[361 Construction of cDNA libraries from single cells

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[36] C o n s t r u c t i o n o f c D N A L i b r a r i e s f r o m S i n g l e C e l l s

By

GERARD BRADY a n d NORMAN N . ISCOVE

Introduction The development of the polymerase chain reaction 1'2 (PCR) has expanded the application of traditional molecular biology to allow examination of both DNA and RNA in samples as small as a single cell. 3'4 The methods developed have generally focused on amplification of known sequences with specific primers, and there has been little attention given to global amplification of expressed genes. 5,6 Here we describe a simple approach, referred to as poly(A) P C R , 6 which was designed for unbiased amplification of cDNA representing all polyadenylated RNA present in a sample as small as a single cell.

General Principle of Poly(A) Amplification The basic principle of the PCR requires that the target sequence be bracketed by known sequences to which the amplification primers can anneal and initiate polymerization. To prepare total cDNA for poly(A) PCR, known sequences are added in two steps (Fig. 1). One end is initially defined through a cDNA reaction using reverse transcriptase and an oligo(dT) primer that will prime via the poly(A) tail present at the 3' end of most mRNA molecules (Fig. 1, step 1). A homopolymer dA tract is then added to the 3' end of the first-strand cDNA using terminal transferase (Fig. 1, step 2). Because the PCR reaction is most efficient for relatively short sequences, amplification of full-length cDNA would result in disproportionate amplification of smaller cDNAs. This bias can be avoided by limiting the length of the initial cDNA strand to around 100-700 bases regardless of the size of the original RNA template. J R. K. Saiki, F. A. Faloona, K. B. Mullis, C. T. Horn, H. A. Erlich, and N. Anaheim, Science 230, 1350 (1985). 2 K. B. Mullis and F. A. Faloona, this series, Vol. 155, p. 335. 3 L. H. Gyllensten, X. Cui, R. K. Saiki, H. A. Erlich, and N. Anaheim, Nature (London) 335, 414 (1988). 4 D. A. Rappolee, A. Wang, D. Mark, and Z. Werb, J. Cell. Biochem. 39, 1 (1989). 5 A. Belyavsky, T. Vinogradova, and K. Rajewsky, Nucleic Acids Res. 17, 2919 (1989). 6 G. Brady, M. Barbara, and N. N. Iscove, Methods Mol. Cell. Biol. 2, 17 (1990).

METHODS IN ENZYMOLOGY, VOL. 225

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

612

GENE IDENTIFICATION Stem 5 t

mRNA

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[36] i.

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FIG. 1. S u m m a r y and outline of poly(A) amplification

PCR amplification of the dA/dT bracketed cDNA is carried out using a single oligonucleotide, known as (dT)-X, which consists of a 5' sequence containing a restriction enzyme site and a 3' dT stretch. Priming of the c D N A in the PCR is initiated via annealing of the dT region of (dT)-X to the homopolymer dA regions present at the termini of the cDNA molecules. The unique 5' sequence provides a convenient means of cloning the amplified product using the included restriction enzyme site, and it increases the stability and precision of primer annealing during PCR. Purification steps have been avoided to minimize loss of starting material and opportunities for contamination. The final protocol is carried out in a single tube and involves only three pippetting steps prior to PCR amplification. The amplified product can be cloned into bacterial vectors and renewed indefinitely through further rounds of the PCR.

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Poly(A) Amplification Protocol The present protocol has been used for several years to amplify many hundreds of samples. During this period only slight modifications and additions have been made to the original method. 6 Most attempted "improvements" have been counterproductive, and for this reason even minor deviations from the given protocol should be avoided. Unless otherwise stated, it is advisable to store stock solutions as small aliquots at - 7 0 °.

cDNA Preparation First-strand cDNA synthesis is carried out using reverse transcriptase in first-strand buffer which is made up from three stock components: cDNA/lysis buffer: 52 mM Tris-HCl, pH 8.3, 78 mM KCI, 3.1 mM MgC1z, 0.52% Nonidet P-40 (NP-40) RNase inhibitors: 1 : 1 mixture of RNAguard (Pharmacia, Piscataway, NJ) and Inhibit Ace (5'-3' Inc.) cDNA primer mix: mixture of nucleotides and dT primer [12.5 mM each dNTP, 6.125 OD260/ml dT24 primer] First-strand buffer should be prepared fresh and used within a few hours. For 100 tzl mix 96/xl cDNA/lysis buffer, 2 ~1 RNase inhibitors, and 2/zl cDNA primer mix freshly diluted 1 : 24 with water. To 4/zl of the first-strand buffer add 1 to 40 cells or up to 1 tzg total RNA (see below) in no more than 0.5/zl. Heat for 1 min at 65° to promote unfolding of the mRNA, cool to room temperature for 3 min to allow annealing of the (dT) primer, and place on ice. To initiate first-strand synthesis, add 0.5/zl of a 1 : I mixture of Moloney murine leukemia virus and avian myeloblastosis virus reverse transcriptases (GIBCO-BRL, Grand Island, NY, or Boehringer-Mannheim, Indianapolis, IN), mixed directly as supplied by the manufacturer. (Both reverse transcriptase enzymes are used to avoid any sequence specificity attributible to either one alone.) Incubate for 15 min at 37°. Stop the reaction by heating to 65 ° for 10 min. Nucleic Acid Preparation for 1--40 Cells: Direct Addition. Provided that the volume does not exceed 0.5 tzl, samples of 1-40 cells may be added directly to 4 tzl of first-strand buffer and stored on ice for a maximum of 1 hr prior to initiating the reverse transcriptase reaction. All biochemical reactions leading to a final poly(A) PCR product can be carried out without further purification. The direct addition procedure relies on NP-40 to lyse the outer cellular membrane and RNase inhibitors to protect the RNA from degradation. Because the NP-40 used does not lyse the nuclear

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membrane, genomic DNA may be recovered by pelleting the nuclei by a 50-sec centrifugation (12,000 g) at 4°. The cytoplasmic RNA present in the supernatant and genomic DNA recovered in the pellet can be processed separately. If there is no requirement for genomic DNA, the entire unfractionated sample can be processed following the poly(A) protocol without any interference from nuclear DNA. Nucleic Acid Preparation for 40-106 Cells: Mini-GT Protocol. Often it is desirable to process and to store large samples such as sections of tissue and cell cultures prior to poly(A) processing. RNA produced by any of the standard methods is readily amplified using the poly(A) PCR procedure. The following protocol provides a convenient method for preparing samples larger than 40 cells.

Mini-GT Protocol for Preparation of Total Nucleic Acids Lyse 40-106 cells either as a pellet, tissue, or in a suspension of no more than I0/zl by adding to 50 tzl GT solution (5 M Guanidine thiocyanate, 0.5% Sarkosyl, 25 mM Sodium citrate, pH 7.0, 20 mM 1,4-Dithioerythritol). Store stock GT solution at room temperature (stable for several months). Vortex the cell lysate for 30 sec (at this point the sample may be processed directly or stored at - 70 ° for several months). Recover total nucleic acids from the sample by ethanol precipitation using glycogen as a carrier, add 25/zl 7.5 M ammonium acetate, 20 ~g glycogen (BoehringerMannheim), and 150/zl ethanol. Mix and leave on wet ice a minimum of 30 min. For samples containing excess extracellular matrix, it is advisable to remove insoluble material after addition of ammonium acetate by centrifuging at 12,000 g at 4° for 5 min and transferring the supernatant. Pellet the ethanol precipitate by centrifuging at around 12,000 g for 30 min in a refrigerated microcentrifuge. To remove residual salts, wash the pellet three times at room temperature with 70% ethanol (each time briefly centrifuge to ensure the pellet is not lost), dry, and resuspend in 5-50 ~1 of 0.5% NP-40 containing a 1 : 100 dilution of the RNase inhibitor Inhibit Ace (5'-3' Inc.). Both DNA and RNA extracted in this procedure can be used as substrates for reverse transcriptase and genomic PCR. Although genomic DNA is present, it does not appear to contribute measurably to the poly(A) PCR product.

Timing of the cDNA Reaction The incubation time of the reverse transcriptase reaction is kept deliberately short to produce abbreviated cDNA molecules that will be efficiently amplified in the final PCR step. Longer incubation times will result

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in longer overall cDNA molecules and consequently poor and nonrepresentative PCR amplification. Oligonucleotide and Nucleotide Concentrations. The final concentrations of dNTPs and oligonucleotides in the cDNA reaction are 0.01 mM and 0.005 OD260units/ml, respectively. The low dNTP concentration effectively limits the rate of reverse transcriptase polymerization and allows convenient addition of a predominantly homopolymer dA tail in a subsequent tailing reaction by the addition of excess dATP. The low dT primer concentration also plays a role in limiting first-strand synthesis and use of the primer as a substrate in the following tailing and amplification reactions. Possible Contaminants. Amplification of cells added directly to the first-strand buffer appears to be relatively insensitive to the cell-suspension medium. Samples have been successfully processed directly from a variety of growth media that have included 10% serum, 1% methylcellulose, as well as buffered salt solutions (including phosphate buffers). Although we have never observed cell-related amplification from growth media and serum alone, a cDNA product has been obtained on occasion from filtered supernatants of dense cell cultures. For this reason appropriate controls should always be included, particularly for single-cell work. Nucleic acids prepared using the mini-GT protocol have invariably proved to be excellent substrates for both genomic and cDNA-directed PCR. Possible contamination from guanidine and ammonium salts is avoided by stringent washing with 70% ethanol. Application. The direct addition approach has led to successful amplification of single primary hemopoietic cells, hemopoietic colonies grown in liquid culture or methylcellulose, fluorescence-activated cell sorted (FACS) populations, various cell lines, single mouse eggs, 2- to 8-cell embryos, blastocysts, and fetal tissue from various stages of development. The mini-GT protocol has proved to be invaluable for examining gene expression and genomic DNA from a multitude of sources. Combined with the poly(A) PCR amplification procedure, it is routinely being used as a simple and more economical alternative to Northern analysis.

Tailing After allowing the heat-inactivated first-strand cDNA reaction to cool on ice for approximately 2 min, add an equal volume 2 x tailing buffer [200 mM potassium cacodylate, pH 7.2, 4 mM CoC12, 0.4 mM dithiothreitol (DTT), and 1.5 mM dATP] containing 10 units Terminal transferase. (Note: The above formula for 2 x tailing buffer is based on the 5 x terminal transferase buffer supplied by Gibco-BRL and can be conveniently made up by mixing 800/zl of 5 × BRL terminal transferase buffer, 30 p.1 of I00

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mM dATP, and 1.17 ml water.) Incubate the tailing reaction for 15 min at 37 ° and heat-inactivate at 65 ° for 10 min. Storage. At this point tailed cDNA preparations from 10 or more cells can be stored at - 7 0 ° for at least 1 month. However, preparations from single cells noticeably deteriorate with storage at - 70 °, and it is advisable to continue with them directly to the PCR step. Timing. Unlike the cDNA reaction, the exact timing of the terminal transferase reaction is not critical, and extended incubation up to 1 hr does not adversely affect the amplified product.

Primary Polymerase Chain Reaction Optimization of Amplification Oligonucleotide and Salt Concentrations. The concentrations of both MgC12 and the amplification oligonucleotide in the primary PCR are critical for efficient amplification of samples equivalent to 1-50 cells. A given set of PCR conditions may allow efficient amplification from 100 cells but fail to produce material from a single cell. Establish optimal PCR conditions by amplifying aliquots equivalent to 1-2 cells from a scaled-up cDNA/tailing reaction. To do this, add 1 /xl containing 50-100 cells to 100/zl first-strand buffer and process to yield 200 /~1 of tailed cDNA following the protocol above. Divide into 4-/zl samples and amplify in a final PCR volume of 20/xl. For MgCI2 titration, prepare a series of 10 x Taq polymerase buffers consisting of 100 mM Tris-HC1, pH 8.3,500 mM KC1, 15-50 mM MgC12 in increments of 5 mM, I mg/ml bovine serum albumin (BSA, Boehringer/Mannheim molecular grade), and 0.5% Triton X-100. For each MgClz concentration, test the amplification oligonucleotide at final concentrations of 1, 2, and 4 OD260 units/ml. PCR Amplification. For every microliter of tailed cDNA add 4/zl of PCR mixture having the following proportions: 10 x Taq polymerase buffer (optimized for 4/xl MgCI2) 25 mM each dNTP 1.5/zl (dT)-X amplification oligonucleotide (see opx/~1 timzation above) Taq polymerase (Perkin-Elmer Cetus, Nor5 units walk, CT) Water Adjust to a final volume of 32/xl Overlay the sample with mineral oil (if required) and amplify using the following cycle profile: 25 cycles consisting of 1 min at 94°, 2 min at 42 °,

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and 6 min at 72 ° linked to a second 25 cycles each having 1 min at 94°, 1 min at 42 °, and 2 min at 72 °. Estimate of Overall Amplification. The efficiency of overall amplification cannot be judged by ethidium bromide staining alone since control samples generated in the absence of added RNA or DNA template frequently yield visible DNA. This is believed to be due to prokaryotic DNA present in commercially supplied Taq polymerase. However, this nonspecific product has not hybridized to any of more than 20 specific eukaryotic probes tested so far. Successful amplification is judged by hybridization to two or more specific cDNA probes. Primer Sequence. The (dT)-X amplification oligonucleotide sequence is 5' ATG TCG TCC AGG CCG CTC TGG ACA AAA TAT GAA TTC 3' plus 24 dT residues. Apart from the EcoRI restriction enzyme site prior to the dT stretch, the unique sequence of the amplification primer given above was chosen arbitrarily from primers available in the laboratory. Several other sequences have also worked, although most of the singlecell amplifications have been carried out with the given sequence. Oligonucleotide Preparation. Different batches of the same oligonucleotide sequence vary markedly in their ability to amplify small samples (< 10 cells). The reason for this is unclear, but oligonucleotides that failed to amplify single-cell samples have frequently been "rescued" by deionization and, in one instance, by adjusting the PCR to include a final concentration of 5 mM EGTA. Commercially available oligonucleotides appear to vary considerably in the purity and percentage of full-length product. Although cost has precluded a systematic analysis of differing sources, oligonucleotides provide by Oligos Etc. Inc. (Wilsonville, OR) have worked well in the single-cell poly(A) PCR protocol without further purification. Deionization. Wash 1 g of mixed-bed resin three times with 30 ml water. Filter to remove excess water and add approximately 0.5 ml of the packed filtered resin directly to the oligonucleotide (in around 0.5-2 ml water). Heat at 65 ° for 5 min and swirl at room temperature for 30 min. Filter out the resin using a 0.22-/~m minifiltration unit and measure the absorption (OD260/280). Make suitable aliquots, lyophilize to dryness, and store at - 7 0 °. Reconstitute when needed with an appropriate volume of water. Buffer Composition. The PCR buffer described appears to be critical for amplification since changes to a higher Tris-HCl concentration or slight changes in the pH of the 2 × tailing buffer and the Taq polymerase buffer have led to dramatic reductions in PCR yield (S. Varmusa and F. Billia, personal communication).

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Storage of PCR Samples. Prolonged storage (>2 months) ofunpurified poly(A) PCR samples at 4 °, - 2 0 °, and - 7 0 ° can lead to deterioration to the point where they can no longer be amplified by additional rounds of poly(A) PCR. It is recommended for precious samples that a portion be stored as a dried ethanol precipitate. A small aliquot of each sample can also be expanded through reamplification and cloning (see below), providing an inexhaustable supply of working material. Reamplification of Poly(A)-AmplifiedMaterial Conditions for successful amplification of a previously amplified sample appear to be less stringent than those used in the primary PCR. Both Taq polymerase, oligonucleotides, and dNTPs may be reduced to around one-fifth of the initial levels. However, as with the primary PCR, it is advisable to titrate the oligonucleotide to establish efficient and economical working conditions. Typically reamplify five samples: Water 440/~1 10 x Taq polymerase buffer 50/zl 25 mM dNTPs 4/xl Amplification oligonucleotide 10D260/ml (final concentration) Taq polymerase 5 units Add 100/M of this mix to 0.5 tM of original sample and amplify through 25 cycles: 1 min at 94 °, 1 min at 42 °, and 2 min at 72°. Reamplification Oligonucleotides. A variety of oligonucleotides may be used during reamplification, including the original primer used for primary PCR, or an oligonucleotide consisting solely of the unique component of the (dT)-X oligonucleotide, or any oligonucleotide having a 3' stretch of dT linked to another sequence. The latter type of reamplification oligonucleotide provides a useful way of introducing new restriction sites, bacteriophage promoter sequences, etc. Dilution of Original Sample. Efficient reamplification appears to be inhibited in part by a product of the primary PCR. This can be overcome by diluting the original sample at least 100 times into the fresh PCR. Sample Analysis Successful amplification will produce samples containing a heterogeneous collection of 3' cDNA fragments representing all of the poly(A) mRNA added to the starting cDNA reaction. On examination by agarose gel electrophoresis (I .5-2% agarise/Tris-borate), 3-5 ~l of the completed PCR sample will produce a strong ethidium bromide-stained distribution

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from 100-700 base pairs in length. Frequently, a biphasic distribution will be apparent that is reduced if the sample is treated with single-stranded nucleases, suggesting that it may reflect the presence of both single- and double-stranded DNA. Genes expressed in the sample can be conveniently examined by conventional Southern blotting procedures as well as dot or slot-blot techniques. However, in our hands, Southern hybridization of amplified material is 10-50 times more sensitive than dot/slot blotting of the same poly(A) amplified sample. This appears to be due to an interfering component of the PCR that separates from the amplified cDNA during electrophoresis.

Bidirectional Southern Transfer A convenient way of providing multiple filters for hybridization analysis is the following adaptation of the bidirectional transfer7 protocol. Add 3-5/~1 of each poly(A) sample directly after PCR to 10/~1 of a suitable gel loading buffer containing bromphenol blue (BPB). Load a 1.5-2% agarose/Tris-borate gel together with size markers and run until the BPB has migrated approximately 3-4 cm. After photographing the ethidium bromide-stained gel, soak it for 30 min in 0.5 M NaOH/1.5 M NaC1, rinse in water, and soak in 1 M ammonium acetate for a further 30 min. Soak two sheets of GeneScreen Plus (Du Pont, Wilmington, DE) for a few minutes in 1 M ammonium acetate. Transfer the DNA by layering on a level surface the following in order: a 3-cm pile of paper towels, one sheet of Whatman (Clifton, N J) 3MM filter paper wetted with 1 M ammonium acetate, one of the soaked GeneScreen Plus filters, the processed gel, the second GeneScreen Plus filter, another Whatman 3MM sheet, and a 3-cm pile of paper towels. Compress the entire sandwich by placing a 0.5 kg weight on top. Although over 90% of the DNA will be transfered within 1-2 h, the transfer setup may be left overnight. The same transfer procedure has been used successfully for nitrocellulose and a variety of commercial nylon membranes. For Hybond-N + (Amersham), the DNA was efficiently transferred directly after denaturation in 0.5 M NaOH, 1.5 M NaC1. DNA was fixed to the membrane following the manufacturer's recommendations. Because the proportion of DNA transferred to top and bottom filters is variable, estimation of relative hybridization intensities must be compared to standards included on the same filter.

Nylon Membrane Hybrid&ation This is based on the procedure recommended for GeneScreen Plus. Prehybridize the filter 2-20 hr at 42 ° in a minimum volume of the following v G. E. Smith and M. D. Summers, Ann. Biochem. 109, 123 (1980).

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mixture: 50% deionized formamide, 1 M NaCI, 1% sodium dodecyl sulfate (SDS), 10% dextran sulfate, 5/xg/ml denatured oligo(dA)/(dT) (optional, see below), and 25/xg/ml denatured herring sperm DNA. Add denatured radiolabeled probe to a final concentration of approximately 2 x 106 counts/min (cpm)/ml and hybridize for 12-48 hr. Washing Conditions. For each probe the stringency of washing necessary to produce strong, clean signals is variable owing to the small size of hybridized fragments and varying A/T richness. Generally, washing for 1 hr at 65 ° in 2 × SSC followed by a second wash for 1 hr further in 0.5 x SSC at 65 ° will produce clear signals. If background is excessive, increase the temperature to 70 ° and/or decrease the final wash to 0.1 × SSC. General Background. The most frequent cause of "spotty" background is the presence of particulate material in the hybridization buffer or from gloves worn when handling the filter (J. Jongstra, personal communication). This can be eliminated by filtering either the complete buffer or each of the individual components and by avoiding powdery gloves. Diffuse darkening of the filter is usually associated with preparation of individual probes and is often eliminated by reisolating the problem probe. Oligo(dA)/(dT). Because 3' sequences are frequently A/T-rich, some probes exhibit an A/T-dependent background. This can be reduced by adding a 20-base oligonucleotide consisting of random additions of either dA or dT at each position to the hybridization mix. Choice of Probes. Because of the 3' nature of the amplified product, detection of an expressed sequence requires that the probe include sequences close to the extreme 3' end of the native transcript. The ideal probe would consist of 400 bases of 3' cDNA sequence directly abutting the polyadenylation site. Where 3' probes are not readily available, the following adaption of the rapid amplification of cDNA ends (RACE) protocol 8 provides a means of generating a usable probe provided some 3' sequence information is available.

Rapid Amplification of cDNA Ends Note that all oligonucleotides used in the procedure are adjusted to 50 OD260/ml. Prepare the cDNA reaction containing the following: cDNA lysis buffer (see cDNA section) 46 ~1 R N A containing gene of interest 1 t~l (10 ng-1 t~g) 25 mM dNTPs 1/xl (dT) + oligonucleotide 1 ~1 s M. A. Frohman, M. K. Dush, and G. R. Martin, Proc. Natl. Acad. Sci. U.S.A. 85~ 8998 (1988).

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Heat for 5 min at 65 °, cool at room temperature for 5 min, add 200 units Moloney reverse transcriptase (GIBCO-BRL), incubate for 30 min at 37°, and heat-inactivate for 10 min at 65°. Set up the PCR as follows: cDNA reaction 10/xl l0 x Taq polymerase buffer 10/xl 25 mM dNTPs 2/xl Unique sequence of the (dT) + oligonucleotide 1.5/zl 24-Base specific-sense oligonucleotide 1/xl Water 75/zl Boil for 5 min, add 5 units Taq polymerase, overlay with mineral oil if necessary, incubate at 72 ° for 30 min, and transfer a thermocycling machine for 25 rounds of 1 min at 95 °, 1 min at 42°, and, depending on the size of fragment, I-5 min at 72 °. Annealing Temperature and Elongation Time. If a large amount of nonspecific material is apparent after amplification, increase the annealing temperature until only specific products remain. Since extended elongation times can also contribute to nonspecific products, the 72° step in the PCR should be kept to the minimum time required for the target sequence.

Cloning into Bacterial Vectors

Additional Polymerization Step After the final round of PCR a varying amount of denatured singlestranded material may be left. To maximize the proportion of clonable double-stranded material, an additional round of polymerization is advisable. For 10 tzl of the original PCR material add 50/xl fresh Taq buffer containing additional nucleotides and oligonucleotide at the same concentration as the starting PCR. Boil the mixture for 2 min, anneal to the primer for 4 min at 42 °, add 5 units Taq polymerase, and incubate for 30 min at 72°. Extract the sample with ether or chloroform to remove residual mineral oil and ethanol-precipitate by adding 30 /zl 7.5 M ammonium acetate and 200/zl ethanol. After 30 min on ice, spin down the DNA and wash two times with 70% ethanol, dry, and resuspend in 10/.d TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA).

Restriction Digestion Bring the entire sample to 100/zl in restriction buffer (50 mM TrisHC1, pH 8.0, 100 mM NaC1, 10 mM MgCI2) and digest with 100 units EcoRI at 37° for 2 hr to overnight. Complete digestion will be apparent by a slight reduction in overall size and the appearance of a 36-bp fragment

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equivalent to the unique component of the amplification oligonucleotide. Because this small fragment contains EcoRI ends, it will compete with the cDNA sequences during the vector ligation step and therefore must be removed either by a sizing column or by elution of the cDNA from an agarose gel. (Note: Taq polymerase is still present in the sample and may interfere with some enzymatic modifications, making it necessary to purify further by proteinase K digestion and phenol-chloroform extraction.)

Ligation and Library Construction For the construction of poly(A) cDNA libraries, it is advisble to titrate the amount of insert against a constant amount of dephosphorylated vector. Under these conditions the absolute number of colonies/plaques produced will increase with increasing amount of insert and will reach a plateau as the number of vector molecules become limiting. Libraries consisting of primarily single inserts will be produced if an insert concentration is used where vector molecules are not limiting. The ligation buffer used for both h and plasmid cloning is a dilution of 5 × LIP [250 mM TrisHC1 (pH 7.6), 50 mM MgC12, 2.5 mM ATP, 5 mM DTT, and 25% (w/v) polyethylene glycol 8000]. Plasmid Vectors. A typical titration series would be as follows: Insert 0, 1, 2, 4, 8 ng Dephosphorylated plasmid 50 ng 5 × LIP 5/.d Water Final volume of 50/zl T4 DNA ligase 40 units Ligate for 1 hr with the tubes in a beaker of 22 ° (room temperature) water. Place the entire beaker and samples in a sealed Styrofoam container and transfer to 4 ° overnight. Transform 10/zl into competent recombinationdefective bacteria and count the resulting colonies to establish a suitable insert concentration for library construction. Lambda Vectors. Set up the equivalent of the following: Insert 0, 1, 2, 4, 8 ng Dephosphorylated h vector 400 ng 5 x LIP 0.6/,1 Water Final volume of 6 tzl T4 DNA ligase 20 units Ligate as described for plasmid cloning and package 1/zl of the ligation into h phage particles (commercial kits are available and suitable). Plate out a range of dilutions (1 : 1000 to 1 : 10) and estimate a suitable insert concentration for library construction.

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Screening and Analysis of Clones Standard molecular procedures can be used for both screening and examination of libraries and individual clones generated from poly(A) PCR material. Because of the abbreviated nature of the amplified material, specific probes must include the extreme 3' sequences in order to detect positive clones in a poly(A)-derived library.

Differential Library Screening Poly(A)-derived PCR material can also be used as a labeled probe to screen either poly(A) or conventional c D N A libraries and as such form the basis of a differential screening approach. Briefly, this would entail producing two replica filters for a c D N A library and hybridizing one with labeled poly(A) c D N A from source A and screening the other with labeled material from source B. Individual clones showing strong hybridization with one and not the other probe represent genes whose expression differed in the two starting samples. Acknowledgments We thank M. Barbara for excellent technical assistance, P. A. Johnson, L. Addy, F. Billio, L. Trumpner, X. Q. Yan, and M. Barbara for critical reading of the manuscript, and B. Vennstrom, R. Gronostajski, and M. Minden for helpful discussions and suggestions. The work was supported by operating grants to N.N.I. from the Medical Research Council and National Institute of Canada. G.B. is a Special Fellow of the Leukemia Society of America.

[37] C o n s t r u c t i o n Chromosome

and Characterization of Yeast Artificial Libraries from the Mouse Genome

By ZOIh LARIN, ANTHONY P. MONACO, SEBASTIAN MEIER-EWERT, and HANS LEHRACH Introduction

The generation of yeast artificial chromosome (YAC) libraries ~ with large D N A inserts provides a powerful tool for contributing to the longrange physical, genetic, and functional mapping of the murine genome. It should now be feasible to construct murine genetic maps that contain I D. T. Burke, G. F. Carle, and M. V. Olson, Science 236, 806 (1987).

METHODS IN ENZYMOLOGY, VOL. 225

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.