The PCR mixture for ds-amplification in one 96-well plate contained following reagents at final concentration: 1 x Tfl-PCR buffer (Epicentre Technologies Co., Madison, WI), 3 mM MgCl2, 210 mM of each of four dNTP, 0.4 mM of both M13 extended sequencing primers (5'-GGGTTTTCCCAGTCACGACG-3' and 5'-CACAGGAAACAGCTATGACG-3'), and 0.3 U/25 ml reaction of Tfl-polymerase (Epicentre Technologies Co., Madison, WI). Into each well containing 25 ml PCR-mixture and 25 ml mineral oil were transferred 2-4 ml of cell suspension from 25- to 50-fold-diluted master-plate cell suspension using 1-mm metal pin array (3 repetitions of the transfer procedure per each plate) (Figure 1, A and B). The 864-well plates contained 10 ml PCR-mixture and 5 ml/well mineral oil per well. Each well was inoculated by 3 transfers using 0.5-mm pins directly from the master-plates cell suspension. For single-strand PCR-amplification were used about 4 ml from a 25-times diluted overnight cell suspension transferred by 3 procedures with 1-mm pin-array or 10 procedures with 0.5 mm pin-transfer and only one of the primers (figure 1 C). All PCR amplifications were performed in a BioOven II and a BioOven III (BioTherm, Fairfax, VA) under the following conditions (for 6-10 plates):
         For 96-well plates: For BioOven II - initial denaturation of 5 min at 94 °C; followed by 27 cycles of denaturation (1.5 min at 94 °C), annealing (0.5 min at 48 °C), and elongation (2 min at 74 °C), and a final elongation of 5 min. For BioOven III - initial denaturation of 5 min at 96 °C; followed by 25 cycles of denaturation (2.5 min at 94 °C), annealing (2.5 min at 48 °C), and elongation (2.5 min at 74 °C), and a final elongation of 5 min at 72 °C.
         For 864-wells plates: initial denaturation of 5 min at 96 °C; followed by 25 cycles of denaturation (5 min at 96 °C), annealing (4 min at 50 °C), and extended elongation (3 min at 78 °C), and a final elongation of 5 min at 74°C.
         If 96-well plates were used for PCR-amplification, 10 ml of the amplified fragment mixtures and 5 ml of oil were transferred by robot into the 864-wells plate before the membrane spotting procedure. Moreover, all plates were bar-coded and all protocol and stock-storage information automatically was transferred in computer databases. Finally, a PCR-amplified inserts' library was created and stored at -20 °C. The top oil layer was created to prevent the liquid evaporation during spotting and (especially) during freezing /melting cycles.

High density array membrane preparation.

         Two Watmann 3MM layers were rinsed in 2 X denaturation solution (DS; 3 M NaCl, 1M NaOH) and then deposited onto a plastic frame (Figure 2). A GeneScreen nylon membrane (DuPont NEN, Boston, MA) wetted in distillated H2O was rinsed in 2x DS for 30 s. After complete-liquid removal, the membrane was deposed onto Watmann layers without air bubbles on the bottom side. Each membrane-replica was labeled with a code name (consisting of the membrane-type signature and membrane-replica number), and the corner nearest the first-spotted DNA-clone was also marked. The membrane was dried slightly under ventilation for 5-10 min. Then the frame with the membrane was mounted onto the robot platform and a spotting procedure was performed using a Biomek 1000 (Beckman Inc., Fullerton, CA) adapted for this purpose (I. Labat, unpublished information). The job-file was transferred into the database. An 864-pin array (0.3 mm pin-diameter) was used for spotting from an 864-well plate onto the membrane by 8 or 10 spotting manipulations on each clone-dot. The spotting procedure for one high-density membrane (31,104 dots) took about 80 min. After spotting, each filter was neutralized in 50 mM sodium phosphate (pH 7.2) for 20 min, dried under ventilation, and baked at 60-80 °C for 60 min. A 30-min UV exposure on low-power (Transilluminator TS-40; UVP, Inc., San Gabriel, CA) provided optimal cross-linking. Membranes were stored between polyethylene shields and only before the first round of hybridizations they were prehybridized in hybridization buffer for 5 min at room temperature.

DOT-hybridization.

         The radioactive labeling reaction (20 ml volume) contained following: 9 ml of corresponding oligonucleotide solution (10 ng/ml), 20 U of T4 polynucleotide kinase (NE BioLabs, cat. # 201L, 1994), 2 ml of 10x kinase buffer, and 60 mCi g-33P-ATP (Amersham Life Sci. or DuPont NEN) for oligonucleotides with 0-1 G/C content or 30 mCi for oligonucleotides with 2-6 G/C content, or 15 mCi for oligonucleotides with 7 and higher G/C content. Reaction mixtures were incubated at 37 °C for 60 min and kinase activity was destroyed by incubation at 60-65 °C for 10 min. The labeling efficiency was detected by deposition of 0.5 ml of labeling mixture on PEI paper (E. Merck, Darmstadt, Germany; cat.# 5579., 1992) and chromatography for 30-50 min in 0.5-1 M KH2PO4. A last modification gave preference to the use of a GF/C glass membrane (Wathmann LabSales Hillsboro, Oregon) as the chromatography carrier because it allows a much higher speed (about 30 s) and absolutely strong separation of labeled DNA/non-incorporated 33P-dNTP signals (Figure 5).
         The hybridization mixtures were prepared by addition of all 20 ml of the labeling mixtures into 3-7 ml of hybridization buffer. All arrayed membranes were initially prehybridized for 5 min in hybridization buffer contained 7% sodium lauroylsarcosine and 0.3 M sodium phosphate, pH 6.8-7.5. Then the access of liquid was removed from each membrane my pressing it between was placed between Wathmann sheets. Membrane was prepared for hybridization by placing it between two polyethylene sheets (Fisher Sci. cat. # 01-812-10, 1994). If necessary, two membranes were placed back-to-back between the same sheet, so that each DNA-containing side of both membranes was free to probe access. Then each hybridization mixture was placed between the two nylon shields so that after closing at the shields, the capillary force distributed the mixture evenly on the top (DNA-containing side) of each membrane; repeated peel-off-&-close improves initial probe distribution. In our case all 20 filter-replicas were arranged as a sandwich [filter membrane(s) between nylon shields/Watmann separating shield/filter membrane between nylon shields] into a plastic box for radiation safety. Then the hybridization was performed in a refrigerator at 8-13 °C. This experimental design allows hybridizing up to 100 probes (each probe with two filters) per person per day in 2-3 plastic boxes. The hybridization was performed for 1-1.5 h at 8-13 °C for oligonucleotides with more than 1 G/C content and at 0-3 °C for oligonucleotides with zero G/C content.
         After hybridization, each membrane was rinsed three times in cold (0-5 °C) 6xSSC buffer (500 mL of SSC for passing of 20 filters) in a plastic box and transferred into another box with 1 L of 6xSSC, where all 20 membranes were collected before washing. Each membrane was washed for a different length of time in 100 mL of 6x SSC at 10-15 °C (for 1 and more G/C contained oligo) or at 0 °C (for zero G/C contained oligo). Because the strength of hybridization depends on many factors (see discussion), the washing process for each filter was controlled by a Geiger-counter: the optimal wash was detected when the total sector-radioactivity was in 50-150 cpm per 50-mm-diameter sector, measured 10 mm from the membrane surface. The washing-time data were collected in a data file so that the next experiments with the same probe(s) did not need monitoring of counts.

Scanning.

         After removal of the washing solution by pressing the membrane between Watmann shields, membranes were exposed in a Phosphoimager cassette at 5-15 °C for 6-12 h and then were scanned on the Phosphoimager SP (Molecular Dynamics, Sunnyvale, CA). Screen images were transferred into a database for further processing using our image analysis program DOTS (J. Jarvis), (6).

Removal of the hybridized oligonucleotide probe from the targets and membrane preparation for the next hybridization.

         All 20 membranes were immersed one by one in 500 mL of hybridization buffer and incubated at 65 °C for 2 h with slow shaking. After washing twice in 1 L of 6x SSC for 2 min, membranes were stored in plastic box with 100 mL of 6x SSC at 4 °C until the next hybridization. Membranes can be reused over 40 times.

RESULTS

         A new experimental design for recombinant DNA clone separation, inserted fragments amplification, high-density membranes preparation and DOT hybridization was developed, basic variant of which has been published before (Drmanac,S & Drmanac,R., 1994; Grujic et al., 1994). Here is presented a detail explanation of a significantly modified and optimized procedure, which guarantees up to a 10-fold increase of sequencing data production, decreases the time for DOT membrane preparation, decreases hybridization- and scanning time by half, and gives a more precise interpretation of the most important experimental steps than has been given before.
         The separation of an individual recombinant clone from a DNA library is the slowest procedure in a large-scale library analysis. In our laboratory, two techniques were routinely used: a manual colony picking with single-well inoculation and a precise limiting dilution of the cDNA-library such that in 10 or 20 ml of diluted library cell culture there was only one bacterial cell. The second procedure is very convenient and much faster, but generates about 20-25% non-precise clone separations (empty wells or two different clones in the same well). The previous data (Drmanac,S. & Drmanac,R., 1994) for the same level of negative results for manual picking (about 25%) have to be reconsidered because so high a percentage of reported negative PCR-reactions can not be generated as a result of the picking procedure, which can reflect only on the quality of clone separation and grow. We detected about 97% acceptable positive PCR-amplifications from picking separated clones and only 70-80% of acceptable results in case of limiting dilutions (figure 1; A and B). One explanation of the previously obtained result is the lower antibiotic concentration, which allows growth of semi-positive recombinants - in fact cell clones with absent plasmid. The usual antibiotic concentration for ampicillin resistance library screening is 100 mg/ml, and it is known that lower concentrations can generate false-positive recombinants, which in case of color selection can be eliminated, but will persist if only the limiting dilution technique has been used. A further improvement of the clone separation procedure can be realized by robotization (Jones et al., 1992; Meier-Ewert et al., 1993; Uber et al., 1991) or by construction of special vector. A cDNA library preparation procedure, based on Stratagene's Lambda-ZAP Express vector, can provide 99% or more recombinant clones (Dyanov, 1995). Analogous to the above is the situation of the results from Tfl-polymerase and Taq-polymerase PCR amplification and primer/dNTP-mixture concentration. Most of the information published by suppliers show that Tfl-polymerase is much higher processive, but less thermostable polymerase than Taq-polymerase. So, about 0.2 units of Tfl-polymerase produced the same amount of amplified product as 1 U of Taq-polymerase. In addition, Tfl-polymerase has slightly higher exonuclease activity. Because of this, the Tfl-polymerase was chosen for fragment amplification for SBH, but it can be replaced by Stratagene Taq-polymerase in the case of 864-well plate PCR, because the cycling time (of some thermocyclers) is too long, Tfl-enzyme stability is lower, and the exonuclease activity is too high (figures 1B and C). A decrease of the cycle time and a Tfl-concentration increase has compensatory effect. The optimal known concentrations of the dNTPs and primers are correspondingly not less than 200 mM and not less than 100 mM (for Taq-polymerase) or 0.400 mM (for Tfl-polymerase). The optimal conditions are library and single colony growth at ampicillin concentrations of 100 mg/ml (higher concentrations can depress the growth of some slowly geminated clones), overnight growth with intensive shaking (300 rpm for 96-well plates) or on a custom vibration platform (for 864-well plates), and Tfl- or Taq-polymerase-mediated insert amplification. The robotics-based inoculation of the PCR mixtures was performed by five transfer manipulations with a 0.3-mm pin-array directly from the overnight cell cultures (master-plates). In addition, optimal cDNA-clone storage and re-use can be occurs at -70 °C in the presence of 20-50% glycerol.

Figure 1. PCR-amplification pattern of different infant brain cDNA clones, grown and amplified in conditions as follows: A - clones, separated by the limiting dilution procedure, grown overnight in 96-wells plates and PCR-amplified in 96-wells plate (Tfl-polymerase); B - clones separated by individual clone picking (ICP) and then proceeded as in A.; C - clones separated by ICP, grown and PCR-amplified in 864-wells plate (Taq-polymerase, Stratagene); D - clones separated by ICP, grown overnight in 864-well plate and PCR-amplified in 96-wells plate; E - the same clones as in D., but amplified in 864-wells plate after a 5 times transfer into the PCR-mixture by 0.5 mm metal pin. F - clones separated by ICP, grown overnight in 864-wells plate and ss-PCR-amplified in 96-wells plate after a 10 time transfer into the PCR-mixture by 0.5 mm metal pin. Two microlitters of each PCR-amplification mixture were loaded into a 1% agarose gel and separated in 1 x TAE buffer at 5 V/cm power. M - indicates position of the molecular weight marker (l phage DNA/BstE II digested); - -> + indicate the direction of fragments movement.

       The use of a plastic frame (Figure 2) for GeneScreen membrane deposition before the spotting procedure increased both the speed and convenience of this procedure. The position of each membrane was fixed and, because of this, it was easily dried, marked, deposited onto the platform, and finally cut to an appropriate hybridization size. This modification was very important because of the very high density of membrane array. Moreover, at the time of spotting was possible to prepare another membrane in a second frame, which saved about 30 min (for 10 membrane replicas, this amounts to about 5 h of working time). In a test (data not shown) we found that minimum of 10 single spots on each dot produce the best reproducibility of hybridization results. Such option allows preparation of at least 50 filter replicas from each 864-well l of mineral oil. The oil additionl of PCR-mixture and 5 µplate containing 10 was designed to prevent evaporation of the PCR mixture in long-term storage, C is recommended forfreeze-defrost cycles, and spotting proceeding. Minus 20 master-plate storage; it is important that the storage temperature be above the oil freezing temperature, because if the oil freezes, evaporation (freezing sublimation) can not be avoided. The high hydrophility and adsorption reactivity of the membrane prevent the undesirable negative influence of the oil layer on the DNA binding to the membrane. Moreover, the oil layer formed onto the membrane surface decreases the evaporation during approximately 1 h of spotting (data not shown).

Figure 2. GeneScreen membrane preparation for spotting. RP - indicate the BIOMEK 1 000 robotics platform; PF - indicate the plastic frame for a double 3MM Wattmann (DW)/GeneScreen membrane (GM) "sandwich" deposition; C - indicate position of the marked membrane corner; MS - the position of membrane signature; 1 - position of the first-dotted DNA-insert; DS - denaturation solution.

Figure 3. Attachment of mixed oligonucleotides to different type membranes. On A to F six different oligonucleotides (5 nM each) were labeled with NEBioLabs T4 Polynucleotide kinase in a total volume of 20 ml and 1 ml of each labeling mixture were deposed onto different type membranes as follows: A, D and E - GeneScreen™ membrane, C - PEI paper, B and F Whatmann GF/C glass membrane. All membranes were first chromatographed in 1 M KH2PO4 - A, B and C. D - membrane was backed at 80°C for 1 hour and then UV-cross-linked for 20 min. and F - non-backed, non-exposed on UV light. G and H presented a Follows membrane with deposed mixture of 20 non-self-complementary oligonucleotides diluted as follows: 50 nM, 10nM, 1 nM, 100 pM, 10 pM (positions from 1 to 5); 1 μl of each dilution mixture was deposed onto membrane. After backing (at 80°C for 50 min) and UV-exposure (for 20 min) they were hybridized for 3 h with 2 different oligonucleotides (NTCATCCANN and GCTCATCGTC), complementary to a corresponding oligonucleotides fixed to the membrane (NNGGATGANN and NTGGTGATGN) D, E, F, G and H were washed two times (20 min each) in 6xSSC at room temperature. The right-sides of fragments G and H shows the results after a measurement of hybridization intensities (numbers 1, 2, 3, 4, 5) - shown is the relation cpm/probe position.

          Several experiments have proven the ability of the GeneScreen membrane to a stabile attachment of mixed oligonucleotide probes (figure 3), which are needed for designing approach to exact quantitative measurement of the probe hybridized to the targets in order to be utilized in future cDNA high-density membranes - several serial dilutions of oligonucleotide mixtures (of non-self-complementary oligonucleotides) and/or longer DNA-fragments with known concentration spotted on the array as concentration measuring tags. A quantitative measurement of the labeled probe, positively hybridized to them can be used as a mass-probe to measure and calculate concentration of the target sites on each clone-dot for any particularly used probe in any particular hybridization experiment - in addition to the relative clone-dot concentration measurement by the mass-probe, complementary to the vector-belong regions of each insert. Such approach will decrease the influence of the fortuitousness in all different hybridization experiments, as well as it allows more precise calculation of the total number of target-sites on any clone for any particular probe.
         In addition, it was detected (data not shown) that a rinse of GeneScreen membrane in NaOH-denaturation solution before the spotting significantly (about 10-fold) decrease the amount of attached oligonucleotides compared to the results obtained on membranes, non-treated with NaOH. The result can be logically explained by a significant decrease of the original positive membrane charge after the hydroxide treatment, which by itself decreases the membrane ability to bind stabile the DNA-molecules. In this sense, more optimal will be to perform clone DNA-denaturation after DNA-fixation onto the membrane because it will allow higher amount of DNA to be attached.
         One high-density membrane was prepared containing 31,104 dots, 27,500 of which represents inserts, PCR-amplified by our several students (see acknowledgements) from a normalized (subtracted) infant brain cDNA library. The membrane pattern was specially designed to allow obtaining of information for hybridization data representativity and reproducibility, equal gradient of probe distribution onto the membrane as well as measurement of hybridization background and computational trustworthiness. By these reasons the membrane pattern contains several empty positions, a duplication of all clones and also duplications (in a sum of 200) of several control DNA-sequences, covered in overlapping order a part of known cosmid sequence (Pizzuti et al., 1992). Twenty replicas of a same pattern were prepared by a Biomek-1000 (Beckman Instruments, Fullerton, CA, USA) robot and our modification of the spotting program. Ten replicas contained 8 spots on each dot and another ten have 10 spots on each dot; all performed by a 0.3 mm metal pin-array, designed to spot simultaneously from all 864-wells of each plate (figure 4-A). All membrane replicas were hybridized with a mass-probe, complementary to the vector-specific linker part of all inserts, in order to measure DNA-concentration relations between all dots, as well as the positions of accidental DNA-absence at some dots. A large-scale hybridization experiments were performed with about 270 specific oligonucleotide probes with sequence formula N0-2OLIGO6-10N0-2 (where N is a random dNTP and "OLIGO" is a 6-10-mer oligonucleotide). About 450 hybridizations were performed in total and about 370 of them delivered high quality result (figure 4. B, C and D). Only one membrane image for each hybridized probe was choused for the computerized analysis.

Figure 4. Dots array pattern on a high-density membrane. Only 1/4- part of the original membrane (22/15 cm) is shown. Position A shows a membrane pattern marked by a 5 times spotting onto each dot; methylene blue was used as a color marker. Position B shows a membrane image of a hybridization result after hybridization with "mass" probe (ACGACGGCCA), hybridized to a target site, presented once in each of the inserts (complementary to a vector-belonging site). Such results were used for a computational measurement of molarities of all dotted clones and calculations of all hybridization results. Position C shows a test-membrane, used for checking probe distribution as well as reproducibility of hybridization results. By using the same membrane pattern inserts were repeatedly spotted onto 8 dots- positions, forming a "square"; the middle of each "square" consist of a empty-dot as a background control. Position D shows a high- quality hybridization result with the E-38 oligonucleotide probe (NCTGCGCAN). On positions B and D are shown images of a membrane with 31 100 dots representing a normalized infant brain cDNA-library, about 200 control clones with known sequence and 3 456 control empty-dots.

         The oligonucleotide probe labeling was performed according to the procedure, presented in the above paper section. Several testing experiments showed that the most important for a high-efficiency probe labeling is the origin of the T4 Polynucleotide kinase. The previously used enzyme from Promega shows higher variation level in its quality and as a result - very high variability in the obtained labeling efficiency. The enzyme supplied from USB showed efficiency lower than the worse results obtained with Promega's enzyme. The enzyme from NEBioLabs is highly recommended as a chipper one, as well as a kinase, which produce very high labeling efficiency in more than 1 000 labeling experiments (figure 5). The labeling efficiency, detected by PEI-paper chromatography showed some difficulties in the correct separation of the labeled probe signal from non-incorporated label seemly due to insufficient oligonucleotide attachment (figure 5 A; “splashes” at area 1 ). Different membranes were tested in their ability to separate precisely the labeled probe from the non-incorporated dNTP (figures 3 and 5). The GeneScreen membrane (figure 3 A) showed a best ability to bind oligonucleotide DNA and to separate the signal without a loss of the signal intensity as a result of membrane thickness, but, because of it less capillary power, the chromatography time was very long and makes it inefficient for the above purpose. The GF/C glass membrane was choused as a best chromatography carrier, which demonstrated both excellent separation and extremely high speed of the chromatography process - only about 15-30 seconds to separate incorporated label from the non-incorporated dNTP (figure 5 B).

Figure 5. Chromatography of T4 Polynucleotide kinase mixture in 1 M KH2PO4: A - shows a separation onto the PEI-paper. B - shows a separation onto the GF/C glass filter. 1 - indicate the labeled oligonucleotide area; 2 -33P-ATP. g - indicate the area of non-incorporated

         All work, from membrane-replicas preparation to hybridization results data storage were performed by single investigator in only two months with a half-use of all lab-equipment capacity. The obtained experimental results showed, that the washing time at a fixed temperature in the range of 10-15°C can vary (for some probes - significantly) from the previously reported calculation type (Drmanac,R., 1990). The nature of the basis events of such observations is not very clear, but most of them are probably connected to the self-complementation and some level of self-aggregation of the oligonucleotide probe in the hybridization solution and conditions. The final experimental data showed that this did not reflect on the quality of miss-mach/full-mach hybrids discrimination if an optimal washing time was chosen. The apparent uncomfortable need of monitoring of each hybridization is necessary only as initial act of the first-step hybridization with each new probe, but in fact it serve as a basis for highly reproducible results in the further experiments using the same oligonucleotide probe.
         Several measurements of the membrane images, obtained from different exposure times of the same hybridized membrane gives a reason to propose that a longer exposure allows better hybridization discrimination of full-matches from the miss-matches as a result of the logarithmic amplification nature of the detected radioactive signal (figure 6). The high importance of this observation is that it allows for much more discriminative rank-scaling of the hybridization data, essential for the precise and reproducible computerized data analysis, according to the previously reported algorithm (21).

Figure 6. Membrane images of a high-density nylon membrane array (31104 dots; 1/2-part of the image presented) obtained after hybridization with A-18 oligonucleotide probe (GCTCATCGTC) and the corresponding diagrams, presenting the cpm/clone-position relation of a measurement of a part of one membrane row from each image. Image A shows a membrane image after 4 h exposure at 4°C; image B - after 14 h exposure. All numbers indicated show some of the areas with highly positive cDNAs hybridized with the target oligo A-18. K indicates 1000 cpm. The grid and small points were created after a computational measurement and optimization of the image by DOTS program - for a better dots'-position visualization and separation.

DISCUSSION

         Practically all procedures of the SBH-methodology (Drmanac, R. et al, 1994), were modified - some of them significantly. As a result was created very compact procedure for large-scale library analysis and characterization by oligonucleotide hybridization. The time- and efforts consuming on any particular methodological step, were decreased significantly and, finally, all data-production cycle was performed twice as quicker, with 3-5-fold data production increase. The reported here procedures were precisely tested and a small-scale experiment was performed with 20 membrane replicas each containing 27,500 cDNA clone inserts from human infant brain cDNA library. In a large scale variant this methodology will allows at least 10-fold increase of sequencing data production.
         In the reported here new approach the hybridization was performed in a plastic (polyethylene) shields. This modification demonstrates several advantages. First, it requires a minimal volume of hybridization mixture. We hybridized a large-size high-density membrane (22/15 cm) with only 3-4 ml of oligonucleotide containing hybridization mixture; in a case of simultaneous use of 2 membranes, each probe was diluted in 5-7 ml of hybridization buffer. Second, because of the minimal volume (of an extremely high probe concentration, more than 20 times higher than the previously used), the capillary forces distribute probe equally in a very thin layer at a very high local concentration over the clone-containing membrane side. This provides an equally distributed hybridization signal, respectively - a high quality and reproducibility of hybridizational- and computational results. The final data demonstrated more than 85% of high-quality hybridization results compared to the 50% good hybridization images obtained by the “standard” proceeding (data not shown). Third, the higher probe concentration produced much higher hybridizational signal for the same standard exposure time and as a result - higher discrimination between the positive and false-positive inserts as well as more precise rank-scaling. Fourth, because of the nature of the hybridization kinetic, this approach decreased the time of the hybridization incubation from a minimum 3 hours (required for the previously reported proceeding) to minimum one hour incubation. Finally, the design of the reported here method allows a simultaneous hybridization of 1-2 membranes with up to 100 probes per day per 1-2 experimentators incubated in a single box in refrigerator and washed in the boxes with 1-2 L of 6 x SSC contained ten or more membranes in the same box. The simultaneous washing practically did not allows cross-hybridization because of the very high range of probe dilution in the washing conditions (300-600 times) - as a result of an extremely delay of hybridization kinetic and, respectively, extremely extended time of a possible expected cross-hybridization. Practically we did not observed such cross-hybridization in all cases when 10 filters were washed in the same box with 1 L of 6xSSC.
         The presented here technique is a higher data-generation productive. It is optimally designed procedure for recombinant cDNA-clones collection, storage, growth, PCR-amplification, dot-spotting, hybridization and phosphor-image creating, most of them robotically performed. In the primary experiment, only one researcher was able to produce hybridization data on 20 filter replicas with 20 oligonucleotide probes per day with a half-use of the existing lab equipment. This corresponded in practice to about 622,080 single dot-hybridization results or about 250,000-1,000,000 sequenced bases per day. I propose that in a further large-scale experiment one person will be able to produce up to 3,110,400 single dot-hybridization data or up to 2-5 million bases per day. The author believed, that the reported here and practically approved methodology (Dyanov & Salbego, 1995; Milosavljevic et al., 1995) can be used successfully and routinely in the SBH-practice. The high quality of the final computer analysis results and the complete cDNA-library characterization, which will be published soon elsewhere, also supported this expectation.

AKNOWLEDGMENTS

         I would like to acknowledge Dr. R. Crkvenjakov for supervising this work in part and for critical suggestions, Dr. M.B. Soares for providing the infant brain cDNA-libraries and R.A. Gibbs (25) for providing the already sequenced clones covered the human distrophin gene used as a controls in our experiments, Z. Strezoska, M. Zeremski, T. Paunesku, D. Grujic, S. Batus, K. Nadas, S. Little, H. Kreuzer and A. Gemell for cDNA clone separation, growth and inserts' PCR-amplification, I. Labat for developing robot-, data storage- and analysis programs, J. Jarvis and A. Milosavljevic for developing data storage- and analysis programs, and David Nadziejka for editorial assistace.
         Work supported by the U.S. Department of Energy, Office of Health and Environment Research, under contract No. W-31-109-ENG-38.

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