided with two or more threaded holes, herein after referred as beam holes, preferably two on the face measuring length of the beam, and one threaded hole ech on the face measuring the height, of the comb beam.
The set. of comb clamps are characterised by rectangular strucatures, which are vertically placed
and hold the comb bean, and are provided by single longitudinal hole, herein after referred as clamp hole, preferably extending upto the half of the height of the comb clamp.
In accordance to the present invention, the comb beam is fitted between the set of comb clamps, with the help of clamp screws and the comb block is fitted on to the comb beam with the help of block screws.
The detailed constructional features and description of the device, disclosed in the present invention its method of use thereof, are described with the help of enclosed figures.
DESCRIPTION OF THE FIGURES
Fig. 1 shows the top-side perspective view of
the comb block in accordance to the most preferred embodiment of the present invention.
Fig. 2 shows the top—side perspective view of
the comb beam in accordance to the most preferred embodiment of the present invention.
shows the top-side perspective view of the comb clamps in accordance to the most preferred embodiment of the present invention.
Fig. 4 shows the top-side perspective view of
the essembled single strip comb, in accordance to the most preferred embodiment of the present invention.
Fig. 5 shows the assay developed by using
piAlsed-field gel electrophoresis adopting the known electrophoretic comb (only four wells have been shown therein)
Fig,, 6 shows the assay developed from the set
of chromosomes already separated by pulsec.l-field gel electrophoresis technique as shown in column 1 of figure 5, which is placed upside (chromosome-( i) ) on left and downside (chromosome-(vi)) on right, side.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly the present invention provides an improved single strip comb for electrophoresis and the methods o! use thereof as stated herein above, primarily comprising a comb block (i), a comb beam (1O) and a set of comb clamps (21).
In accordance to the most preferred embodiment of the present invtention, the comb block (1) is characterised by six faces (2,3,4,5,6 and 7) and atleast two longitudinal all through holes (8). The faces, 2 and 3 sire rectangular and opposite and parallel
to each other said measure the length of the comb block. Similarly, faces 4 and 5 measure height of the comb block and the faces 6 and 7 measure the width or thickness of the comb block. The holes (8) are provided on faces 2 and 3.
In accordance to the most prefereed embodiment of the present invention, the faces 4 and 5, are provided with horizontal grooves (9).
In accordance to the most preferred embodiment of the present invention, the comb beam (10) is characterised by six faces (11 .„ 12, .1.3,14 ,15 and 16} and atleast two threaded holes (17) provided on face 1.1 or 12) measuring the length of the comb block (1O) and one hole (19) on each face, 13 and 14, measuring the height of the comb beam (10). The set of faces 11 and 12,13 and 14,15 and 16, preferably rectangular in shape and parallel and opposite to each other. The faces, 15 and 16 measure the width or thickness of the comb beam (10). The holes (17) are preferably extending upto the depth 2O.
In accordance to the most preferred embodiment of the present invention, the set of comb clamps (21) is also characterised by six faces (22,23,24,25/26 and 27) and a longitudinal all through hole (28) provided on the rectangular, and parallel and opposite faces 22 and 23, measuring the length of the comb clamps (21). The another set of two rectangular, and opposite and parallel faces 24 and 2.5, and 26 and 27, measure? the height of the comb clamp (21) and the width/thickness of the comb clamps respectively.
In accordance to the most preferred embodiment of the present invention, the height (face 4 or 5) of comb block is more than height (Face 24 or 25) of comb clamps (21) and thickness (face 6 or 7) of the comb block (1) is preferably equal to the thickness (face 1.5 or 16) of the comb beam (10).
In accordance to another preferred embodiment of the present invention, the length (face 11 or 12) of comb beam (1C) is preferably about 1.60 to 2.OO times, more preferably 1.70-1.80 times more than the length (face 2 or 3) of the comb block (1).
In accordance to the most preferred embodiment oi the present invention, the comb block is not. provided with any cuts on the faces ( 6 and 7) measuring the thickness, which in turn does not generate mutple wells,, rather generate only one well of length essentially equal to the length of the comb block (1) and of width essentially equal to the width/thickness of the comb block (1), which in turn is suitable to hold the sample, that is a set of chromosomes separated by PFGE or PFE technique, to further develop the assay in order to resolve the smear of DNA fragment according to their belongingness to the individual or particular chromosome., and which further in turn facilitate the study of in vitro DNA damage and or repair caused by irradiating means to the chromosomes irrespective of the cell cycle stage as described herein above.
In accordance to the most preferred embodiment of the present invention, the comb block (i) is screwed on to the comb beam (10) with the help of set of block/beam screws (28) passing through block holes 8 and beam holes 17. The comb beam (10) is
screwed onto the comb clamps (21) having one comb clamp on each side/face (13 and 14) of the comb beam (10) with the help of clamp screws (3)) passing through clamp holes (28) and beam holes (19).
The nome-fnc lature, such as comb block, comb beam, comb clamp,, block holes, beam holes, beam/block screws and clamp screws etc. have been used to explain the device and its assembling procedure. Mere changing the nomenclature? or the dimentions of the parts, particularly bomb beam (10) and comb clamps (2.1) may fall within the scope of this invention. However, the ratio of length (face 2 or 3) of the comb block According to the most preferred embodiment of this invention, the thickness (face 6 or 7) of the comb block (1) is preferably between 8 mm to 14 mm, more preferably between 10 mm to 12 mm, and the length (face 2 or 3) of the comb block is preferably equal to 90 mm to 110 mm.
The device as enclosed herein, can be made of any polymeric material preferably of acrylamide.
The comb block (1) is provided with horizontal grooves (9) on both faces 4 and 5, which measure the height of the comb block (i). The horizontal graduation or grooves (9) are provided to adjust. the horizontal balancing of the comb block. (1) with respect to comb beam (10) and hence providing an advantage of accuracy of paralleling and height adjustment. The longitudinal holes 8 and 28 are provided to help in adjusting the height of block (i) and clamp (2.1) respectively with respect to the agarose gel and electrophoresis tray respectively.
METHOD OF USE
i) The horizontal grooves (9)
provided on both the sides (faces 4 and 5) of the comb block (1) are placed at exactly aligned position with the comb beam (10). The holes (8) are matched with the holes (17).
ii) The comb block (1) is then screwed on to
the comb beam (1O) with the help of screws (29) and the
comb block (1) is maintained only slightly and uniformly
raised preferably O.5 mm from the base of gel casting
tray.
iii) The comb beam (1O) with the comb block (1) is screwed on to the comb clamps (21) with the help of clamp screws (30) and the height of the comb beam (10) is maintained at about 2.0 to 3.0 cm from the base of the horizontal gel casting tray and is maintained parallel to the base of gel casting tray.
iv) The surface and the margins of the comb block are polished well.
v) Thee final adjustment of the height of comb block (1) from the gel surface or the horizontal gel casting tray is made with the help of clamp screws (30) so that the comb block (1) surface is maintained clean and polished.
vi) The gel is poured in usual fashion and is allowed to set.
vii) The final gel, so formed has a single large well in which the? unirradiated (column 1 of figure 5) strip (35) of sample already developed by using the known method of pulsed field gel electrophoresis and known comb, as described herein above, is placed after irradiation and is electrophoresed and desired separation/resolution of the smear of chromosomal DNA fragments form the individual chromosomes ( i to vi) is achieved, figure 6.
It is observed in figure 5 that the unirradiated colums, that is column nos. i and 2 (31, 32) show clear separation of chromosomes ( 1 to vi) but. no DNA fragment and hence no mear formation. However, the irradiated columns, that is column nos. 3 and 4 (33, 34) do not show clear separation of chromosomes and as well DNA fragments and the smear (36) is formed therein. In the figure 6, which shows the assay developed from the set of chromosomes (35), already developed by PF6E technique, of column 1 (31), after irradiating the same, it is observed that. the DNA fragments (37) of individual chromosomes have been separated/resolved clearly even after irradiating the set of chromosomes, separated by PFGE of figure-5.
It is understood from the foregoing
description that, the device developed and disclosed
herein above, definitely has the advantage of overcoming
the disadvantages/drawbacks of the prior art as
described herein above.
The additional advantage is provided to have the accurate adjustment of horizontal balancing of the comb block with respect of ground level, agarose gel surface and comb beam.
The further additional advantage of the presently disclosed device is that the comb beam's height can also be adjusted with respect to the grond level, even after adjusting the height of the comb block which in turn has an advantage of not disturbing the comb block with respect to comb beam.


WE CLAIM
1- An improved single strip comb for
electrapharesis comprising a comb block, a comb beam and a set of comb clamps.
2. An improved single strip comb for
electrophoresis as claimed in claim i wherein said comb
block, comb beam and comb clamps have each six faces.
3. An improved single strip comb for
electrophoresis as claimed in preceedincg claims wherein
said comb block has atleast two longitudinal all through
holes and horizontal grooves.
4. An improved single strip comb far
electrophoresis as claimed in claim 3 wherein said
horizontal grooves are provided on said faces measuring
the height, of the said comb block.
5. An improved single strip comb for
electrophoresis as claimed in claims 1 & 2 wherein said
comb beam has atleast two threaded htsles provided on
said face measuring the length of the comb block, and
one hole on said faces measuring the height of the comb
beam.
6. An improved single strip comb for
electrophoresis as claimed in preceeding claim 5 wherein
said holes of the comb beam preferably extend upto the
depth.
7. An improved single strip comb for
electrophoresis as claimed in preceeding claims 1 & 2
wherein said set of comb clamps has all through hole
provided an said faces measuring the length of said comb
clamps.
8. An improved single strip comb for
electrophoresis as claimed in preceeding claim 7 wherein
the? height of said comb block is more than the height of
said comb clamps and the thickness of the comb block is
preferably equal to the thickness of the comb beam.
9. An improved single strip comb for
electrophoresis as claimed in claims 7 £ 8 wherein said
length of said comb beam is preferably about 1.6O to
2.00 times, more preferably 1.70-1.80 times more than
the said length of said comb block.
10- An improved single strip comb for
electrophoresis as claimed in proceeding claims wherein said comb block generates only one well of length equal to the length of the said comb block and of width essentially equal to the aid width/thickness of the said comb block.
11. An improved single strip comb for
electrophoresis as claimed in preceeding claims wherein
said thickness of the comb block is preferably between 8
mm to .14 mm, more preferably between 10 mm to 12 mm, and
the length is preferably equal to 90 mm to 11O mm.
12. An improved single strip comb for
electrophoresis as claimed in preceeding claim wherein
the ratio of the length of said comb block to the
thickness of the comb block is more preferably between
8.0 : 1.0 to 9.O : 1.0 and that of the height to the
thickness is between 3.O : 1.0 to 4.0 : 1.0 and that of
the length to the height is between 2.0 s 1.0 to 3.0 :
1.0.
13. An improved single strip comb for
electrophoresis as claimed in preceeding claims, wherein said comb beam is screwed onto the said comb clamps having one said comb clamps on each side of the said comb beam with the help of said clamp screws.
1.4. An improved single strip comb for
electrophoresis substantially as herein described and i 1 lustrated .

Documents:

678-del-1997-abstract.pdf

678-del-1997-claims.pdf

678-del-1997-correspondence-others.pdf

678-del-1997-correspondence-po.pdf

678-del-1997-description (complete).pdf

678-del-1997-drawings.pdf

678-del-1997-form-1.pdf

678-del-1997-form-19.pdf

678-del-1997-form-2.pdf

678-del-1997-form-3.pdf

678-del-1997-gpa.pdf


Patent Number 215301
Indian Patent Application Number 678/DEL/1997
PG Journal Number 11/2008
Publication Date 14-Mar-2008
Grant Date 25-Feb-2008
Date of Filing 18-Mar-1997
Name of Patentee THE CHIEF CONTROLLER , DEFENCE RESEARCH & DEVELOPMENT ORGANISATION, MINISTRY OF DEFENCE, GOVT.OF INDIA.
Applicant Address B-341, SENA BHAWAN, DHQ P.O. NEW DELHI-110011.
Inventors:
# Inventor's Name Inventor's Address
1 DR. (MRS) MADHU BALA, SCIENTIST 'C' INSTITUTE OF NUCLEAR MEDICINE & ALLIED SCIENCES, DELHI-110054
PCT International Classification Number G01N 27/26
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA