The development and application of automated gridding for efficient screening of yeast and bacterial ordered libraries

GENOMICS

12, 534-541

(1992)

The Development and Application of Automated Gridding for Efficient Screening of Yeast and Bacterial Ordered Libraries D. R. BENTLEY, C. TODD, J. COLLINS, J. HOLLAND, I. DUNHAM,

5. HASSOCK, A. BANKIER,*

AND F. GIANNELLI

Division of Medical and Molecular Genetics, United Medical and Dental Schools of Guy’s and St. Thomas’s Hospitals, 8th Floor, Guy’s Hospital Tower, London Bridge, London SE1 9RT, United Kingdom; and ‘Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, United Kingdom Received

July 1, 1991;

revised

An automated gridding procedure for the inoculation of yeast and bacterial clones in high-density arrays has been developed. A 96-pin inoculating tool compatible with the standard microtiter plate format and an eight-position tablet have been designed to fit the Biomek 1000 programmable robotic workstation (Beckman Instruments). The system is used to inoculate six copies of 80 X 120-mm filters representing a total of -20,000 individual clones in approximately 3 h. Highdensity arrays of yeast artificial chromosome (YAC) and cosmid clones have been used for rapid large-scale hybridization screens of ordered libraries. In addition, an improved PCR library screening strategy has been developed using strips cut from the high-density arrays to prepare row and column DNA pools for PCR analysis. This strategy eliminates the final hybridization step and allows identification of a single clone by PCR in 2 days. The development of automated gridding technology will have a significant impact on the establishment of fully versatile screening of ordered library resources for genomic studies. Q 1992 Academic Press, Inc.

INTRODUCTION

The isolation and analysis of yeast or bacterial clones from high-complexity genomic or cDNA libraries are essential parts of the study of molecular genetics in humans and other organisms. Traditional screening procedures have involved the hybridization of radioactive probes to filters prepared from libraries plated at random (Grunstein and Hogness, 1975; Gergen et al., 1979). Subsequent isolation of individual clones has required repeated rescreening and colony or plaque purification to isolate the clone or clones of interest for further studies. As the interest in studying large numbers of clones that represent, for example, chromosome regions or multigene families has increased, the rate-limiting rescreening step has become prohibitive to the isolation of sufficient numbers of clones for large mapping projects. To eliminate the need to purify each clone after identification, some studies have involved the transfer of individual clones into microtiter plate wells directly after construction of the library (Coulson et al., 1986; Olson et osss-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

534

October

3, 1991

al., 1986; Kohara et al., 1987; Evans and Lewis, 1989; Nizetic et al., 1991). The investment of time and labor in transferring every clone was compensated for by the elimination of multiple rescreening steps. Screening of ordered libraries of high complexity by hybridization has been severely compromised by the difficulty of manually preparing filters containing all the clones. One alternative that has been used with yeast artificial chromosome (YAC) libraries has been to prepare DNA from pools of clones and screen them by the polymerase chain reaction (PCR), so that only a small number of clones from the library (corresponding to a positive DNA pool) are stamped out manually and analyzed by hybridization in the final step (Green and Olson, 1990). To screen high-complexity ordered libraries efficiently by hybridization, it is necessary to make use of the precision and speed available from automation to develop an effective procedure for the preparation of high-density ordered arrays of clones from stored microtiter plate wells. Nizetic et al. (1991) have developed a customized system for gridding cosmid libraries of flowsorted chromosomes, which has also been applied to YACs (M. Ross and H. Lehrach, personal communication). Here we report the design and construction of an inoculation tool and a tablet to enable a commercially available robotic workstation (Biomek 1000, Beckman) to be used for preparation of high-density grids of yeast (e.g., YAC) or bacterial (e.g., cosmid or plasmid) clones. This makes it easy to screen ordered, high-complexity libraries by hybridization and is of particular value in establishing a shared resource, as filters can be readily exported by mail. In addition, the high-density grids are useful for preparing DNA pools representing high-density rows and columns for an improved PCR screening strategy.

MATERIALS

AND

METHODS

YAC and cosmid libraries. The Washington University YAC library (a gift from Maynard Olson) (Burke et al., 1987; Brownstein et aZ., 1989; Burke and Olson, 1991) consists of approximately 60,000 clones, which were stored in sealed microtiter plate arrays in YPD (20

DEVELOPMENT

AND

APPLICATION

g/liter bactopeptone, 10 g/liter yeast extract, 2% glucose, pH 5.8) containing 20% glycerol at -70°C. The library was replicated into three copies prior to freezing using a manual 96-pin replicator. One copy served as an archival stock, whereas the other two copies were used as primary working and backup stocks. Further working copies were made from the backup stock. During the replication process the YAC clones were grown for 2 days at 30°C on double selective AHC medium (1.7 g/liter yeast nitrogen base, 5 g/liter ammonium sulfate, 10 g/liter acid-hydrolyzed casein, 20 mg/liter adenine hemisulfate, 2% glucose pH 5.8) and then transferred to microtiter plates containing YPD for another 2 days before addition of glycerol and freezing. Prior to gridding out, the working stocks were allowed to thaw for 4 h. The cosmid library was constructed from partial Mb01 digests of DNA from a human cell line of karyotype 49,XXXXX cloned in the cosmid vector Lorist 6 (Gibson et al., 1987) as described in Holland et al. (manuscript in preparation). Cosmids were arrayed in microtiter plates, grown at 37”C, and stored at -70°C in 2~ TY (15 g/liter bactotryptone, 10 g/liter yeast extract, 5 g/liter sodium chloride, pH 7.4) supplemented with 3.6 mM dipotassium hydrogen phosphate, 1.3 mh4 potassium dihydrogen phosphate, 2 n&f trisodium citrate, 1 mM magnesium sulfate, 12% glycerol, and 30 yg/ml kanamycin (Knott et al., 1988; Coulson and Sulston, 1988). Gridding procedure. The gridding was performed using a Biomek 1000 (Beckman Instruments). The Biomek 1000 is fully programmable in three axes using the Biotest software package (Beckman). In this study, subroutines were written using version 1.60. The standard Biomek tablet has four locations for microtiter plates or other plasticware of equivalent size. The tablet moves in one horizontal axis. An arm positioned above the tablet moves a detachable tool in the second horizontal and the vertical axis. A 96-pin gridding tool was constructed to fit to the robot arm (see Fig. la). An eight-position tablet was attached to the standard Biomek tablet (see Fig. lb). Details of the design of these components are given under Results. Tools and tablets can be manufactured and supplied on request. The Biotest software package is available from Beckman Instruments, Inc. (Fullerton, Ca). Further details on programming using Biotest version 1.60 are available on request. YAC and cosmid clones were gridded out onto sterile 80 X 120-mm nylon filters (Hybond N, Amersham; available precut to size) placed on nutrient agar rectangular petri plates (Dynatech) compatible with microtiter plate format. Agar plates were poured to uniform thickness (50 ml agar per plate). Nylon filters were marked while dry with an Edding 1800 pen. The ink used was permanent even through the hybridization and washing steps. The gridding tool was treated prior to each session by sonicating for 30 min with the tips immersed in 50% Trisept to remove cellular debris, followed by 3 min in water. The tool was then air-dried for 30 min at 37°C before fitting to the robot. The robot was set up with a microtiter plate containing ethanol in the sterilizing location, the thawed working microtiter plate in the master location, and up to six filter/petri plates in the replica locations of the eight-position tablet (see Fig. lb). The gridding cycle consisted of a 15-s sterilization in ethanol, followed by air-drying for 10 s, then immersion to the bottom of the master plate, and finally inoculation onto the filter. The last two steps were repeated until the same plate of cells had been transferred to each filter and the cycle ended with the tool in ethanol for sterilization. The next master plate was positioned and the cycle repeated until all the required grid positions up to 16 had been inoculated. Other gridding subroutines for preparation of arrays of lower densities (nine cycles to form 3 X 3 arrays; four cycles to form 2 X 2 arrays) were also used. Filter growth and lysis. YACs were grown on filters on YPD agar containing 50 pg/ml ampicillin for 2 days at 3O’C. The filters were spheroplasted by being placed on Whatman 3MM paper soaked in SCE (1 M sorbitol, 0.1 M trisodium citrate, pH 5.8, 0.01 M EDTA) containing 4 mg/ml Novozyme (Novo Biolabs) and 14 mMfi-mercaptoethanol and incubation overnight at 37°C in a sealed container. The filters were denatured on 3MM soaked in 0.5 M NaOH, 1.5 M NaCl for 20 min and then dried on 3MM for 10 min. Filters were neutralized by submerging in 0.5 M Tris-HCl, pH 7.4,1.5 M NaCl for 5 min and then

OF

AUTOMATED

GRIDDING

535

in 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl for 5 min. Filters were incubated in 50 mM Tris-HCI, pH 7.4,0.15 M NaCl, 250 pg/ml proteinase K (Sigma Type XI-S) for 1 h at 37’C and then washed once in 50 mM Tris-HCl, pH 7.4,0.15 M NaCl for 5 min, and once in 50 mM Tris-HCl, pH 7.4, for 5 min. Finally, the membranes were dried on 3MM, baked at 80°C for 10 min, UV crosslinked, and stored dry (modified from Larin et al., 1991). Cosmids were grown for 1 day at 37°C on filters on L broth agar containing 30 pg/ml kanamycin. Cosmid filters were prepared by lysis on Whatman 3MM paper soaked as follows (Coulson and Sulston, 1988): 10% SDS for 4 min, denaturation on 0.5 M NaOH, 1.5 M NaCl for 5 min, and then neutralization twice on 0.5 M Tris-HCl, pH 8,1.5 M NaCl for 5 min. The membranes were then washed in 2~ SSC, 0.1% SDS (1X SSC is 150 mM NaCl, 15 mM trisodium citrate) for 5 min and then in 2~ SSC for 5 min. The membranes were UV crosslinked and stored dry. Preparation of probes and hybridization. DNA probes were labeled with [a-“P]dCTP (3000 Ci/mmol, Amersham) either by a refined hexamer labeling method (Feinberg and Vogelstein, 1984) as described by Hodgson and Fisk (1987) or by the PCR. For PCR labeling the required DNA segment was amplified in a lo-al reaction mix containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl,, 170 pg/ml BSA (bovine serum albumin), 200 pM each dNTP, 1 PM each oligonucleotide primer, 0.5 unit AmpliTaq (Perkin-Elmer/Cetus), and 25-100 ng of template DNA (Saiki et al., 1985, 1988). Primers were either specific to the sequence required or vector specific to amplify the cloned insert. PCR was carried out in a Perkin-Elmer/Cetus thermocycler with an initial denaturation step of 5 min at 94”C, followed by 30-35 cycles of 93°C for 1 min, 1 min at the annealing temperature appropriate to the primer pair, and 2-3 min at 72°C. The PCR product was separated by agarose gel electrophoresis, and an aliquot (l-10 ng) was reamplified for 20-30 cycles in a reaction mix as above except that it contained 100 WM each of dTTP, dGTP, and dATP and 1.6 PM [ol-32P]dCTP. Radiolabeled probe DNA was purified from unincorporated nucleotides by separation over Sephadex G50 spin columns and denatured at 95°C immediately prior to hybridization. Filters were hybridized overnight at 65°C in 6X SSC, 10X Denhardt’s (0.2% Ficoll400,0.2% polyvinylpyrrolidone, 0.2% BSA), 50 mMTrisHCl, pH 7.4, 10% dextran sulfate, 1% N-lauroyl sarcosine containing probe DNA at 5-0.5 X 10” cpm/ml. A ?S-labeled background probe was included in the hybridization to allow visualization of the colony grid. Host background probe was prepared by labeling either total DNA from yeast strain AB1380 containing pYAC4 (a gift from E. P. Green) or Lorist 6 vector DNA as appropriate with [?S]dATP by the hexamer labeling method. Background probe was stored at -20°C and used as required. The amount of ‘?S-labeled probe included in the hybridization was determined empirically for each batch of probe made. The filters were washed twice in 2X SSC for 10 min at room temperature, twice in 0.2X SSC, 1% N-lauroyl sarcosine at 65°C for 30 min, and then for 10 min at room temperature in 0.2X SSC. Autoradiography was performed for 4 h to overnight at -70°C with preflashed film (Kodak X-Omat S) with two intensifying screens. Preparation of YAC library DNA pools for PCR screening. For primary pools the cells from a high-density grid of 16 microtiter plates were scraped off a membrane after 2 days of growth. Rows and columns were prepared by cutting the appropriate strips from the highdensity grids and pooling the cells as detailed in Fig. 3 (see also Kwiatkowski et cd., 1991). Cell suspensions were processed as follows (modified from Anand et al., 1990). The yeast cells were washed once in 50 mM Tris-HCl, pH 8, and resuspended in 2.5 ml SCE/g wet wt containing 1 mg/ml zymolyase 20-T (ICN) and 14 mM o-mercaptoethanol. The cell suspension was mixed with an equal volume of 2% LGT agarose (Seakern, Marine Colloids, Inc.) dissolved in SCE, and the mixture was allowed to gel on ice in disposable plastic Pasteur pipets. Once set, the cell suspension was extruded into SCE containing 1 mg/ml zymolyase 20-T and 14 mM &mecaptoethanol and was incubated overnight at 37°C. The spheroplasts were then washed in 1% lithium dodecyl sulfate, 100 mM EDTA, 10 mM Tris-HCl, pH 8, for 1 h, and then overnight at 37°C. The DNA was washed three times for

536

BENTLEY

30 min at 50°C in 10 n&f Tris-HCl, pH 8,0.1 mM EDTA (TE) and then three times for 30 min in TE at room temperature. For PCR screening, the DNA in agarose was diluted to 10 ng/pl and 40 ng was used in each lo-p1 PCR reaction. The PCR was performed as described above on both primary pools, rows and columns, and human DNA. Controls included reactions containing DNA from AB1380 transformed with pYAC4 and TE (i.e., no DNA). The final PCR steps were performed directly on the yeast colonies from a highdensity grid as previously described (Huxley et al., 1990).

RESULTS

A system for inoculating large numbers of yeast or bacterial clones from microtiter plates onto filters in high-density arrays using the Biomek 1000 robotic workstation was developed. The workstation, originally designed for automated liquid handling as in ELISA applications, was adapted for gridding as follows. A 96-pin inoculating tool was constructed (see Fig. la). The pins were set in a block of Teflon to allow freedom of movement in the vertical direction. Thus, each pin self-adjusts on making contact with a solid surface to accommodate any irregularities in the surface and ensure 100% successful inoculation of nylon filters on nutrient agar plates. The ability to grid out filters on agar prevents filter dehydration. The pins were machined to have ends of small diameter. The optimum size for producing high resolution and for retaining the mechanical rigidity necessary for thousands of repeated operations was found to be 0.6 mm. The pins are of sufficient length (50 mm) to permit transfer to or from microtiter plate tubes as well as microtiter plates. For gridding, two of the four locations on the standard Biomek 1000 tablet were occupied by rectangular nutrient agar plates containing the filters for inoculation, and the other two positions held the microtiter plate of clones and the ethanol sterilization plate. To increase the number of replicas that could be produced during each cycle, a new tablet with eight positions for microtiter plates was fitted onto the existing tablet (see Fig. lb). This system can be used to inoculate up to six copies of high-density grids of up to 4 X 4 arrays, i.e., 1536 clones spaced at centers of approximately 1.5 mm (see Figs. 2a and 2~). Six copies of 13 grids (each copy therefore representing 19,968 clones) may be made in one afternoon. Tests to optimize the sterilization step were carried out to ensure an effective but simple sterilization. Immersion of the pins in ethanol for 15 s, followed by a 10-s evaporation step, was sufficient to ensure sterilization. This was tested by removing the microtiter plate of clones at alternate cycles. Thus, the pins came into contact with the surface of the agar plate directly after sterilization. This was repeated on a complete filter, so that the sterilization of each of the 96 pins was tested eight times (or a total of 768 events). No growth of yeast cells was observed in any of the alternate positions (2, 4, 6, 8 . . . 16) in the grid (Fig. 2b). The same conditions were effective for sterilization of bacteria (results not shown). Two further precautions were taken to ensure sterility. First, the robot was stationed in a laminar flow hood,

ET

AL.

and the inoculating tool was routinely sonicated before use, as described under Materials and Methods. This prevents the accumulation of cell debris on the pins that would otherwise compromise the sterilization step. Second, to ensure sterility of the library stocks, gridding was performed only from replica plates that were prepared from the archive (master) plates manually using a flame-sterilized inoculating tool. Replica plates were repeatedly frozen at -70°C and thawed as required. To optimize the hybridization procedure, modifications to the preparation of filters and probes were made. The spheroplasting step was found to be critical to producing good hybridization signals. fl-Mercaptoethanol was found to be a more reliable reducing agent than DTT in part because of easier storage. After efficient spheroplasting, colonies were seen to “flatten down” onto the filters. In addition, more reliable signals and a better background grid were produced when filters were not wiped to remove cellular debris but instead were agitated in the neutralization and proteinase K solutions during the lysis procedure. Reliable identification of the correct position of each positive clone in a high-density array was made easier by including 35S-labeled DNA from a yeast strain transformed with pYAC4 in each hybridization. The low intensity of signal obtained from the 35S isotope was sufficient to show the position of every colony of the grid in an overnight exposure without masking the signal derived from a 32P-labeled-specific probe (see Fig. 2d). To improve the signal obtained when very short DNA fragments (as small as 100 bp) were used as probes to identify YACs, a PCR-labeling procedure was developed for use in place of the random hexanucleotide priming method of Feinberg and Vogelstein (1984). This method is therefore readily compatible with the use of STSs for genomic mapping. Primers that were specific either for the locus or for vector sequences flanking a cloned insert were used to amplify the appropriate DNA fragment. Following electrophoresis of the PCR products on a horizontal agarose or acrylamide minigel, an aliquot of the fragment was transferred to a second PCR, which contained [cu-3”P]dCTP (3000 Ci/mmol; 50 &!i per 10 ~1 PCR: see Materials and Methods). Typically, autoradiographic results were obtained between 4 h and overnight with these probes. The result of a hybridization using this method of probe labeling is shown in Fig. 2d. The Washington University YAC library was gridded on 38 filters and screened with a 194-bp probe specific for human y-glutamyl transpeptidase, a multigene family (Pawlak et al., 1988) with at least four loci that have been localized to chromosome 22 (Bulle et al., 1987). Thirty positive clones were identified, one of which is shown at grid reference FlO, position 15 (Fig. 2d; see Fig. 2c for details of grid numbering). All positive clones were subsequently confirmed by colony PCR analysis using an STS (data not shown). The ability to prepare high-density arrays of gridded YACs made it also possible to develop an improved strategy for PCR screening that permits isolation of a posi-

DEVELOPMENT

AND APPLICATION

OF AUTOMATED

GRIDDING

537

FIG. . 1. (a) 96-pin gridding tool. (b) Biomek 1000, fitted with eight-position tablet. The microtiter plates containing the glycerol stocks of 96 clones and the ethanol for sterilization are located on the right at the front and back, respectively. The remaining positions are occupiced by rectan .gular petri dishes containing nylon filters on nutrient agar.

BENTLEY

ET

AL.

FIG. 2. (a) A 4 X 4 array of YAC colonies (1536 in total) from the Washington University YAC library. (b) A sterilization test grid showing the presence of colonies after inoculation of alternate positions (1, 3, 5, 7, . . . 15). The sterilized pins made contact with the filter at the remaining positions; no growth of cells is observed at any of these positions. (c) Diagram of the arrangement of clones in the grid. Each square is defined by a grid reference that corresponds to the microtiter plate grid. Square Al is shown expanded to illustrate the arrangement of clones from well Al of each of the 16 microtiter plates used to form the grid. In the first cycle of gridding, the inoculating tool deposits an inoculum in the top left-hand position (marked 1 in the figure). The second inoculum is placed adjacent to the first, at position 2, and so on as numbered. (d) Autoradiograph of the hybridization screen of the filter with the PCR-labeled 194-bp probe for the y-glutamyl transpeptidase gene sequence (a multigene family). The positive clone is located at grid reference FlO, position 15. A “S-labeled background probe was included in the hybridization to visualize the colony grid.

tive clone in as little as 2 days using three consecutive PCR experiments (see Fig. 3a). Primary DNA pools were prepared using cells harvested directly from each high-density grid. Secondary DNA pools were prepared from high-density rows and columns, in which copies of the high-density grids were cut into strips (eight rows A-H containing 192 clones each, or 12 columns 1-12 containing 128 clones each), the cells were harvested, and DNA was extracted from each strip. To reduce the number of individual DNA preparations, these strips were paired such that 40 (16 rows and 24 column) preparations represented 4 of the 4 X 4 arrays (see Fig. 3b).

B

A Whole

library

I I 16 ond

PCR analysis was carried out as follows (see Fig. 3a). First, the 38 primary pools were screened by PCR to identify which pool(s) contained a positive clone. Second, PCR was performed on the appropriate set of eightrow and 12-column pools. For example, if a positive clone was identified in the primary pool corresponding to filter 1 (see Fig. 3b), the secondary screen was carried out using row pools A to H and column pools 1 to 12. This resulted in identification of a set of 16 candidate clones. For example, a 288-bp STS specific for human a-IV-acetylgalatosaminidase (Wang and Desnick, 1991) was used to screen the Washington University YAC li-

clones column

Single

for

clone

PRIMARY POOLS

SCREEN

OF 1536

CLONES

16

SECONDARY 6 ROW AND

POOLS

OF

ROWS

SCREEN 12

COLUMN

POOLS

each row intersection

I

CLONE COLONY

IDENTIFICATION

24

POOLS

OF COLUMNS

PCR

identified

FIG. 3. (A) Flow diagram of the PCR screening scheme. (B) Diagram of the mode of pooling of high-density rows and columns from four filters. This generates 16 and 24 row and column pools, respectively, but only 8 row and 12 column pools are used for the secondary screen because the appropriate filter is identified previously in the primary screen. Thus, for example, if a positive clone is identified in filter 4, the secondary screen is performed using row pools I-P and 13-24 only.

DEVELOPMENT

AND APPLICATION

OF AUTOMATED

539

GRIDDING

a 1 2 3 4 5 6

H

8 9 10 11 12

888888X8’%888888gsS8888 8888=8%88%888888888

Rl R2 R3 R4

C.

FIG. 4. Results of a secondary screen and clone identification using a 26%bp STS for cu-N-acetylgalactosaminidase. The primary screen had previously resulted in identification of positive clones on four filters, including BB3. (a) PCR analysis of the rows and columns for filter BB3 demonstrated that the positive clone is among the 16 at grid reference E12. (b) Analysis of the 16 candidates by PCR of four rows (Rl-R4) and columns (Cl-C4) demonstrated that the positive is at position 3, which corresponds to the clone El2 in microtiter plate B35. (c) Colony PCR of the 16 individual candidates demonstrated that the positive is at position 3, which is in agreement with the result in (b). CONTROL: Negative control, with no DNA in the PCR. GENOMIC: Positive control, with human genomic DNA (100 ng CGM-1 DNA per lo-p1 reaction) in the PCR.

brary, and positives were identified in four primary pools including BB3. PCR analysis of the rows and columns corresponding to filter BB3 resulted in identification of a set of 16 clones at position El2 (see Fig. 4a). Final identification of the positive clone was accomplished either by pooling live cells from each colony into four rows of 4 and four columns of 4 for PCR analysis (eight PCRs in total: Fig. 4b) or by performing colony PCR of each candidate. In both of these options the final clone was identified in position 3 of the 16 (as shown in the diagram in Fig. 4b), which corresponded to plate B35. Occasionally, two positives may be present on the same plate. This leads to the identification of up to two

positive rows and two positive columns, and hence up to 64 candidate clones. To avoid unnecessary PCR analysis, it may be preferable to perform PCRs on four pools, one of each of the 16 colonies at each row and column intersection, before proceeding to the final stage. The approach described above improves the rate of isolation of clones because it is based exclusively on PCR, the final hybridization step that was used previously having been eliminated. Thus, five clones can be isolated from the library in 2 days by 3 consecutive PCR experiments using a total of 178 lo-p1 PCS (38 PCRs in the primary screen, 20 PCRs in the secondary screen for each clone, and 8 in the final identification of each clone

540

BENTLEY

-see Fig. 3a) plus controls. The same approach may be used to screen other libraries, taking into account the factors that may determine the optimal screening strategy in each case. For example, in libraries that are enriched for particular chromosomes and/or have larger inserts, such as the 48,XXXX libraries of Anand et al. (1990) or Larin et al. (1991), the probability of two positive clones being present in the same number of colonies is increased. To decrease this probability, the density of each primary pool may be reduced by gridding the library in arrays of lower density. DISCUSSION The efficient screening of complex libraries stored in microtiter plates is important in the study of large genomes. Previously, the preparation of filters for hybridization was performed manually. This was a disadvantage because the process was time consuming and because it was only possible to make grids of low density (384 clones per 8 X 12-cm filter, 150 filters per copy of the Washington University YAC library). Automation of the gridding procedure has resulted in the ability to prepare grids of fourfold higher density, thus reducing the number of filters required to represent a library by a factor of 4 and making it easy to perform primary hybridization screens of high-complexity libraries. The method is now in routine use in the laboratory, and more than 100 YAC clones have been isolated by hybridization to date. In our experience, the ability to choose either hybridization or PCR as a screening method as appropriate to each case has significantly increased the success rate of isolation of YACs. The choice of method depends on the number of probes being used, whether they are unique, whether PCR primers are available, and the number of positives expected. Thus, for example, the use of hybridization in primary screens is essential with pooled probes or probes specific for multigene families, in which large numbers of positive candidates are identified. This was demonstrated in the example shown here, in which 30 positive clones for GGT were identified in the Washington University YAC library. To screen for a single gene when no STS is available, it is faster to perform a hybridization screen than to generate an STS de novo. By contrast, if there is already an STS, a single screen by PCR is faster and avoids the use of radioactive isotopes. PCR screening is important in conjunction with the use of an STS approach to genome mapping (Olson et al., 1989), especially with STSs that are derived from uncharacterized segments of DNA isolated at random (Cole et al., 1991; Green et al., 1991) and may contain repetitive sequences and thus be unusable for hybridization. The previous PCR screening protocol (Green and Olson, 1990) still retained a final hybridization step. We have used strips cut from the high-density grids to prepare additional DNA pools for a row-and-column PCR strategy. This is similar in principle to the approach re-

ET

AL.

ported by Kwiatkowski et al. (1991) in which crude yeast cell lysates of rows and columns were prepared from low-density (2 X 2) filters. We experienced some variation in the ability of these preparations to sustain PCR reliably after prolonged storage. Instead, we prepare DNA from rows and columns of high-density grids; as a long-term source of reagents for screening, and incorporate a higher order of pooling (see Fig. 3B) to reduce the number of preparations to a minimum. The row and column strategy, when combined with colony PCR, permits elimination of the hybridization step, thus avoiding the use of radioactivity and reducing the time required to identify the final clone in the appropriate pool from 2-3 days to 1 day. These advantages must be weighed against the threefold greater number of PCRs that are performed relative to the original strategy of Green and Olson (1990). In conclusion, the development of an automated procedure using a commercially available robotic workstation for gridding high-density arrays of clones has made it easier to screen high-complexity yeast or bacterial ordered libraries by hybridization or PCR. The ability to choose either approach for primary screening maximizes the versatility and use of such resources for genome mapping and analysis of related problems in human genetics. ACKNOWLEDGMENTS We are grateful to Richard Keightley and Beckman Instruments, Ltd., for provision of the Biotest software and for discussions. We also thank M. Olson, D. Schlessinger, B. Brownstein, and the staff of the Center for Genetics in Medicine at the Washington University School of Medicine for their generous assistance in transfer of the YAC library. This work was supported by the MRC Human Genome Mapping Project, the Generation Trust, and the Spastics Society. Note added in proof. The results shown were obtained exclusively with the 96-pin tool manufactured to the design described in the text. A different 96-pin tool (the HDR tool) has been developed by Beckman Instruments, Inc., which is intended to perform a similar operation, but is not at present compatible with the &position tablet reported here.

REFERENCES Anand, R. A., Riley, J. H., Butler, R., Smith, J. C., and Markham, A. F. (1990). A 3.5 genome equivalent multiaccess YAC library: Construction, characterisation, screening and storage. Nucleic Acids Res. 18: 1951-1956. Brownstein, B. H., Silverman, G. A., Little, R. D., Burke, D. T., Korsmeyer, S. J., Schlessinger, D., and Olson, M. V. (1989). Isolation of single-copy human genes from a library of yeast artificial chromosome clones. Science 244: 1348-1351. Bulle, F., Mattei, M. G., Siegrist, S., Pawlak, A., Passage, E., Chobert, M. N., Laperche, Y., and Guellaen, G. (1987). Assignment of the human gamma-glutamyl transferase gene to the long arm of chromosome 22. Hum. &net. 76: 283-286. Burke, D. T., Carle, G. F., and Olson, M. V. (1987). Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 8066812. Burke, D. T., and Olson, M. V. (1991). Preparation of clone libraries in yeast artificial chromosome vectors. In “Methods in Enzymology,” (C. Guthrie and G. R. Fink, Eds.), Vol. 194, pp. 251-270. Academic Press, San Diego.

DEVELOPMENT

AND

APPLICATION

Cole, C. G., Goodfellow, P,N., Bobrow, M., and Bentley, D. R. (1991). Generation of novel sequence tagged sites (ST&.) from discrete chromosomal regions using Alu-PCR. Genomics 10: 816-826. Coulson, A., and Sulston, J. (1988). Genome mapping by restriction fingerprinting. In “Genome Analysis, A Practical Approach” (K. Davies, Ed.), pp. 19-39, IRL Press, Oxford. Coulson, A., Sulston, J., Brenner, S., and Karn, J. (1986). Toward a physical map of the genome of the nematode Caenorhbditis ekgans. Proc. Natl. Acad. Sci. USA 83: 7821-7825. Evans, G. A., and Lewis, K. A. (1989). Physical mapping of complex genomes by cosmid multiplex analysis. Proc. Natl. Acad. Sci. USA 86: 5030-5034. Feinberg, A. P., and Vogelstein, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity: Addendum. Anal. Biochem. 137: 266-267. Gergen, J. P., Stern, R. H., and Websink, P. C. (1979). Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 7: 21152136. Gibson, T. J., Rosenthal, A., and Waterston, R. (1987). LoristG, a cosmid vector with BamHI, NotI, ScaI, and Hind111 cloning sites and altered neomycin phosphotransferase gene expression. Gene 53: 283-286. Green, E. D., Mohr, R. M., Idol, J. R., Jones, M., Buckingham, J. M., Deaven, L. L., Moyzis, R. K., and Olson, M. V. (1991). Systematic generation of sequence-tagged sites for physical mapping of human chromosomes: Application to the mapping of human chromosome 7 using yeast artificial chromosomes. Genomics 11: 548-564. Green, E. D., and Olson, M. V. (1990). Systematic screening of yeast artificial chromosome libraries by the use of the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87: 1213-1217. Grunstein, M., and Hogness, D. S. (1975). Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72: 3961-3965. Hodgson, C. P., and Fisk, R. Z. (1987). Hybridisation probe size control: Optimised “oligolabelling.” Nucleic Acids Res. 15: 6295. Huxley, C., Green, E. D., and Dunham, I. (1990). Rapid assessment of S. cerevesiae mating type by the PCR. Trends Genet. 6: 236. Knott, V., Rees, D. J. G., Cheng, Z., and Brownlee, G. G. (1988). Randomly picked cosmid clones overlap the pyrB and oriC gap in the physical map of the E. coli chromosome. Nucleic Acids Res. 16: 2601-2612.

OF

AUTOMATED

GRIDDING

541

Kohara, Y., Akiyaina, K., and Isono, K. (1987). The physical map of the whole E. coli chromosome: Application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50: 495508. Kwiatkowski, T. J., Jr., Zoghbi, H. Y., Ledbetter, S. A., Ellison, K. A., and Chinault, A. C. (1991). Rapid identification of yeast artificial chromosome clones by matrix pooling and crude lysate PCR. Nucleic Acids Res. 18: 7191. Larin, Z., Monaco, A. P., and Lehrach, H. (1991). Yeast artificial chromosome libraries containing large inserts from mouse and human DNA. Proc. Natl. Acad. Sci. USA 88: 4123-4127. Nizetic, D. N., Zehetner, G., Monaco, A. P., Gellen, L., Young, B. D., and Lehrach, H. (1991). Construction, arraying and high density screening of large insert libraries of the human chromosomes X and 21: Their potential use as reference libraries. Proc. Natl. Acad. Sci. USA 88: 3233-3237. Olson, M. V., Dutchik, J. E., Graham, M. Y., Brodeur, G. M., Helins, C., Frank, M., Maccollin, M., Scheinman, R., andFrank, T. (1986). Random clone strategy for genomic restriction mapping in yeast. Proc. Natl. Acad. Sci. USA 83: 7826-7830. Olson, M., Hood, L., Cantor, C., and Botstein, D. (1989). A common language for physical mapping of the human genome. Science 245: 1434-1435. Pawlak, A., Lahuna, O., Bulle, F., Suzuki, A., Ferry, N., Siegrist, S., Chikhi, N., Chobert, M. N., Guellaen, G., and Laperche, Y. (1988). Gamma-glutamyl transpeptidase: A single copy gene in the rat and a multigene family in the human genome. J. Biol. Chem. 263: 99139916. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985). Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354. Wang, A. M., and Desnick, R. J. (1991). Structural organization and complete sequence of the human a-N-acetylgalactosaminidase gene: Homology with the ol-galactosidase A gene provides evidence for evolution from a common ancestral gene. Genomics 10: 133-142.