METHOD OF COMPUTER-AIDED AUTOMATED DIAGNOSTIC DNA TEST DESIGN

Abstract: The invention encompasses a semi-automatic and/or automatic computer-aided technique for designing comprehensive DNA diagnostic tests for mutations in disease genes through searching for optimized conditions for PCR amplification and for optimal melting behavior in denaturing gradient gels and for optimal distribution in two-dimensional electrophoresis display of the mutational target fragments.

Description: METHOD OF COMPUTER-AIDED AUTOMATED DIAGNOSTIC DNA TEST DESIGN The present invention relates to techniques lor designing comprehensive DNA diagnostic tests for mutations in disease genes; being more particularly directed to computer-aided searching for optimized predicted conditions for Polymcrasc Chain Reaction (PCR) amplification, including multiplex PCR amplification, optimal melting behavior in denaturing gradient gels and optimal distribution by two-dimensional electrophoresis of the mutational target fragments, thereby to provide an overall design for actual diagnostic tests. BACKGROUND OF THE INVENTION As is well-known, a strand of DNA is comprised of four different nuclcotidcs, as determined by their bases: Adcninc. Thyminc, Cytosinc and Guaninc, respectively designated A. T, C, G. For each strand of DNA, there is a homologous strand in which A pairs with T, and C pairs with O. A specific sequence of bases which codes for u protein is referred to as a gene, and that gene is segmented into regions which arc responsible for protein compositions (cxons) and regions which do not contribute to protein composition (nitrons). An exon can vaiy in length from about 30 bascpairs to thousands of bascpairs (bp). For purposes of the present invention, however, primary concern resides with the coding regions of the gcnc(cxons), though the test procedures of the invention can be easily appropriately modified, where desired, to screen for carcinogenic mutations in the introns, also, and for other purposes as well. DNA diagnostic testing of genes with mutational defects is important for ascertaining information with regard to individual susceptibility to particular diseases, classification ol the disease in a thciapcutically iclcvant sub-gioup, the cai ncrship (and potential tian fci ) oi picnatal picscucc of buth defects, and othci important pui poses ' I he gold standaid in DNA diagnostic testing for the pi csc cc of mutations is DNA .sequencing, which involves the complete decoding ol the gene This, howcvci, is limc- consuming and inefficient I imc-consuming because despite numcious ongoing attempts to simplify and gicatly accclciatc the pioccss, all cuπcntly used routine sequencing .systems aic still based on the piinciplc oiiginaliy developed by Sangcr and colleagues (as dcsciibcd. loi example, in MolccuLii Pathology, l lcim and Silveiman, 1990. pages 7- 10), and only partially automated I hey aic inefficient because, instead of simply dctci mining dilfciences between entiic DNA li.igmcnls, every DNA fiagmcnt must be completely decoded bcfoic any dilfciences Horn noi mal (mutations) aic icvcalcd. The fact that the exact location ol a disease-causing mutation can be dilfcicnl horn one individual lo the next, mot covet, piccludcs the possibility of only testing foi licqucntly occuiπng known mutations Indeed, the many diffcicnt mutations that may convert a healthy gene into a diseased gene makes it necessary in each case to inspect the entire gene at all possible positions for mutations (tcimcd gene scanning) lalhci than foi only a few ficqucntly occuinng ones (mutation sciccning) Mutation screening methods arc relatively simple and cost-efficient Outside DNΛ sequencing, potential gene scanning systems aic scaicc and thcii cost-efficiency is questionable. Recently, a method was disclosed by one of the applicants of the picscnt patent application foi compaiativc scanning of 100-600 bascpaiis (bp) gene fragments by multiplex PCR amplification followed by two-dimensional elcctrophorctic separation in polyaciylamidc gels on the basis of both size and bascpair sequence, "Multiplex Co- amplificatiυn of 24 Rctinoblasloma Gene Cxons Altei Pic-amplification By Long-Distancc PCR," Jan Vim and Daizonu Li, Nuclei ιc Acids Rescaich, 1996, Vol 24, No l , p 538-9, " fwo-Dimcnsional DNA 1 ypmg", Ian Vqg, Molcculai Biotechnology, pages 275, on, Vol 4, 1995. and in copendmg US patent application Serial No 08/471 ,249, filed June 6, 1995 for Method Of And Appaiatus foi Diagnostic DNA Testing The lattci can be accomplished by denaturing giadicnt gel clcctiophorcsis (DGGE) This multiplexing technique foi analyzing picdctctmmcd gene cxons dciivcd from DNA, involves adding pπmcr pairs suiioundmg successive gioups of thc gene cxons followed by effecting longdistance PCR amplifications thcieof in a common tube oi vessel (multiplex long-PCR) to achieve relatively long tcsulting amphcons, adding furthci pπmci pans surrounding each ol the gene cxons oi parts thcieol, and then effecting multiplex PCR amplifications thcieof m the common tube oi vessel with iclativcly short resulting amplicons, and clcctt ophorctically scpaiatmg the gene fi agmcnts By size separation, mutations icpicscnting deletions oi insertions varying from scvcial to many bascpaiis can be detected In DGGE. point mutations, such as bascpair substitutions arc also detectable This is due to the tendency of doublc-sti andcd DNA fiagmcnts to melt at a point in the giadicnt wheic the tcmpciatuic equals the melting tcmpciatuic of thc lowest-melting domain of thc fiagmcnt In the pioccss of PCR amplification, the mutational target fiagmcnts (c g , gene cxons) ate sut ioundcd by piimcus, i.e., short (about 20 base pans) singlc-stiandcd DNA fiagmcnts Pi imeis aic chosen in such a way that they aic complementary (bind to) positions al the lclt and l ight boundaiics oi ends of thc taigct fiagmcnts By using appiopnatc enzymes that extend each piimci inwatds, towards each other, the mutational taigct can be copied 1 his can be icpcatcd a gicat numbci of times in a so-called ihcrmocyclci - - a machine that fiist heats up the DNA sample, thereby scpaiatmg the single stiands followed by cooling down, which icsult in annealing of the pnmci.s to their taigct sequences, and the subsequent enzymatic extensions of thc ptimcrs by polymcrasc enzymes The net icsult is an amplification of thc fiagmcnt in between the primers of typically one million times This piovidcs enough taigct DNA to detect the fragment by using a DN A-bindmg dye, altei elecliophoietic scpaiation, without the use of radioactive tiaccis. the test of thc DNA being now a iclativcly small amount and invisible. The positioning o thc pnmcis is cutical because, for such short sequences, there is ample opportunity to bind clscwhcic in the complex DNA molecules that form the starting matciial of thc test This would lead to the copying of other fiagmcnts than the ones of intcicsl Positioning of pnmcis is even moic cutical in dcnatuiing gradient gel electrophoresis. wheic each fiagmcnt must have an optimal melting tcmpcratuic in older to allow all possible mutations to be detected It is common pi cticc to couple one of thc two pnmcis suiiounding a gene taigct fiagmcnt to a GC-πch clamp sequence of about 30 bascspaii long This clamp is very stable and functions as the highest melting domain, that is, the part of thc DNA molecule that keeps the fiagmcnt togcthci. This is important because once a fiagmcnt migiates of f the gel it can no longci be detected In PCR-DGGE it is |ust as important, lutthcπvtoic. that the taigct fiagmcnt consists of one single domain (flat thiouiihout the gene fiagmcnt) that has a lovvci stability than the GC-clamp. In that case, it will melt cailicr than the clamp, icsulting in a structuic that is partially doublc- sti andcd (the clamp) and partially singlc-stiandcd (the taigct fiagmcnt) a so-called blanched strυctut c. Such a fiagmcnt will be gicatly ictaidcd in the gel. T ypically, the exact position whei c such a fiagmcnt melts (and thus halts its migiation) is completely dependent on its sequence. With a fiagmcnt of, say, 500 bascpairs, one single bascpair diffcicncc will lead to a migiational diffcicncc that can be employed by detecting mutations in such fragments as a migiational diffcicncc with a control (wildtypc) fiagmcnt. I lcncc, in conti ast to DNA sequencing, DGGE docs allow comparative scanning of whole fiagmcnts for mutational diffcicnccs without the need to completely decode each molecuc. To cany out DGGE in two dimensions (2-D) lathci than in one, increases the efficiency of thc system as well as its icliability. Indeed, in a 2-D gel, many moic fiagmcnts can be analyzed simultaneously than by a 1-D separation. It is more icliablc because every fiagmcnt can be defined by both its melting tcmpciaturc and its size. A disadvantage, however, resides in its inci cased complexity, icquiring cxtiemc attention to the design of thc test. Since the design of a DGGE test itself is not trivial (primers must be chosen in a way that the amplified fi agmcnts icpicscnt a single domain, as before discussed), the 2-D piinciplc adds a dimension in complexity as well as in icsoluliυn. A typical design of PCR primcis, moicovci , must take into considci tion many other vaiiables inhcient to the PCR piocess, such as ptimer annealing temperatures. As disclosed in the above-cited Vijg papcis and the said patent application, the (vvo-stcp PCR piocess enables many diffcient cxons to be amplified simultaneously in the same reaction. In this method, first, groups of taigct fiagmcnts (e.g.. groups of cxons) arc amplified as large 5-40kb amplicons, for example, by long-distance PCR (an improved form of PCR in which more efficient polymcrasc enzymes arc employed that generate longer fiagmcnts). Second, with these amplicons as templates, large numbers of individual target sequences can now be amplified simultaneously in the same reaction vessel under a single set of experimental conditions. Normally it is very difficult to find one set of reaction conditions under which multiple fragments specified by multiple primer pairs arc amplified simultaneously (i.e., multiplex PCR). Probably because the prc-amplification by long-distance PCR increases the amount of taigct sequences relative to the rest of thc complex genomic DNA, flexibility with respect to the subsequent PCR amplification of thc target sequences is much greater than normal, which permits extensive multiplexing; i.e., co-amplification of different target fragments in the same reaction. This method of extensive multiplexing greatly economized the process of template preparation in genetic testing over earlier techniques for inspecting for mutations after PCR amplifying the many distributed cxons of thc often very large disease genes. As an example, it is now possible, to generate a collection of as many as 25 fiagmcnts corresponding to 26 cxons of the tumor suppresser gene RB I in one single two-step PCR reaction. After the two-step multiplex PCR amplification, a single 2-dimcnsional clccliophorctic separation, as the third step, is sufficient to resolve all these fragments and detect all possible mutations as variations in fragment spot position. With this system, a genetic testing method has become available that is both highly accurate (in detecting all possible mutations) and cost-efficient. The only icmaining diawback involves the absence of a rapid design of an optimal test lot one oi mote individual genes involved in a particular disease. 1 he human genome, indeed, contains an estimated numbci of 100,000 diffcient genes, many of which might ultimately prove to be involved in one oi mote diseases To design a set of PCR pnmcis foi many diffcient genes and/oi gene combinations that fulfill criteria for optimal (multiplex) PCR, optimal dcnatui ing giadicnt clectiophoi etic separation and 2-D distribution is not ti ivial As shown in later-described Fig. I , the computer-assisted test design of thc invention must piovide, for an optimal 2-D genetic test for one or multiple genes with predicted primci and GC-clamp i cspcctivc positions and lengths for meeting optimal melting ci itct ia, PCR ci itci ia nd 2-D spot disti ibution criteria. T he present invention is dii cctcd to solving this pioblcm and, through a computer- assisted proccduic, to scmi-automatically and/or automatically design multiplex PCR/2-D clcctrophorctic tests for one or mot e genes. OBJECTS OF INVENTION An object of thc invention, accoidingly, is to piovide a new and impiovcd method of and appaiatus for computci -aided tapid design of a genetic test based on multiplex PCR amplification of the target sequences, followed by clectiophoietic separation of the fiagmcnt mixtui c in two dimensions- size and bascpaii sequence. A further object is to piovide for supciior pci foi mancc of multiplex PCR/2-D electrophoresis tests Ihiough the hcictolbic unavailable rapidity in computer software- aided selecting of optimal sets of conditions among the laigc number of possibilities available. Other and further objects will be dcsciibcd hereinafter and arc mote particularly delineated in the appended claims. DESCRI PTION OF THE DRAWINGS ' I he invention will now be dcsciibcd in connection with the accompanying dia ings in which: Fig. I, as cailicr mentioned, is a block diagram ofthc underlying computer-assisted system for optimal 2-D genetic test design for one or multiple genes; Fig. 2 rcpicscnts experimentally clcctiophorctically obtained mutation displays of exemplary l -D and 2-D DGGE tests on illustrative BRCA- I gene fiagmcnts; Figs. 3, 4 and 5 arc melting graphs (melting tcmpciatuic vs. bascpair offset) respectively of unacceptable multiple domain and acceptable left and right end GC- clampcd single domain profiles, taken for an cxcmplaty rctinoblasloma gene RB Exon 21 , Fiagmcnt # 1 ; Figs. 6A and 6B present flow diagrams of the present-day sequence of process slcps required for determining appropriate primeis and GC clamps for electiophoretic testing of designated gene fiagmcnts; Figs. 7A and 7B arc similar flow charts of thc steps involved in scmi-automatically, with operator assist, rapidly designing appropriate test primers, GC clamps and melting profiles and providing simulated electiophoretic gene separation displays in accordance wilh the computer-aided software techniques of thc present invention; Figs. RA and RB arc flow charts similar to Figs. 6A and 6B for full automation; Fig. 9 shows a prcfcitcd computer display screen with the various monitoring and display icons, boxes or windows and options available to the investigator,. Fig. 10 shows this screen with inputs for a particular desired gene— in this illustration, P53 involved in colon and other cancers, assuming no prior knowledge of appropriate primers, temperature gradients, etc.; Figs. 1 I A and M B aic similar views with different test values of pi imcrs, GC clamp, etc. producing unsatisfactory (simulated) melting profiles with multiple domains. Fig. I I A with the clamp located on the right, and Fig. I I B, on the left; Fig. 12 shows a satisfactory melting profile for corrected test values of primer, GC clamp, etc.. with Figs. 1 1 A, 1 1 13 and 12 showing vaiiation of predicted melting cui ves of cxons of thc gene obtained with only slight variations in the positioning of one or more of PCR pi imcis, lengths of pi imcrs and GC-clamp, type of GC-clamp, and length of the fiagmcnt, which serves to illustrate the real and qualitative features of thc invention with respect to the final test design, and with Fig. 12 containing an additional column read-out of temperatures to the l ight of thc graph; Fig. 13 illustrates displays of sequences with melting temperature; Fig. 1 shows a predicted 2-D display that the design of thc invention will enable to be ptoduccd in actual 2-D electrophoresis operation in accordance with the design; and Fig. 15 is a time giaph for test design for gene RB I comparing the prior manual pioccdurcs with those of thc invention. SUIVirVI ARY OF THE INVENTION In summary, the invention cmbiaccs a method of computer-aided genetic test design that involves capture of a gene sequence file, e.g.. fiom Genbank; indicating the start and ends of dcsireil taigct sequences; generating optimal melting piofiles, PCR conditions and 2-D distributions by varying ( I ) fiagmcnt size (gap-size), (2) GC-clamp, and (3) primer size; and icpoiting the selected optimal primer sets for being synthesized. From a moic detailed viewpoint, the invention cmbiaccs a computer-assisted method for gcnciating the design of optimized and compichcnsivc genetic DNA diagnostic tests based on PCR/2-D electiophoicsis foi sciccning mutations in genes, that compi iscs, inputting a computci micioptoccssor with a dcsiicd gene exon fragment ATGC lctlci sequence; piogiamining and contiolling the microprocessor first to select a trial start and end of thc sequence; then, selecting and positioning trial piimcr letter paiis to define the opposite boundai ics of thc cxons to be tested; adding a trial GC clamp to at least one piimcr. gcnciating ttial melting piofiles oi the trial pi imcts and GC clamp to dctciminc whcthci the piofilc consists of a lclativciy high-melting GC clamp peak and a substantially single fiat domain of iclativcly low-melting value for the fragments; in the event that the trial piofilc has multiple and not a satisfactory single flat domain, changing one or both of other dial piimcr sequences and GC clamp sizes and generating said profiles theicfiom until such a satisfactoiy single fiat domain is attained, and for primcts of each paii having substantially similai annealing temperatures suitable for PCR annealing: compaiing the pi imci letter sequence with olhci known gene sequences in the context of the total lcttci sequence of thc gcuc exon to insuic the uniqueness of thc sequences and to avoid ovci lap with any other than the selected gene fiagmcnt; simulating a 2-D distribution of thc fiagmcnts defined by the piimci pans by separation along one dimension by size and along an orthogonal dimension by melting tempeiature of thc fragment; selecting a giadicnt of inci casing melting tcmpciatuics along the orthogonal dimension to allow an optimal spicad of thc fiagmcnts thcicalong. and reporting the selected primei sets and tempeiature giadicnt, theieby pioviding an optimal design for an actual PCR/2-D clcctiophorcsis test. Picfcncd and best mode designs and details aic hereinafter fully described. DESCRIPTION OF PREFERRED EMBODIMENTS T he present invention, as bcfoic stated, involves a pioccdurc for the semi- automated and/or automated computei -aided design of optimized genetic screening tests based on PCR/2-D clcctiophoicsis, and ptefcrably multiplex PCR 2-D clcctiophorcsis Brief Summary Of Preferred Diagnostic Procedural Techniques In accoi dance with the pioccdurcs useful also with the computer-assisted semi- automated and automatic fcatuics of thc invention, two DNA fiagmcnts of, for example, I l-50kb aic constructed which aic homologous to an 18 to 25 base sequence in the intronic sequence both preceding and following an exon; the leading and trailing primers. Because many mutations occur in the ttansition fiom intron to exon, these priming sites, as bcfoic explained, aic scpaiatcd fiom the exon by a gap of at least, say, 10 base pairs, and usually not mote than about 40 base pairs. For each "multiplex" test herein described, optimal primers foi all of thc fragments would have comparable annealing tcmpciaturcs, as picviously noted. If the pnmcis all anneal within a few degrees centigrade of one another, each fiagmcnt should be equally amplified in the multiplex PCR Once pi imcrs aic selected, the exon is amplified from template DNA by PCR 1 he DNA sequence which is amplified was cailici rcfcricd to as a PCR fragment oi an amplicon, including the exon, the p mei sequences, the intionic gaps between the exon and pi imcr sequences, as well as usually a 30-60 base pan GC clamp, for example, which will be discussed latci in icfcicncc to the computer software progiammmg of thc present invention With such operation, the PCR fiagmcnt is at least about 86 base pairs longer than the exon itself Following a .successful multiplex PCR, millions of copies of thc dcsiicd fiagmcnts exist T hese can be viewed on a gel following clcctiophorcsis on both size and dcnatui ing giadicnt gels; it being important that each amplicon has its own unique spot on the 2-Dimcnsιonal mapping In othci words, the fiagmcnt should have a size and melting tcmpcratuic unique to all othci fiagmcnts included in the same test. The computei softwaic of thc invention, accoidingly, is designed, as latci detailed, to enable the user more icadily to attain a test in which these ci itcπa arc optimally met. With tcgard gcncially to the software design for achieving the puiposcs of thc invention, an optimal test design would suic that each fiagmcnt would have a diffcient size as well as a diffctcnt Tin oi melting tempeiature Using the later-described piogram, the fiagmcnt length can be va cd by adjusting the length of thc piimcr and by adjusting the position of thc primei Primei length for short PCR, as before described, generally langcs fiom about 18-25 bases pairs; and piimct position is usually between about 10 and 30 base paiis fiom the beginning and end of thc exon. This is also described as the bctorc- mentioned gap of, say, l()-30bp. To determine the T m o thc fiagmcnt, the computer uses the Let man Piogram (dcsci ibcd, for example, in "Computational Simulation of DNA Melting and Its Application to Dcnatui ing giadicnt gel Elcctiophoicsis," Leonard S. Lciman and Silverstein, Methods In Enzymology. pages 482-501. Vol. 155, 1987), which will produce a thcoictical Tin foi a fragment. %UF (uica-foimamidc) is a chemical gradient from which the actual melting tcmpciatuic Tin in degtccs C can be determined. Tin, moreover, is completely dependent upon base sequence. An Λ-T bond is less stable than a C-G bond. If a fiagmcnt has a high C-G content, thcicforc, it will melt lower in the gel (high Tin). Likewise, a fiagmcnt with a high Λ-T content will have a lower melting tcmpciatuic and will melt sooner (lower %UF) in the gel. Because the bases have an effect on neighboiing bases, a single mutation, called a point mutation, can alter the To ofthc entire fragment enough that it can be detected by dcnatuiing gradient gel electrophoresis. For example, a point mutation in which a single T is changed to a G, is enough to change the melting temperature of thc cntiie fragment from, for example, say 56% UF to 58% UF. If an A oi a T changes to a C or a G, the mutant will have a higher Tin than the latcr- desctibed healthy wild-type, due to the inci cased stability ofthc C-G bond. By similar icasoning. if a C oi a G is mutated to an A or a T, the mutant will melt at a lower %UF to To. At the end of thc PCR. a special cycle is introduced in which the DNA is denatured and then allowed to bond with a complementary strand. Because cvciy individual possesses two double stianded allclcs oi copies of thc same gene, one from the mothci and one from the father, four combinations of allclcs can occui during this cycle. If a mutation is present in one of thc allclcs, it is rcfcircd to as the mutant strand. The foui possible combinations would then be wild-type with wild-type, mutant with mutant, (these two aic icfc cd to as homoduplcxcs) and wild-type with mutant and mutant with wild - type (icfcncd to as hctcioduplcxcs) Using (he terminology mothci (M ) foi the maternal allclc and father (F) for the paternal allclc, the four stiands can be called MM and FF for the homoduplcxcs. and MF and FM foi the hctcioduplcxcs. When a mutation is picscnt. the hctcioduplcxcs will be notably less stable than cithci of thc homoduplcxcs. if, for example, a point mutation from a T to a G has occuncd, as above described, in the hetcroduplex strands, there will be a destabilizing mismatch of A with C and T with G. The mismatch will cause the hctcioduplcxcs to become far less stable than cither of thc homoduplcxcs. The presence of a mutant allclc will accoidingly pioducc foui spots in a 2-D pattern. The mutant and wild-type homoduplcxcs aic scpaialcd due to the natuic of thc mutation, and the two hctcioduplcxcs will melt consideiably highci in the gel due to thcit destabilizing mismatch. In the above example of a T to G point mutation, the wildtypc homoduplex will melt at say, the before-mentioned 56% UF, the mutant homoduplex will melt at 58% UF, and the two hctcioduplcxcs will melt consideiably above, but at melting points different from each othci . Thus, in the example of a BRCA- I mulatioii/hctcroduplcx shown in Fig. 2, two control samples with the same polymorphism and a sample with a 1 -bp insertion mutation detected by l -D DGGE (photographs A to the left) and by 2-D DGGE (photographs B to the right) illustrate how mutations arc revealed in the system by the appearance of four bands and spots, respectively, instead of one, and also show how the mutations or polymorphisms can be clearly distinguished from one another. In accordance with the picscnt invention, the computer helps dctcπninc a test in which each fragment produces a distinct spot on the 2-D pattern in which a hetcroduplex at that position can be easily detected. The earlier mentioned Lcrman's Program ("Melt 87") calculates the melting temperature of each base pair. Then a program "Slide Write" (described in the Advanced Graphics Software, Inc. Manual "SlidcWritcΦ Plus for Windows™," 1994) is used to constmct a melting graph in which the different melting domains of thc fragment can easily be viewed. The generation of a CG clamp at one end of thc fragment can influence the remainder of thc strand to act as a single melting domain. Without such clamp, there arc generally varying or multiple levels or domains for the fragment (Fig. 3). In the gel. a fiagmcnt with a GC clamp-induccd single melting domain will melt, but the GC clamp itself, which melts considerably higher, will remain intact, as shown by the left-hand spike or peak in Fig. 4. Without a GC clamp, the fiagmcnt will simply split into two single stiands and run off the gel. With the intact GC clamp, one strand enters a pore in the gel and the other enters another pore in the gel, and the intact GC clamp inhibits the fiagmcnt from moving any further in the gel. On the computer, a GC clamp of, for example, 30, 35 or 38bp can be selected. The user can also choose to which primer the clamp should be added; Fig. 5 showing the clamp spike or peak to the right. The optimal melting pattern for a given fragment is aii ivcd at thiough t al and error by selecting diffcient pnmcr lengths and positions and then trying diffcient types and positions of GC clamp Operation Befoi e The Present Invention Bcfoic the picscnt invention, the usci would cntci the DNA sequence Then Lcπnan's piogram would begin asking questions Aftci the usci had answered all the questions, the computer would calculate the melting tcmpciatuic After the melting temperature was aiπvcd at, the outcome had to be ttansfctrcd to another program which would construct a melting map of thc fiagmcnt If the fragment was not optimal, the user would return to the Lcπnan Piogiam and alter the p mcr position/length manually, and dclciminc whcic they had been placed on the fit st tun Once the user had achieved adequate melting maps foi each of the fi gmcnts, a 2-D pattern could be const! uctcd If each spot did not have a unique position on the 2-D pattern, the user would have to start all ovci with whichcvci fiagmcnts ovci lapped. Using this system, it took about foui weeks to design a test for RB I , though still sub-optimal With the piogiam of thc invention, a moic optimal test was designed in about foui hours Foi moic genes participating m the same test, design problems using MEL T 87 became increasingly scvcic The point comes at about 30 fragments when it is no longer possible to design manually according to the Lcrman program Fig 15 plots the time in hours as a function of numbci of fiagmcnts for test design foi RB I of piioi techniques and those ol the invention Curve A depicts data taken dining the construction of thc two-dimensional DGGE lest for RB 1 with fully manual softwaic 160 man hoius wcic necessary to design a test which could adequately test the 27 cxons of thc rctinoblasloma gene. Manual lest design has an empirically determined capacity limit of about 40 fiagmcnts. Curve B, on the other hand, exhibits the drastic improvement in both time efficiency and test capacity experiences with semi-automated test design of thc invention. With such semi-automated test design, the 27 exon RB I test was redesigned in approximately two and a half hours. Curve C, furthermore predicts the exponential improvement fully automated test design software of thc invention can have on the construction of two-dimensional multiplex tests, extending the capacity of two-dimensional multiplex gene testing even to the theoretical resolution limit of 2200 fragments on a 1 cm x 15cm denaturing gradient gel. The program of thc invention, furthcm.orc, is able to calculate the Tm or annealing temperatures of thc primers which also greatly Facilitates the development of a megaplex PCR. In the past, the user had to consult yet another program for this information. Lastly, the program herein also searches the gene sequence to insure that each primer sequence is unique so that only the desired PCR fiagmcnt will be amplified. For this purpose, only the long-PCR fiagmcnts have to be searched through. Before this feature, the user would have to conduct a time-consuming blast search on the Internet in order to insure the uniqueness of thc primers. A typical scries of these earlier time-consuming steps for finding appropriate primers, GC clamps, etc., and melting profiles, is presented in the Flow Charts of Figs. 6A and 6B in connection with the illustrative case of rctinoblasloma genes (RBG), selected as at I , Fig. 6A. After finding the number of cxons and starting with the first (2), the beginning and end locations of the exon arc sought (3). If the number of base pairs is not greater than, foi example, 500 letters (ΛTGC), the exon string is increased by at least, for example, 18 on each end. to create primers (5); but if there arc more than 500 bascpairs, they arc broken up into smaller sections, with overlap as at (4). GC clamp sequences arc added on the header and trailei (6) with shipping and icoidei ing to leave 6 gioups of scpatatcd ATCG sequences, and with a new exon file. Melting profiles aic then tun six times, three with thicc types of GC clamps on ihe leading pi imcr and three on the trailing piimcr, at (7); and then corresponding graph profiles arc generated at (8), Fig. 6B, such as arc shown in Figs. 4 and 5. If the melting profile for those primer sequences produces a satisfactory fiat single domain at (9), as in Figs. 4 and 5, using a search of thc world wide web (www), uniqueness is determined at ( 1 ). If an unsatisfactory multiple or other unsatisfactory domain profile is obtained, however, as in Fig. 3, the primer sequence length and/or GC clamp size (or an added GC clamp) arc varied, or the splitting of thc exon fragment ( I I ). The latter variations arc also pcrfonncd ( 1 I ) if lack of uniqueness amongst the data base is found at ( 10). T his is then followed by reformatting ( 12), i ccording new GC clamp positions, etc., and tc-muning the melting profiles with variation of start/end positions, etc., as required ( 1 ). A determination of substantially the same annealing temperature for the primers, as icquircd for PCR sequencing, is then made ( 14), with variations as at ( 1 1 ) effected if the primer temperatures arc not substantially matched. The melting temperatures arc then calculated from the percentages of urea fotmamidc by the formula ( 15), enabling the making of a melting temperature versus number of ba.scpairs for all cxons complete ( 1 ). Should the present exon of interest be located too near any of the othci s. the vaiiations of step ( I I ) aic employed to piovide greater separation. If, however, there is no crowding, a table entry for the exon, including its numbci, GC clamp location, pi imcr letter code and total length of letter code, and melting temperatures Tm ai c all rccoidcd ( 1 7). If thcic ate more cxons ( 18), the beginning step 3, Fig. 6A, and subsequent steps arc i cpcatcd; othei wisc, the pioccdurc for exon primer dctcimination is completed ( 19). Operation in Accordance With The Invention The computer-aided softwaic-contiollcd micioproccssor programming that underlies the semi-automatic or automatic exon piimci determination pioccdurc of thc picscnt invention, greatly speeds this design process, and with much more facility and flexibility. A semi-automatic vcision is shown in the micropioccssor software implemented Flow Chart of Figs. 7 A and 7B, latci dcsci ibcd, contiolling the computer display screen of Figs. 9 to 14. The screen display formal, as shown in Fig. 9, provides boxes or icons, such as "window" displays, an automatic plotting or graphing display region for simulated dial melting tcmpciatuic piofiles over all the gene fiagmcnts, and an automatically picscnlcd simulated 2-D gene fiagmcnt spot disti ibution presentation (2-D) of thc type attainable by actual 2-D clcctrophoiclic scpaiation, as described in the said Vijg papers. Description Of Display Screen In the lower right of thc screen is the 2-D pattern-distribution display area, labeled "Migration Ch," in which the melting temperature (in %UF) of thc fragments is plotted as a function of thc size of thc fragments. The purpose of this migration chart/pattern is to insuic that no two fiagmcnts will occupy the same place in the pattern, and thus mutations in all of thc fragments will be detectable Above the Migiation Ch atca, undci the title "Gene View", thcic is presented the window for displaying the sequence ol the cntiie gene. The usci can load the gene sequence into the ptogram so that it is latci possible lo scioll thiough the gene in this window lo locate the cxons, as in capital letters, and the nitrous as in lower case lcttcis. The Gene View makes it easy to locate the beginning and end of thc cxons and indicates to the user the total length of thc fiagmcnt (i.e. exon, gaps, primers, and GC clamp). On the left-hand lowci and ccntial portion of the sciccn, there is provided a "melting tcmpciatuic vs base pan offset" mapping aica, and between this mapping aica and the Migiation Ch aica, thctc is a tabular column space in which the individual melting tcmpciatuics Tin of each base pan is punted and scrollable In the uppct center, boxes foi indicating the selected number and fragment and the "exon length" arc piovidcd. defining the selected exon/fiagment. The number the user enters hcic refers to the fiagmcnt fot which the user is designing a test. Sometimes an exon is too long to analyze in a single fiagmcnt and thus must be divided into different fragments. Fot example if exon 3 of a given gene needed to be divided into three fragments, the user would number the fiagmcnts 3.1 , 3.2, and 3.3. These numbers would read exon 3 fragment I , exon 3 fiagmcnt 2, and exon 3 fragment 3. To the left and light of this box, arc windows or boxes labeled "gap", which arc the boxes that tell the user the length of thc gap between the end of thc pnmcr and the exon sequence. The box to the left indicates the gap between the end of thc leading piimci and the beginning of the exon, while the box to the right indicates the gap between the end o thc exon and the beginning o thc ailing pi imcr sequence To the left and right of thc gap-length boxes, arc boxes labeled "leading pi imci" and "trailing piimcr", respectively. The numbcis here repicsent the length of thc piimcrs, usually between 18 and 25 base paiis in length, as bcfoic described. On the left and right of thc pi imcr boxes, Ihcrc arc also provided left and right boxes labeled "GC Clamp". The user can click on these boxes to select whether to place the GC clamp on the leading or trailing pi imcr. Beneath the GC clamp box is the woid "type", referring to the type of GC clamp that best suits a given fragment. The numbci s 1 , 2 and 3 arc assigned to correspond to the cai licr-desci ibcd illustrative 30. 35 and 38 bascpair clamps. Below the primer boxes, arc boxes "anl" for indicating the Tm of thc primer sequences, which arc icfcncd to as the annealing temperatures of thc primers. This function is especially useful in designing the before-described multiplex tests in which the annealing tcmpci atu cs of thc primeis of thc different fragments should optimally be within a few degrees of each other, as cailicr slated. Diicctly under these "anl" boxes, in tuin, further boxes arc provided in which the words "unique" or "not unique" appear. This refers to whether or not the given primer sequence occurs somewhere else in the gene. The function of this is to ensure that each amplicon has a set of primeis which do not share sequence homology with any other 18- 25bp sequence so that only the desired fragments will be amplified. The box that reads "Melt" at the top line of thc display, when selected, commands the computer to calculate the Tin and construct a melting map of thc fragment for the trial parameters which the user has indicated (primer lengths, etc.). Figs. 10- 12. If the melting map is optimal, as in Fig. 12, and the fiagmcnt occupies a unique location on the 2-D migration map, the user clicks on the box marked "Save Primers" at the top-line right. This will save all of thc information concerning a given fragment; i.e. length of primers, gap length, GC clamp type and location. The information will be saved under whichever heading the user selected in the box designated the exon "fragment". The box Reset Primers will reset the computer to the standard conditions of, for example, a 30 bp gap length and 18 bp primer length with no GC clamp. "Restore Primers" recalls saved information concerning a given primer. The user types the exon fiagmcnt information in the exon fiagmcnt box and clicks on the Restore Primer box. If the user types in 3.2, for example, the computer will recall the primer positions, gap lengths, GC clamp location and type of exon 3, fragment 2. Seiui-Automatic Operation Returning, now, to the semi-automatic version of operation in accordance with the invention, the operator inputs or controls, by a conventional "mouse" or the like, the sequencing of thc program. The general procedure for the computer-aided design of optimized genetic screening tests based on such PCR 2-D electrophoresis (and including multiplex operation, as bcfoic discussed) is as follows: Preliminary 1. Retrieve the gene sequence code (e.g., from Genbank). While the gene sequence code is a four-letter code (that is. a particular combination of A\s, T\s. C's and G's). the complete code that specifics a gene can be as long as 100,000 letters. Only small parts, however, each not more than 100-600 letters long, arc considered important for making the test. These parts arc the cxons, and indications arc given as to where they start and where they end in the total letter code Small parts before and after each exon (splice sites) arc also included as well as so-called regulatory regions close to the actual gene or some distance apart. 2. Find the cxons in the letter code or specify other target fragments. 3. Position primers so as to surround the cxons. Such primers, as bcfoic explained, arc small parts of thc letter code, i.e., about 20 letters, that define the boundaries of thc cxons to be tested. Primers should be completely outside the exon. from 5 letters outside it up to 100 letters outside. Where required, as previously explained, it is allowed to split cxons in two: that is, position four primers rather than two, so as to define an exon by two fragments rather than one (for very large cxons even more fiagmcnts arc allowed). In uch case, however, the fiagmcnts must necessarily overlap by about 10 letters so as to assure that the exon is completely covered. At least one of thc two primers defining a fragment should be coupled to a GC-clamp. Melting Criterion 4. Run each fragment defined by its primers on a conventional melting profile generating program such as the before-mentioned Mclt87 (or a derivative) program so as to generate melting curves. Each fragment starts with the first letter of its left primer and ends with the last letter of its right primer; so it includes the GC-clamp. Ideally, each melting curve, as previously explained, should consist of one high-melting domain (the GC-clamp) and one low-melting domain (the exon or part of it); and when this is not the case, the program should tty other primer positions and/or split cxons in half, and/or change the size o thc GC-clamp. and/or add two, instead of one GC-clamp. I'CR conditions 5. Compare the annealing temperature of thc selected primers. They should be almost the same (at least the primers of one pair). The annealing temperatures of primers can be detennined by using the conventional PCR-dcsign program. 6. Compare the primer letter codes among each other and with the total letter code of the gene, so as to avoid overlap with any other than the defined fragments. Two-Dimensional Distribution 7. Distribute the fragments defined by their primers over two dimensions; one dimension being the size (say, left to right) and the other dimension being the melting temperature (say, top to bottom) of the fragment. Avoid clustering, though some is unavoidable. The easiest way to separate clustered fragments is to change their size, which can be done by changing the position of the primers and or changing the size of thc GC-clamp (a 5-10-lcltcr change is usually enough to separate fiagmcnts of thc same size). Select a gradient of increasing melting temperature so as to allow an optimal spread of thc fiagmcnts from top to bottom of thc gel. RBI Example As a first example, the above steps will now be schematically translated into the scheme, programmed under "Windows", for the before-mentioned tumor-supprcsscr gene RBI, a large gene, and with a test design that requires extensive optimization. Like many other genes, the sequence of this gene is available in Genbank from which it may be retrieved and copied into a file to be subjected to the procedure representing the invention. First, the start and endpoints o thc cxons or coding sequences of this gene arc indicated by typing in the respective equences until the program indicates uniqueness on the display. Then, the system starts by assuming 18-mcr primer, gaps between primers, and cxons of 30 bp and a defined GC-clamp of 30 bp long. A melting profile of this configuration as well as the PCR annealing temperatures of thc primers is automatically obtained. Either by observation of thc screen or automatic monitoring; a decision is taken about the suitability of thc melting curve. If not entirely satisfactory, one or more of thc following parameters arc varied: gap-size, primcr-sizc, GC-clamp, until a satisfactory profile and set of annealing temperatures is obtained. If this proves to be impossible, the fragment is split in two or more fragments until optimal melting behavior is attained. Referring more specifically to the previously mentioned Flow Chart of Figs. 7A and 7B, the illustrative RB gene is located (21 ) and file copied (22). The primer window. Fig. 9, is activated (23), injecting a trial primer letter sequence The trial GC sequence is modified at (26), if required. Automatic monitoring can cause variation of thc primer start and end positions as icquiicd (28. 29) and doing so while monitoring uniqueness (30). melting temperature mapping, and display separation at Migration Ch, Fig. 9. If propci trial values arc available, the melting profile genctation is checked (32), and the graph viewed (33) to insuic a satisfactoiy single domain ovci the gene fragments. If there arc no mote cxons (34), the resulting piimcr table for all cxons, including their exon numbers, GC clamp locations, piopci letter codes, total length of letter code, and Tin values arc icportcd by pi int-out (35), completing the test design (36). In the event, however, that a warning is highlighted (31 ) of any of non-unique starting and/or ti iling piimcrs. piimcr annealing temperatures, or spot migration crowding or ovcilap, and/or in the event moic cxons arc present (34), repetition of variation of exon numbers (24) and GC clamp sequences (26) is effected lo provide proper values or conditions. Automatic Version For fully automatic test design where the setting of trial values is effected by known types of feedback control in response to deviation from rules for a standard single domain melting profile, for example, predisposed cxons arc imported in the file header when enabled (42), Fig. 8A, after automatic features (40) and the gene file (41) have been established. As previously discussed, fiagmcnts less than, say, 500 long arc selected (43), and short close cxons arc combined (44). If desired, optimum break points on the melting curve of thc entire gene may be located and/ot unique lengths of fragments selected. Trial piimcis arc then inputted for all cxons (45) which, fiom experience, preferably start, for example, with each equal to 18 base pairs long, and with 30 base pair lengths for split ends The annealing temperatures aic then plotted (46) with selection (47) of thc highest values, and the increase of all other primei lengths to achieve substantially the same annealing tcmpciatui c (48). As before described, the primer lengths arc adjusted (49), Fig. 8B, foi uniqueness, consistent with substantially common annealing tcmpciaturcs - say, foi example, within 5°C of one another. Starting with a fiist exon fiagmcnt (50), a pic-set number (such as six) ti ial melting graphs arc run for three types of trial GC clamps (51 ), and the closest fit to the desired standard single domain piofilc shape (52) is selected (53), with the fit being measured, for example by one of RMs or sigma deviation fiom a icclanglc. If the profile design rules arc not met, an ci lor flag is raised or highlighted. For additional cxons (53), steps (51 ) and (52) arc repeated. If the cxons meet minimum display spicad migration rules at migration Ch, Fig. 9, the design is complete (57). If, however, any cxons violate minimum display spicad migration rules (54), it is determined whcthci another melting profile can be selected to meet the required domain and spot migration spicad rules (55). and, if not, different GC clamp lengths arc tried or an cnoi flag again laiscd (56). P. 53 Gene Example In the successive displays of Figs. 10- 14, another example is presented of thc carrying out o thc proccduics of thc invention for another impairment gene- -the P53 gene Starting with a numbci 5, fiagmcnt I , Fig. 10, for a gene sequence ttcccatcaag ccctagggctcct ("gene view" box), ti ial values of exon length 184, gaps 30 leading and trailing pi imcr lengths 18, and annealing tempeiature of 67.3, and no GC clamp, the multi- domain unsatisfacloiy melting tempcraluie profile was produced at the lower left. Adding a light-hand GC clamp of 30. Fig. 1 1 A. did not adequately impiovc the melting piofilc. Substituting a 30 GC clamp at the left ptovided some impiovcmcnt in reducing the multiple domain excursions. Fig. 1 1 B. Reducing the gap length to 10, Fig. 12, howcvci, ptovided a satisfactoiy flat domain following the GC clamp left-hand peak. As bcfoic described, tables of pi imct sequences arc displayablc with predicted average melting tcmpciatuic as an expanded View Primer Table, Fig. 13: and the design- predicted resulting 2-D clcctrophorctic fiagmcnt spot "Migiation Ch" chart may be displayed and cnlaigcd as in Fig. 14 Othci ariangcmcnts of display boxes and picscntations arc, of course, possible with the same general underlying software piogiamming concepts and methodology of thc invention, and further modifications will occur to those skilled in this ait, such being considcicd to fall within (he spit it and scope of thc invention as defined in the appended claims

Claims: What is claimed is: 1 . A computer-assisted method for generating the design of optimized and comprehensive genetic DNA diagnostic tests based on PCR/2-D clcctiophorcsis for screening mutations in genes, that comprises, inputting a computer microprocessor with a desired exon fragment ATGC letter sequence; programming and controlling the microprocessor first to select a trial start and cud of thc sequence: then, selecting and positioning trial primer letter pairs to define the opposite boundaries of thc cxons to be tested; adding a trial GC clamp to at least one primer; gcnciating trial melting profiles for the trial primers and GC clamp to determine whether the profile consists of a relatively high-melting GC clamp peak and a substantially single flat domain of relatively low- melting value for the fragments; in the event that the trial profile has multiple and not a satisfactory single flat domain, selecting one or both of other trial primer sequences and GC clamp sizes and gcnciating said profiles therefrom until such a satisfactory single flat domain is attained, and for primers of each pair having substantially similar annealing temperatures suitable for PCR annealing; comparing the primer letter sequences with other known gene sequences in the context of thc total letter sequence of thc gene exon to insure the uniqueness of thc sequences and to avoid overlap with any other than the selected gene fiagmcnts; simulating a 2-D distribution of thc fiagmcnts defined by the primer pairs by separation along one dimension by size and along an orthogonal dimension by melting temperature of thc fragment: selecting a giadicnt of increasing melting temperatures along the orthogonal dimension to allow an optimal spread of thc fragments thcrcalong: and reporting the selected primer sets and temperature gradient, thereby providing an optimal design for an actual PCR/2-D clcctiophorcsis test. 2. A method as claimed in claim I and in which, in the event of indicated clustering of the fragments along the one dimension, changing their size by changing the position of thc primers and/or changing the size ofthc GC clamp to separate fiagmcnts of similar size 3. A method as claimed in claim I and in which the sequence of thc gene is obtained from a data base such as Genbank and is copied into a file for use in carrying out said method. 4. A method as claimed in claim I and in which, if a trial melting profile is obtained that docs not have a satisfactory single domain, varying one or more of primer size, gap size between cxons. and GC clamp size until a satisfactory profile and set of annealing temperatures is obtained. 5. A method as claimed in claim 4 and in which the varying of thc size of thc GC clamping size is effected by using two instead of one GC clamp. 6. A method as claimed in claim I and in which the annealing temperatures of thc primers arc determined by a PCR-dcsign program. 7. A method as claimed in claim 1 and in which, if a satisfactory melting profile is not obtained, the exon fragment sequence is split into parts. 8. A method as claimed in claim 7 and in which the exon fragment sequence is split in two with two pairs of primers so as lo define the exon by two fiagmcnts with some letter ovci lap so as to assure that the exon is completely covered. 9. A method as claimed in claim I and in which the primer pair letter sequences that define the boundaties of thc cxons to be tested arc positioned outside the exon letter sequences and arc small compaicd with the exon letter sequences. 10. A method as claimed in claim 9 and in which the primer letter sequences arc of thc older of about 20 letters. 1 1 . A method as claimed in claim 9 and in which the piimcrs arc positioned from about 5 to up to about 100 letters outside the exon Icttci sequences. 1 2. A method as claimed in claim I and in which the melting profile is displayed to a monitoring operator on a computer display screen, and the selecting of each of start and end of thc exon fragment letter sequences, primer pair letter sequences and GC clamp arc manually entered by the operator and displayed on the screen. I 3. A method as claimed in claim I and in which the characteristics o thc melting profile aic automatically monitoicd, and start end Icttci sequence primer pair letter sequence and GC clamp arc automatically varied as required until a predetermined satisfactoiy single domain melting profile is attained. 14. A method as claimed in claim 1 and in which the fragment distribution is displayed on a computer display screen. 15. A method as claimed in claim 14 and in which, upon an opciator monitoring Ihc fiagmcnt disttibution display screen and noting undue fiagmcnt clustering, the operator cntcis vai iations in one oi moic of thc position of thc piimei and the size of thc GC clamp to scpaiatc the fiagmcnts and to display the changes on the sciccn. 16. A method as claimed in claim 14 and in which the fiagmcnt distribution is automatically monitored, and upon detection of undue fragment clustering, vaiying one or mote of thc position of thc pi imcrs and the size of thc GC clamp to separate the fragments. 17. A method as claimed in claim I and in which the tcinpcratuic gradient distribution is displayed upon a computer display sciccn. 18. A method as claimed in claim I and in which an actual PCR/2-D clcctiophorcsis is caπ icd out with an actual gene coiicsponding to said desitcd gene using the reported primei sets and temperature gradient. 19. A method as claimed in claim 18 and in which the actual PCR operation is carried out after adding primer paiis to successive groups of thc gene cxons by effecting longdistance PCR amplifications thcieof in a common tube ot vessel to achieve relatively long resulting amplicons; then adding further primer paiis to each of thc gene cxons and effecting multiplex PCR amplifications thcieof in the common tube or vessel with relatively short resulting amplicons: and then 2-D clccltophoictically scpaialing the gene fiagmcnts. 20. A method as claimed in claim I and in which the leporting comprises visual display upon a computei display screen, under the control of thc programmed microprocessor, of: a) the simulated profiles of thc melting temperature for the gene fiagmcnts: b) 2-D maps of thc simulated melted fragments: c) a table of thc primer sequences; d) readout of thc annealing temperatures of thc primers: c) verification of thc uniqueness of thc ptimcr-gene sequences; and 0 continuous display of thc gene sequence during the whole procedure 2 1. A method as claimed in claim 20 and in which the melting temperature is presented as a percentage of urca-formamide 22. A computer-assisted method for generating the design of optimized and comprehensive genetic DNA diagnostic tests based on PCR/2-D clcctiophorcsis for screening mutations in genes, that comprises, programming and operating a computer microprocessor to input a desired gene exon fragment ATCG letter sequence: indicating the start and end ofthc desired sequence; generating simulated optimal conditions for PCR primer annealing sequences generating an optimal electrophoresis 2-D fragment separation simulation including a simulated melting profile by vaiying trial fragment sequence size GC clamp sequence size and primer sequence size to achieve a single domain profile of relatively low melting temperature for the gene fragments, clamped by a relatively high melting temperature sequence; and reporting the selected primer sets for being synthesized. 23. A method as claimed in claim 22 and in which the reporting comprises displaying said primer and GC clamp sequences, melting temperature values and 2-D fiagmcnt size and melting temperature profile distribution on a computer display screen. 24. A method as claimed in claim 23 and in which the sequences and said values aic displayed in window or box type displays.. 25. A method as claimed in claim 23 and in which the letter size variations aic effected by an operator viewing the trial values in window or box type displays on the display sciccn. 26. A method as claimed in claim 22 and in which the said vaiying is effected automatically in response to prc-sct melting profile characteristics. 27. A method as claimed in claim 22 and in which the variations arc effected in accordance with the steps of thc Flow Charts herein. 28. Computer-aiding apparatus for generating the design of optimized and comprehensive genetic DNA diagnostic tests based on PCR/2-D electrophoresis for screening mutations in genes, having, in combination, a computer microprocessor being softwaic-piogtammcd to effect the steps of method claim 20. 29. Computer-aiding apparatus as claimed in claim 28 and in which the steps of thc programming include those of method claim I .

Inventors: BISHOP ROBERT, VIJG JAN, BISHOP, ROBERT, VIJG, JAN
Applicants: BELTRONICS INC, BISHOP ROBERT, VIJG JAN, BELTRONICS INC., BISHOP, ROBERT, VIJG, JAN
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1