- Email: [email protected]
Constructing yeast libraries
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[3] Constructing Yeast Libraries By HAOPING LIU
Introduction Having a good library is crucial for gene cloning in Saccharomyces cerevisiae. Different types of libraries are used for different applications. The most frequently used approach in cloning a yeast gene is complementation of a recessive mutant with a yeast genomic library on a low-copy-number yeast vector. Literally, hundreds of yeast genes have been cloned this way. Another popular approach that has efficiently identified many yeast genes is by functional cloning from overexpression libraries, based on the suppression of a recessive mutant by gene overexpression or other phenotypic consequences associated with overexpression. Overexpression libraries include high-copy-number yeast genomic libraries or cDNA libraries under the control of a strong inducible promoter. Libraries under the control of inducible promoter allows the isolation of genes that are toxic at elevated level and of low abundant genes that are not expressed to a level high enough from a high-copy-number vector. Other approaches of functional cloning in yeast include phage library screens, two-hybrid interactions, and insertional mutagenesis, some of which are described elsewhere in this volume.la-c The easiest way of obtaining a library is by mail. The last volume of this book listed several S. cerevisiae libraries, ld Since then, many new libraries have been constructed, and some of them are listed in Table I. These libraries are constructed with more recent multipurpose yeast expression vectors. Therefore, they should give higher yield in DNA preparation and be more convenient in molecular manipulation of the cloned genes. Table I also lists some Candida albicans genomic libraries. Although numerous yeast genomic libraries are available, many circumstances require the construction of a new library. Methods for de novo construction of yeast genomic libraries and considerations involved in the process have been described in the previous volume in this seriesJ d Here I would like to describe two specific strategies that have been used successfully for the construction of several yeast libraries. One strategy should be applicable to the construction of any genomic library, and the other for any cDNA libraries.
la M. Fromont-Racine, J.-C. Rain, and P. Legrain, Methods EnzymoL 350, [29], 2002 (this volume). lb j. E Gesa, T. R. Hagbun, and S. Fields, Methods Enzymol. 350, [28], 2002 (this volume). lc A. Kumar, S. Vidan, and M. Soryder, Methods Enzymol. 350, [12], 2002 (this volume). ld M. D. Rose and J. R. Broach, Methods Enzymol. 194, 195 (1991).
METHODSIN ENZYMOLOGY,VOL.350
Copyright2002,ElsevierScience(USA). All fightsreserved. 0076-6879/02 $35.00
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CONSTRUCTING YEAST LIBRARIES
i
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ii i i i .i.i! i !iiii
73
74 Construction
BASIC TECHNIQUES of Yeast Genomic
[31
Libraries
Cloning Strategy
To clone a gene with dominant alleles or a gene whose activity is absent from the strains used for the construction of available yeast libraries, it is necessary to construct a new genomic library d e n o v o . A major concern in constructing a genomic library is to prevent vector self-ligation, so that most o f the clones in the library contain a genomic D N A fragment. This vector self-ligation can be efficiently reduced to minimal by using a cloning strategy where partially filled S a u 3 A I genomic D N A fragments are ligated to partially filled X h o I or S a l I ends o f a vector, as shown in Fig. 1. Partial S a u 3 A I digestion o f yeast genomic D N A generates fragments with Y-GATC as the overhang ends. Filling the ends with d G and d A leaves only 5 ' - G A as the overhang ends. S a l I or X h o I restriction of vector D N A generates Y - T C G A as the overhang ends. Filling in the ends with dT and dC leaves only 5'-TC for base pairing. Therefore, the 5'-TC overhang of the vector can only pair with the 5 ' - G A overhang o f genomic D N A fragments. Neither the inserts nor the vector can self-ligate. This strategy has been used successfully for the construction of several yeast genomic libraries 3,4,15 and is highly recommended for anyone interested in constructing a new genomic library. P r e p a r i n g C u t Vector
Currently used yeast vectors are described elsewhere in this volume. Yeast centromeric plasmids should be used to avoid cross-suppressing clones if the goal is to clone a gene by complementing a recessive mutation in yeast or to isolate a dominant allele o f a gene from a particular mutant strain. The vector should have a selectable marker usable for the transformation o f the recipient strain. The vector 2 H. Liu, J. Krizek, and A. Bretscher, Genetics 132, 665 (1992). 3 C. M. Thompson, A. J. Koleske, D. M. Chao, and R. A. Young, Cell 73, 1361 (1993). 4 H. Liu, C. A. Styles, and G. R. Fink, Genetics 144, 967 (1996). 5 E Hieter, personal communication (1992). 6 C. Coo.nellyand E Hieter, personal communication (1992). 7 H. V. Goodson, B. L. Anderson, H. M. Warrick, L. A. Pon, and J. A. Spudich,J. Cell Biol. 133, 1277 (1996). 8 H. Takagi, M. Shichiri, M. Takemura, M. Mob_d,and S. Nakamori, J. Bacteriol. 182, 4249 (2000). 9 S. W. Ramer, S. J. Elledge, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 89, 11589 (1992). 10K. Thevissen, B. E Cammue, K. Lemaire, J. Winderickx,R. C. Dickson, R. L. Lester, K. K. Ferket, E Van Even, A. H. Parret, and W. E Broekaert, Proc. Natl. Acad. Sci. U.S.A. 97, 9531 (2000). 11A. M. Gillum, E. Y. Tsay, and D. R. Kitsch, Mol. Gen. Genet. 198, 179 (1984). 12A. K. Goshorn, S. M. Grindle, and S. Scherer, Infect. Immun. 60, 876 (1992). 13M. E. Fling, J. Kopf, A. Tamarkin,J. A. Gorman, H. A. Smith, and Y. Koltin, Mol. Gen. Genet. 227, 318 (1991). 14A. Rosenbluh, M. Mevarech, Y. Koltin, and J. A. Gorman, Mol. Gen. Genet. 200, 500 (1985). 15H. Liu, J. Kohler, and G. R. Fink, Science 266, 1723 (1994).
[3]
CONSTRUCTINGYEASTLmRARIES
Genomic DNA
~ Sau3A I partial digestion 5'-GATC
Jf Sal I ~
~TAG-5' -
Fill-in with dATP and dGTP 5'-GATC AG
75
G
Jr Fill-in with dCTP and dTTP
,~TAG-5'
CAGCTAG'
CTAGCTG
~
Transformation
Genomic library
FIG. 1. A strategyfor constructinggenomiclibraries. must have a unique SalI or XhoI site, which is preferably located in the middle of the polylinker to allow the efficient recovery of inserts. Once the vector is chosen, follow the following steps to prepare cut vector. 1. Digest the vector DNA with Sail or XhoI to completion. Use overnight digestion followed by adding more of the enzyme for 2 additional hr of digestion to ensure that the cleavage is thorough. Extract DNA with phenol and precipitate with ethanol. 2. Fill in the ends with dCTP and dTTP in a 100-/zl reaction. 25/zg DNA 10/zl of 10x Restriction buffer for SalI or XhoI 5/zl of 0.5 mM dCTP 5/zl of 0.5 mM d T r P 10-15 U Klenow fragment Bring the reaction to 100/zl with H20. Incubate at 30 ° for 30 min. 3. Ligate overnight and purify linear DNA away from the ligated circular vector. Precipitate DNA and set up a ligation reaction with T4 DNA ligase for
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overnight. This will ligate any molecules whose ends are not filled in. Then, purify linear DNA away from ligated circular DNA by agarose gel electrophoresis, and extract linear DNA from gel slices with QIAEX gel extraction kit (from Qiagen, Valencia, CA). Resuspend the DNA to 100 ng/#l. This ligation step significantly reduces the background of vector self-ligation.
Preparation of Fragmented Yeast Genomic DNA For yeast genomic DNA isolation, we have used the procedure described in the previous volume of this series. 16 An outline of a scaled-up preparation with the procedure is given here. 1. Grow 250 ml yeast culture in YPD overnight to saturation. 2. Spin down cells, resuspend in 25 ml of SE buffer (0.9 M sorbitol, 0.1 M EDTA, pH 8.0). 3. Spin down cells, resuspend in 10 ml SE buffer, add 10/xl of 2-mercaptoethanol (2-ME). 4. Add 2.5 ml of 2 mg/ml Zymolyase 100K (ICN, Costa Mesa, CA) in SE buffer. Incubate 45 min at 37 ° until spheroplasts are formed. 5. Spin 5 min at 5000 rpm. Resuspend gently in 20 ml TE. 6. Add 4.5 ml TSE solution [1.2 ml 2 M Tris, pH 7.6, 1.2 ml 10% sodium dodecyl sulfate (SDS), 3 ml 0.5 M EDTA, pH 8.0], mix, and incubate for 30 min at 65 °. 7. Add 4 mi of 5 M potassium acetate, then place on ice for at least 60 min. 8. Spin 20 min at 10,0OO rpm. Transfer the supernatant and precipitate by adding at least two volumes of ethanol. 9. Spin 5 min at 8000 rpm. Rinse pellet with 70% (v/v) ethanol. 10. Resuspend pellet in 12.5 ml TE (let the pellet resuspend overnight). 11. Spin out debris. Transfer supernatant and add 65 /zl of 10 mg/ml RNase A. Incubate for 30 min at 37 °. 12. Add 13 ml 2-propanol, mix gently, spin, rinse pellet with 70% ethanol. Air-dry. Resuspend DNA in 1 ml TE overnight. To obtain an optimal Sau3AI partial digestion ofgenomic DNA, we recommend using different dilutions of Sau3AI in the digestion reactions. For example, use 1 : 1OO, 1:50, 1:25, and 1:10 dilutions of 4U//zl Sau3AI (in 50% glycerol)in the following reaction: 50/zl genomic DNA of 1/zg//zl 50/zl 10x Sau3AI restriction buffer 16p. Philippsen, A. Stotz, and C. Scherf, Methods Enzymol. 194, 169 (1991).
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390 #1 H20 10 #1 Sau3AI Incubate the reactions at room temperature. Remove 250 #1 at 15 min and let the remaining reactions to incubate for another 15 min. Stop digestions by phenol/chloroform extraction. Precipitate DNA. To determine which is the optimal reaction of partial Sau3AI digestion, fractionate the Sau3AI digested DNA on a 0.7% agarose gel. Run the rest of the appropriate reactions on an agarose gel and cut out the region that contains DNA fragments of desired size. The size of inserts is determined based on the ease of screening or selection method that will be used with the library. Use a QIAEX gel extraction kit (Qiagen) to extract DNA from gel slices. Determine the amount of DNA. Fill in the Sau3AI overhangs with dATP and dGTP by Klenow, using the same reaction condition as described for the SalI cut vector. Extract with phenol/ chloroform twice. Precipitate DNA with ethanol. Wash and dry. Resuspend DNA to obtain a DNA concentration around 300-500 ng//zl.
Assembly of Library We have used the following condition for ligation reactions (5/zl): 1 #1 genomic DNA fragments at 300-500 ng//zl 1 #1 cut vector DNA at 100 ng//zl 0.5/zl 10× T4 DNA ligase buffer 0.5/zl T4 DNA ligase, about 4 units 1.5/zl H20 Incubate overnight at 16° . A control ligation reaction without the genomic DNA fragments should be set up at the same time. We recommend the use of electrocompetent Escherichia coli or commercially available competent cells in library transformation, because high transformation efficiency is important for boosting the size and complexity of the library. Transformation efficiency from the reaction with genomic inserts should be at least 10-50 times higher than the control. If the control ligation gives a transformation efficiency that is too high, the filling of vector overhangs with dC and dT may have not occurred properly. If the ligation with genomic inserts does not give high transformation efficiency, the dA and dG filling of genomic fragments may be a problem. Whether a ligation reaction has proceeded properly can be examined by running the remaining ligation reaction on an agarose gel next to the cut vector
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DNA. Successfully ligated DNA should show a smear above the position of the cut vector. The quality of a genomic library is determined by its coverage of the genome and the percentage of clones carrying a genomic insert. The second standard can be assessed by randomly picking some clones to perform restriction digestions with enzymes that release the insert from the vector. The average size of inserts, the percentage of clones with inserts, and the number of colonies from initial transformation can be used to estimate the size of the library in terms of its genome coverage. C o n s t r u c t i o n o f Y e a s t cDNA L i b r a r i e s Cloning Strategy The currently used yeast cDNA library is constructed from mRNA expressed in Mata cells grown in YPD medium and is therefore limited to genes transcribed in this cell type and growth condition. A new cDNA library needs to be constructed if one is interested in identifying genes expressed in other cell types or growth conditions, such as meiosis or starvation and other stress conditions, with a cDNA library. The major consideration in designing a cloning strategy for the construction of a cDNA library is unidirectional ligation of cDNA inserts into the vector. The restraint in ligation orientation is essential to render the expression of cDNA inserts under the control of a promoter from the vector as well as to eliminate vector self-ligation. Directionality is obtained typically by introducing two different restriction endonuclease sites at the ends of cDNA, which is initiated by using a primer-adapter to initiate first strand synthesis. The SuperScript Plasmid System for cDNA synthesis and plasmid cloning from Invitrogen Corporation features this strategy in their design (Fig. 2), and the system has been used successfully in constructing a yeast cDNA library. 2 A NotI primer-adapter, which contains 15 dT and restriction sites of NotI and other enzymes, is used to prime the first strand cDNA synthesis at the poly(A) tail of mRNAs (Fig. 2). The product of the first and second strand reactions is blunt-ended cDNA, to which SalI adapters are added. The SalI adapter is duplex oligomers that are blunt-ended at one terminus and contains a 4-base overhang at the other terminus. Only the blunt-ended 5'-end of the SalI adapter is phosphorylated, which eliminates self-ligation of the adapters during ligation to the cDNA (Fig. 2). The SalI adapter also contains an additional MluI site which can be used, together with Nod, to release the cDNA insert from the vector. After the addition of SalI adapters to both termini, the NotI terminus is exposed by NotI digestion. Thus, the 3' end of the cDNA is identified with NotI, and the 5t end with SalI. The cDNA can then be ligated to a SalI- and NotI-digested vector, with the SalI site positioned at the terminus with an inducible promoter (Fig. 2).
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CONSTRUCTING YEAST LmRARIES
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InlRNA -AAAAAA TTTTTTCCCGCCGGCG
~ First strand synthesis "AAA.~A "TTTTI"rCCCGCCGGCG
i, Second strand synthesis ~GGGCGGCCGC ~CCGCCGGCG
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addition
~ -'~C~l-~ TTTTTTCCCGCCGGCC
Sal I
~111
JFNot I digestion,
ro moter
size fractionation
~ Sail & Not I digestion
~ ' q " I " P ~ C C GCC GG Sal I
~ Transformation
cDNA library FIG.2. A strategyfor constructingcDNAlibraries (adaptedwith permissionfrom the SuperScript Plasmid Systemfor cDNAsynthesis and plasmidcloning of InvitrogenCorporation).
Preparation of Cut Vector A YCp vector is usually used to construct a cDNA library. The vector must contain an inducible promoter, preferably with a high level of expression under the induction condition and completely off under a noninducing condition. If the cloning strategy in Fig. 2 will be used for this construction, the vector must also contain a unique NotI site in the polylinker region and one Sail or XhoI site between the NotI and the inducible promoter. To prepare the cut vector, digest the vector DNA with NotI to completion. Monitor the digestion on an agarose gel. Digest the NotI cut vector with SalI and gel-purify the linear DNA. Extract the cut DNA from gel slices with a QIAEX gel extraction kit. Because the NotI and SalI sites are close to each other in the polylinker region of the vector, it is difficult to determine whether the second digestion is complete.
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To monitor the completeness of second digestion, we have end-labeled 3% of NotI-digested DNA with 3Zp and then digested the DNA with the second enzyme, SalI. The extent of SalI digestion can be checked by digesting with an enzyme that cuts the vector backbone. Two 32p-labeled bands are expected if the second digestion has been complete. A partial SalI digestion should give three 32p bands. Alternatively, transformation efficiency of ligated SalI-digested vector can be used to assess whether the second digestion is complete. Low transformation efficiency is expected if the second digestion has occurred successfully. As described in the previous section for preparing cut vector, ligating the NotI- and SalI-digested vector, followed by gel purifying the linear unligated DNA, can be used to purify double digested vector DNA away from single restricted vector. Total RNA Extraction and mRNA Purification Construction of a good cDNA library begins with the preparation of highquality mRNA. Generally, 5/xg of mRNA is sufficient to construct a cDNA library containing about 106 clones in E. coll. Since only 1.3 to 1.4% of a total RNA population in yeast is mRNA, it is necessary to start with at least several milligrams of total RNA. To avoid RNase contamination, it is important to use sterile disposable plasticware and diethyl pyrocarbonate (DEPC)-treated H20 throughout RNA purification. The hot acidic phenol method 17 works well for the extraction of total RNA from yeast. For a scaled-up preparation, about 101° (500 ml culture) S. cerevisiae cells in mid-exponential phase are collected in 50-ml polypropylene tubes, washed once with ice-cold water. Resuspend cells in 12 ml TES (10 mM Tris-Cl, pH 7.5, 10 mM EDTA, 0.5% SDS). Add 12 ml acid phenol (unbuffered liquefied phenol from Sigma, St. Louis, MO) to the tube and vortex vigorously for 10 sec. Incubate for 30 to 60 min at 65 ° with occasional vortexing. Centrifuge and transfer the aqueous (top) phase to a clean 50-ml tube, add 12 ml acid phenol, vortex vigorously, and centrifuge 10 min at 3000 rpm in a swing-bucket rotor in a SorvaU table top centrifuge. Transfer aqueous phase to a new tube, add 1/10 volume of 3 M sodium acetate, pH 5.3, and 2 volumes of ice-cold 100% ethanol, and precipitate. Centrifuge at top speed for 30 min at 4 °. Wash the RNA pellet with 70% ice-cold ethanol. Centrifuge, air-dry the RNA pellet, and resuspend the pellet in 0.5 ml H20. Fully dissolve RNA before measuring OD260. The yield should be around 5 mg. mRNA is isolated from total RNA by oligo(dT) cellulose column chromatography) 8 For 5 mg RNA, use 0.75 ml of oligo(dT)-cellulose in a column. About 17F. M. Ausubel, R. Brent, R. E. Kingston, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (eds.), in "Current Protocols in Molecular Biology," Vol. 1. John Wiley & Sons, New York, 1987. 18 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.
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1/10 of the RNA is expected to be recovered from the first round of purification. Then, the oligo(dT) column is regenerated with 0.1 M NaC1 and equilibrated. The eluted poly(A) mRNA can be loaded onto the oligo(dT) column directly without the need for precipitation for the second round of purification by adjusting the NaC1 concentration of the eluted mRNA to 0.5 M. About 50% of the applied RNA is expected to be recovered in the elution. After three cycles of purification through the oligo(dT) column, most of the tRNA and rRNA should be removed, and the material should be highly enriched in mRNA. Quality of total RNA and purified mRNA should be evaluated by a RNA gel. Because SDS is in the loading buffer of the oligo(dT) column and can interfere with the cDNA synthesis, it is necessary to precipitate and wash the mRNA twice after the column elution to reduce the content of SDS in the mRNA.
cDNA Synthesis and Fractionation For cDNA synthesis and fractionation, we recommend using the SuperScript Plasmid System from Invitrogen Corporation. The system contains all necessary reagents sufficient for three experiments, each converting up to 5 #g of mRNA to fractionated ready-to-ligate cDNA. The instruction manual from Invitrogen, Carlsbad, CA, is very explicit, and if followed closely, one should succeed in making a cDNA library without too much trouble. The important steps for preparing cDNA inserts are highlighted below. First-Strand Synthesis. First strand cDNA synthesis from mRNA is achieved with SuperScript II RT (reverse transcriptase) in the following 20/zl reaction. The engineered RT enzyme SuperScript II does not contain RNase H activity, but still exhibits an improved polymerase activity. 1. Add 2/zl of Nod primer-adapter to 1 to 5/zg of mRNA; dilute as needed to make the final reaction 20/zl (including RT and 8-#1 reagents that will be added in step 2). 2. Heat to 70 ° for 10 min and quick-chill on ice. Spin briefly and add: 4/zl of 5 x First strand buffer 2 #1 of 0.1 M D T T 1 #1 of 10 mM dNTP mix 1/zl of [ot-32p]dCTP (1/zCi//zl) 3. Incubate at 37 ° for 2 min to equilibrate the temperature. Add SuperScript II RT (use 1/zl of RT for each/zg of mRNA used in the reaction). 4. Incubate at 37 ° for 1 hr. Place the tube on ice to terminate the reaction. 5. To determine the yield of first strand synthesis, remove 2 tzl from the reaction and add it to 43 tzl of 20 mM EDTA (pH 7.5) and 5/zl of 1 /zg//zl yeast tRNA. From the diluted sample, spot duplicate 10-/zl aliquots onto glassfiber filters. Dry one to determine the specific activity (SA) of the dCTP
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reaction. Wash the other filter three times, 5 min each, in a beaker containing 50 ml of ice-cold 10% TCA with 1% sodium pyrophosphate, and once in 50 ml of 95% ethanol. Dry the filter to determine the yield of first strand cDNA. The specific activity (SA) is determined by dividing the counts per rain from the 10-#1 aliquot from the unwashed filter with the quantity (in pmol) of the same nucleotide in the 10-/zl aliquot: SA(cpm/pmol dCTP) =
cpm/10/.tl 200 pmol dCTP/10/zm
Once the SA is determined, the amount of cDNA synthesized in the first-strand reaction can be calculated from the amount of acid-precipitable radioactivity. Amount of cDNA(/zg) = (acid-precipitablecpm) x (50/zl/10/zl) x (20 ~1/2/zl) x (4pmol dNTP/pmol dCTP) (SA cpm/pmol dCTP) x (3030 pmol dNTP//xg cDNA) "3030" is the amount of nucleotide equivalent to 10/zg of single-strand DNA. The yield of first-strand synthesis can be calculated by dividing the amount of cDNA synthesized by the amount of starting mRNA. We have had around 38% yield from 3.1 /zg of yeast mRNA. A lower yield of first-strand synthesis does not necessarily indicate that a library cannot be made, since each ligation reaction only requires 10 ng of cDNA. It is critical, however, that the size distribution of the cDNA products be similar to that of the mRNA. Shorter cDNA products may indicate the danger of RNase contamination. Second-Strand Synthesis
1. On ice, add the following reagents to the remaining 18-#1 first-strand reaction: 93/zl of H20 30/zl of 5 x Second strand buffer 3/zl of 10 mM dNTP mix 1 izl E. coli DNA ligase (10 U//xl) 4 Ixl E. coli DNA polymerase I (10 U//xl) 1 Ixl E. coli RNase H (2 U//xl) Final volume is 150/~1. 2. Incubate for 2 hr at 16°. 3. Add 2/zl (10 units) of T4 DNA polymerase, continue incubating at 16° for 5 min. 4. Place the reaction on ice, and add 10/zl of 0.5 M EDTA.
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5. Extract with 150/zl phenol: chloroform: isoamyl alcohol (25 : 24 : 1). Precipitate the cDNA with 70 /xl of 7.5-m ammonium acetate and 0.5 ml ethanol (-20°). Wash with 0.5 ml of 70% ethanol (-20°). Air-dry at 37 ° for 10 min. 6. To determine the quality of synthesized cDNA, run some (5 /zl leftover from the aqueous phase of each phenol extraction is enough) second-strand reactions on an agarose gel and dry the gel. Visualize by exposing the gel to an X-ray film. Compare the range of cDNA smear to that of the starting mRNA to determine the quality of cDNA.
SalI Adapter Addition and NotI Digestion 1. Set up a 50-#1 ligation reaction to add SalI adapter to the cDNA from step 5 of second-strand synthesis. cDNA 25/zl H20 10 #1 5× T4 DNA Ligase buffer 10/zl SalI Adapters 5/zl T4 DNA Ligase 2. Incubate at 16° overnight. 3. Extract with phenol : chloroform :isoamyl alcohol. Precipitate, wash, and air-dry DNA. 4. Set up a 50-/,1 Nod digestion. 5. Incubate for 2 hr at 37 °. 6. Extract with phenol : chloroform : isoamyl alcohol. Precipitate, wash, and air-dry DNA.
Fractionation by Column Chromatography. To ensure the quality of your cDNA library, it is crucial to remove all residual SalI adapters and the NotI fragments released by restriction digestion with NotI. This is accomplished by filtrating the cDNA through a size fractionation column provided by the SuperPlasmid System. 1. Dissolve the NotI digested cDNA in 100 #1 TEN buffer (10 mM Tris-HC1 pH 7.5, 0.1 mM EDTA, 25 mM NaC1). 2. Wash a cDNA fractionation column (provided in the Invitrogen SuperScript Plasmid System) with 0.8 ml TEN buffer four times; let the column drain completely for each wash. 3. Load the 100/zl cDNA to the column; collect flow-through into tube 1. 4. Add 100 #1 TEN buffer to the column and collect into tube 2. Let it drain completely.
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5. From the second 100/zl TEN wash, collect single drop fractions (-'~35/zl) into each microcentrifuge tube. Continue adding 100-/zl aliquots of TEN until a total of 18 drops have been collected into tubes 3 through 20, one drop per tube. 6. Measure the volume in each tube with a fresh tip. Identify the fraction whose cumulative elution volume (sum from tube 1 to this fraction) is closest to but not exceeding 550/zl. Discard all tubes after this fraction. If fractions beyond 550 ~1 are used, the library will contain "empty" clones, which come from ligation of the short NotI-primer-adapter-SalI fragment released from the NotI digestion. 7. Place the remaining tubes in a scintillation counter and obtain Cerenkov counts for each tube in the tritium channel without using scintillation fluid. Counts should increase after reaching 400 to 450/zl of elution volume. Calculate the amount of cDNA in each fraction by using the following equation with the SA determined for the first strand synthesis: Amount of ds cDNA (ng) (Cerenkov cpm) x 2 x (4pmol dNTP/pmol dCTP) x (1000ng//zg ds cDNA) SA(cpm/pmol dCTP) × (1515 pmol dNTP//zg ds cDNA) .
Determine the cDNA concentration for each fraction by dividing the amount of cDNA in each fraction with the volume. If the cDNA concentration is higher than 0.71 ng//zl, 10 ng cDNA from that fraction can be used directly for ligation with the vector without precipitation. Otherwise, the cDNA has to be concentrated. To maximize the average insert size in the cDNA library, cDNA from the earliest fraction should be used for ligation. Often, the earliest fraction does not have enough cDNA and is not concentrated enough to be used directly. In this case, fractions may have to be pooled together and precipitated to obtain the 10 ng of cDNA needed for ligation.
Ligation of cDNA to Vector and Transformation Set up ligation reactions with 10 ng of cDNA and 50 ng of SalI- and NotI-cut vector in 20/zl reactions, as following: 14/xl of 10 ng cDNA in TEN buffer 1/_tl of Notl-SalI cut vector DNA (50 ng//zl) 4 ~1 of 5 x T4 DNA ligase buffer 1/zl of T4 DNA ligase (1 unit//zl ) Incubate for 3 hr at room temperature.
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Ligation reactions from each column fraction should be kept separately as different pools of library. A negative control ligation with the same amount of cutvector DNA, but without cDNA, should be used. In addition, we suggest setting up a control ligation reaction using cDNA from one of the later fractions (less valuable) with the Nod- and SalI-cut pSPORT vector DNA provided in the SuperScript Plasmid System. The transformation efficiency of this reaction will inform you whether a ligation problem is caused by cDNA or by the cut vector. In order to generate a sufficiently large eDNA library, it is important to use the most competent E. coli cells in transformation. Electroporation usually offers higher transformation efficiency. With a successful ligation, we typically obtain around 10 6 transformants per ligation from electroporation of ElectroMAX DH10B cells from Invitrogen (transformation efficiency at 109-101° transformants/#g of supercoiled plasmid). If the transformation efficiency is 100-fold lower, the library size will be on the borderline for genes of low abundance. Transformation efficiency is also a good indication of whether the ligation reaction has worked or not. We expect that the ligation with cDNAs should give 50-100 times more transformants than the negative control without cDNAs. If not, there may be problems with ligation reactions, which can come from either cDNA or the cut vector. If the ligation reaction with the pSPORT vector gives 100-fold higher transformation efficiency than the ligation with the cut vector, the vector preparation may have a problem. If the transformation efficiencies of both ligations are low (<105 transformants/ligation, assuming that supercoiled plasmid yields >109 transformants//zg) the ligation reactions may not be proceeding properly and cDNA is likely the cause of the problem. Since synthesized cDNA has been visualized on an agarose gel and length of cDNA should not affect transformation efficiency, the first and second strand syntheses are not likely to cause the problem. On the other hand, problems in SalI adapter addition and NotI digestion will not be easily detected during the process and will affect the ligation efficiency dramatically. To dissect which of the two steps has gone wrong, the ligation reaction can be checked by running the remaining ligation reaction on an agarose gel next to the cut vector DNA. Successfully ligated DNA should show an upward smear from the position of the cut vector. However, the same upward smear will be seen if either NotI or SalI end of the inserts has ligated to the vector. To pinpoint the cause of a failed ligation, we have digested the ligation reaction with either SalI or NotI and run the digestions on an agarose gel next to the ligated DNA and cut vector DNA. If NotI digestion, for example, can convert the upward smear to a band same size as the cut vector, the SalI end must have not ligated properly. Alternative, the adapter addition step can be monitored by nondenaturing polyacrylamide gel electrophoresis during the process before the final ligation step. If 10/zl of the adapter ligation reaction is run on a 12% acrylamide gel and the DNA is visualized by ethidium bromide staining, the
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ligation of adapters to each other, mostly at their blunt ends but occasionally at the sticky ends, will be evidenced by a ladder of fragments of 16, 32, 60, 88 bp, etc. Although this does not verify that the SalI adaptors have ligated to the cDNA, it does show that the ligation reaction is successful. To examine Nod digestion, 4/zl of NotI digestion can be run on the same 12% acrylamide gel together with SalI ligation reaction. After visualizing the SalI adapter ligation ladder with ethidium bromide, the gel can be dried and exposed to X-ray film to visualize a NotI-SalI fragment of 40 bp, which should be between dimers and quadruples of the SalI adapters.
Determining Quality of Library A good cDNA library has three key characteristics that set it apart from the mediocre counterparts: (1) it should be large enough to contain genes of all genes expressed, including the ones expressed at low abundance, (2) it should contain full-length cDNA inserts, and (3) over 90% of its clones should contain cDNA inserts at the expected orientation. Either a complementation in a yeast mutant strain or a colony hybridization screen with the library could be performed to determine whether the library contains cDNAs for mRNAs of low abundance. The last two stipulations can be assessed by restriction digestion of some randomly picked clones with SalI and NotI and by sequencing each insert with a primer from the promoter in the vector. Acknowledgment I thank Craig M. Thompson for sharing his protocol and expertise in making a yeast genomic library. I thank Gerald R. Fink and Anthony Bretscher for providing laboratory space and support during this work.
BASIC TECHNIQUES
[31
[3] Constructing Yeast Libraries By HAOPING LIU
Introduction Having a good library is crucial for gene cloning in Saccharomyces cerevisiae. Different types of libraries are used for different applications. The most frequently used approach in cloning a yeast gene is complementation of a recessive mutant with a yeast genomic library on a low-copy-number yeast vector. Literally, hundreds of yeast genes have been cloned this way. Another popular approach that has efficiently identified many yeast genes is by functional cloning from overexpression libraries, based on the suppression of a recessive mutant by gene overexpression or other phenotypic consequences associated with overexpression. Overexpression libraries include high-copy-number yeast genomic libraries or cDNA libraries under the control of a strong inducible promoter. Libraries under the control of inducible promoter allows the isolation of genes that are toxic at elevated level and of low abundant genes that are not expressed to a level high enough from a high-copy-number vector. Other approaches of functional cloning in yeast include phage library screens, two-hybrid interactions, and insertional mutagenesis, some of which are described elsewhere in this volume.la-c The easiest way of obtaining a library is by mail. The last volume of this book listed several S. cerevisiae libraries, ld Since then, many new libraries have been constructed, and some of them are listed in Table I. These libraries are constructed with more recent multipurpose yeast expression vectors. Therefore, they should give higher yield in DNA preparation and be more convenient in molecular manipulation of the cloned genes. Table I also lists some Candida albicans genomic libraries. Although numerous yeast genomic libraries are available, many circumstances require the construction of a new library. Methods for de novo construction of yeast genomic libraries and considerations involved in the process have been described in the previous volume in this seriesJ d Here I would like to describe two specific strategies that have been used successfully for the construction of several yeast libraries. One strategy should be applicable to the construction of any genomic library, and the other for any cDNA libraries.
la M. Fromont-Racine, J.-C. Rain, and P. Legrain, Methods EnzymoL 350, [29], 2002 (this volume). lb j. E Gesa, T. R. Hagbun, and S. Fields, Methods Enzymol. 350, [28], 2002 (this volume). lc A. Kumar, S. Vidan, and M. Soryder, Methods Enzymol. 350, [12], 2002 (this volume). ld M. D. Rose and J. R. Broach, Methods Enzymol. 194, 195 (1991).
METHODSIN ENZYMOLOGY,VOL.350
Copyright2002,ElsevierScience(USA). All fightsreserved. 0076-6879/02 $35.00
[3]
CONSTRUCTING YEAST LIBRARIES
i
×~
<
eq
ii i i i .i.i! i !iiii
73
74 Construction
BASIC TECHNIQUES of Yeast Genomic
[31
Libraries
Cloning Strategy
To clone a gene with dominant alleles or a gene whose activity is absent from the strains used for the construction of available yeast libraries, it is necessary to construct a new genomic library d e n o v o . A major concern in constructing a genomic library is to prevent vector self-ligation, so that most o f the clones in the library contain a genomic D N A fragment. This vector self-ligation can be efficiently reduced to minimal by using a cloning strategy where partially filled S a u 3 A I genomic D N A fragments are ligated to partially filled X h o I or S a l I ends o f a vector, as shown in Fig. 1. Partial S a u 3 A I digestion o f yeast genomic D N A generates fragments with Y-GATC as the overhang ends. Filling the ends with d G and d A leaves only 5 ' - G A as the overhang ends. S a l I or X h o I restriction of vector D N A generates Y - T C G A as the overhang ends. Filling in the ends with dT and dC leaves only 5'-TC for base pairing. Therefore, the 5'-TC overhang of the vector can only pair with the 5 ' - G A overhang o f genomic D N A fragments. Neither the inserts nor the vector can self-ligate. This strategy has been used successfully for the construction of several yeast genomic libraries 3,4,15 and is highly recommended for anyone interested in constructing a new genomic library. P r e p a r i n g C u t Vector
Currently used yeast vectors are described elsewhere in this volume. Yeast centromeric plasmids should be used to avoid cross-suppressing clones if the goal is to clone a gene by complementing a recessive mutation in yeast or to isolate a dominant allele o f a gene from a particular mutant strain. The vector should have a selectable marker usable for the transformation o f the recipient strain. The vector 2 H. Liu, J. Krizek, and A. Bretscher, Genetics 132, 665 (1992). 3 C. M. Thompson, A. J. Koleske, D. M. Chao, and R. A. Young, Cell 73, 1361 (1993). 4 H. Liu, C. A. Styles, and G. R. Fink, Genetics 144, 967 (1996). 5 E Hieter, personal communication (1992). 6 C. Coo.nellyand E Hieter, personal communication (1992). 7 H. V. Goodson, B. L. Anderson, H. M. Warrick, L. A. Pon, and J. A. Spudich,J. Cell Biol. 133, 1277 (1996). 8 H. Takagi, M. Shichiri, M. Takemura, M. Mob_d,and S. Nakamori, J. Bacteriol. 182, 4249 (2000). 9 S. W. Ramer, S. J. Elledge, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 89, 11589 (1992). 10K. Thevissen, B. E Cammue, K. Lemaire, J. Winderickx,R. C. Dickson, R. L. Lester, K. K. Ferket, E Van Even, A. H. Parret, and W. E Broekaert, Proc. Natl. Acad. Sci. U.S.A. 97, 9531 (2000). 11A. M. Gillum, E. Y. Tsay, and D. R. Kitsch, Mol. Gen. Genet. 198, 179 (1984). 12A. K. Goshorn, S. M. Grindle, and S. Scherer, Infect. Immun. 60, 876 (1992). 13M. E. Fling, J. Kopf, A. Tamarkin,J. A. Gorman, H. A. Smith, and Y. Koltin, Mol. Gen. Genet. 227, 318 (1991). 14A. Rosenbluh, M. Mevarech, Y. Koltin, and J. A. Gorman, Mol. Gen. Genet. 200, 500 (1985). 15H. Liu, J. Kohler, and G. R. Fink, Science 266, 1723 (1994).
[3]
CONSTRUCTINGYEASTLmRARIES
Genomic DNA
~ Sau3A I partial digestion 5'-GATC
Jf Sal I ~
~TAG-5' -
Fill-in with dATP and dGTP 5'-GATC AG
75
G
Jr Fill-in with dCTP and dTTP
,~TAG-5'
CAGCTAG'
CTAGCTG
~
Transformation
Genomic library
FIG. 1. A strategyfor constructinggenomiclibraries. must have a unique SalI or XhoI site, which is preferably located in the middle of the polylinker to allow the efficient recovery of inserts. Once the vector is chosen, follow the following steps to prepare cut vector. 1. Digest the vector DNA with Sail or XhoI to completion. Use overnight digestion followed by adding more of the enzyme for 2 additional hr of digestion to ensure that the cleavage is thorough. Extract DNA with phenol and precipitate with ethanol. 2. Fill in the ends with dCTP and dTTP in a 100-/zl reaction. 25/zg DNA 10/zl of 10x Restriction buffer for SalI or XhoI 5/zl of 0.5 mM dCTP 5/zl of 0.5 mM d T r P 10-15 U Klenow fragment Bring the reaction to 100/zl with H20. Incubate at 30 ° for 30 min. 3. Ligate overnight and purify linear DNA away from the ligated circular vector. Precipitate DNA and set up a ligation reaction with T4 DNA ligase for
76
BASIC TECHNIQUES
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overnight. This will ligate any molecules whose ends are not filled in. Then, purify linear DNA away from ligated circular DNA by agarose gel electrophoresis, and extract linear DNA from gel slices with QIAEX gel extraction kit (from Qiagen, Valencia, CA). Resuspend the DNA to 100 ng/#l. This ligation step significantly reduces the background of vector self-ligation.
Preparation of Fragmented Yeast Genomic DNA For yeast genomic DNA isolation, we have used the procedure described in the previous volume of this series. 16 An outline of a scaled-up preparation with the procedure is given here. 1. Grow 250 ml yeast culture in YPD overnight to saturation. 2. Spin down cells, resuspend in 25 ml of SE buffer (0.9 M sorbitol, 0.1 M EDTA, pH 8.0). 3. Spin down cells, resuspend in 10 ml SE buffer, add 10/xl of 2-mercaptoethanol (2-ME). 4. Add 2.5 ml of 2 mg/ml Zymolyase 100K (ICN, Costa Mesa, CA) in SE buffer. Incubate 45 min at 37 ° until spheroplasts are formed. 5. Spin 5 min at 5000 rpm. Resuspend gently in 20 ml TE. 6. Add 4.5 ml TSE solution [1.2 ml 2 M Tris, pH 7.6, 1.2 ml 10% sodium dodecyl sulfate (SDS), 3 ml 0.5 M EDTA, pH 8.0], mix, and incubate for 30 min at 65 °. 7. Add 4 mi of 5 M potassium acetate, then place on ice for at least 60 min. 8. Spin 20 min at 10,0OO rpm. Transfer the supernatant and precipitate by adding at least two volumes of ethanol. 9. Spin 5 min at 8000 rpm. Rinse pellet with 70% (v/v) ethanol. 10. Resuspend pellet in 12.5 ml TE (let the pellet resuspend overnight). 11. Spin out debris. Transfer supernatant and add 65 /zl of 10 mg/ml RNase A. Incubate for 30 min at 37 °. 12. Add 13 ml 2-propanol, mix gently, spin, rinse pellet with 70% ethanol. Air-dry. Resuspend DNA in 1 ml TE overnight. To obtain an optimal Sau3AI partial digestion ofgenomic DNA, we recommend using different dilutions of Sau3AI in the digestion reactions. For example, use 1 : 1OO, 1:50, 1:25, and 1:10 dilutions of 4U//zl Sau3AI (in 50% glycerol)in the following reaction: 50/zl genomic DNA of 1/zg//zl 50/zl 10x Sau3AI restriction buffer 16p. Philippsen, A. Stotz, and C. Scherf, Methods Enzymol. 194, 169 (1991).
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390 #1 H20 10 #1 Sau3AI Incubate the reactions at room temperature. Remove 250 #1 at 15 min and let the remaining reactions to incubate for another 15 min. Stop digestions by phenol/chloroform extraction. Precipitate DNA. To determine which is the optimal reaction of partial Sau3AI digestion, fractionate the Sau3AI digested DNA on a 0.7% agarose gel. Run the rest of the appropriate reactions on an agarose gel and cut out the region that contains DNA fragments of desired size. The size of inserts is determined based on the ease of screening or selection method that will be used with the library. Use a QIAEX gel extraction kit (Qiagen) to extract DNA from gel slices. Determine the amount of DNA. Fill in the Sau3AI overhangs with dATP and dGTP by Klenow, using the same reaction condition as described for the SalI cut vector. Extract with phenol/ chloroform twice. Precipitate DNA with ethanol. Wash and dry. Resuspend DNA to obtain a DNA concentration around 300-500 ng//zl.
Assembly of Library We have used the following condition for ligation reactions (5/zl): 1 #1 genomic DNA fragments at 300-500 ng//zl 1 #1 cut vector DNA at 100 ng//zl 0.5/zl 10× T4 DNA ligase buffer 0.5/zl T4 DNA ligase, about 4 units 1.5/zl H20 Incubate overnight at 16° . A control ligation reaction without the genomic DNA fragments should be set up at the same time. We recommend the use of electrocompetent Escherichia coli or commercially available competent cells in library transformation, because high transformation efficiency is important for boosting the size and complexity of the library. Transformation efficiency from the reaction with genomic inserts should be at least 10-50 times higher than the control. If the control ligation gives a transformation efficiency that is too high, the filling of vector overhangs with dC and dT may have not occurred properly. If the ligation with genomic inserts does not give high transformation efficiency, the dA and dG filling of genomic fragments may be a problem. Whether a ligation reaction has proceeded properly can be examined by running the remaining ligation reaction on an agarose gel next to the cut vector
78
BASIC TECHNIQUES
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DNA. Successfully ligated DNA should show a smear above the position of the cut vector. The quality of a genomic library is determined by its coverage of the genome and the percentage of clones carrying a genomic insert. The second standard can be assessed by randomly picking some clones to perform restriction digestions with enzymes that release the insert from the vector. The average size of inserts, the percentage of clones with inserts, and the number of colonies from initial transformation can be used to estimate the size of the library in terms of its genome coverage. C o n s t r u c t i o n o f Y e a s t cDNA L i b r a r i e s Cloning Strategy The currently used yeast cDNA library is constructed from mRNA expressed in Mata cells grown in YPD medium and is therefore limited to genes transcribed in this cell type and growth condition. A new cDNA library needs to be constructed if one is interested in identifying genes expressed in other cell types or growth conditions, such as meiosis or starvation and other stress conditions, with a cDNA library. The major consideration in designing a cloning strategy for the construction of a cDNA library is unidirectional ligation of cDNA inserts into the vector. The restraint in ligation orientation is essential to render the expression of cDNA inserts under the control of a promoter from the vector as well as to eliminate vector self-ligation. Directionality is obtained typically by introducing two different restriction endonuclease sites at the ends of cDNA, which is initiated by using a primer-adapter to initiate first strand synthesis. The SuperScript Plasmid System for cDNA synthesis and plasmid cloning from Invitrogen Corporation features this strategy in their design (Fig. 2), and the system has been used successfully in constructing a yeast cDNA library. 2 A NotI primer-adapter, which contains 15 dT and restriction sites of NotI and other enzymes, is used to prime the first strand cDNA synthesis at the poly(A) tail of mRNAs (Fig. 2). The product of the first and second strand reactions is blunt-ended cDNA, to which SalI adapters are added. The SalI adapter is duplex oligomers that are blunt-ended at one terminus and contains a 4-base overhang at the other terminus. Only the blunt-ended 5'-end of the SalI adapter is phosphorylated, which eliminates self-ligation of the adapters during ligation to the cDNA (Fig. 2). The SalI adapter also contains an additional MluI site which can be used, together with Nod, to release the cDNA insert from the vector. After the addition of SalI adapters to both termini, the NotI terminus is exposed by NotI digestion. Thus, the 3' end of the cDNA is identified with NotI, and the 5t end with SalI. The cDNA can then be ligated to a SalI- and NotI-digested vector, with the SalI site positioned at the terminus with an inducible promoter (Fig. 2).
[3]
CONSTRUCTING YEAST LmRARIES
79
InlRNA -AAAAAA TTTTTTCCCGCCGGCG
~ First strand synthesis "AAA.~A "TTTTI"rCCCGCCGGCG
i, Second strand synthesis ~GGGCGGCCGC ~CCGCCGGCG
JFSall adapter %--I
addition
~ -'~C~l-~ TTTTTTCCCGCCGGCC
Sal I
~111
JFNot I digestion,
ro moter
size fractionation
~ Sail & Not I digestion
~ ' q " I " P ~ C C GCC GG Sal I
~ Transformation
cDNA library FIG.2. A strategyfor constructingcDNAlibraries (adaptedwith permissionfrom the SuperScript Plasmid Systemfor cDNAsynthesis and plasmidcloning of InvitrogenCorporation).
Preparation of Cut Vector A YCp vector is usually used to construct a cDNA library. The vector must contain an inducible promoter, preferably with a high level of expression under the induction condition and completely off under a noninducing condition. If the cloning strategy in Fig. 2 will be used for this construction, the vector must also contain a unique NotI site in the polylinker region and one Sail or XhoI site between the NotI and the inducible promoter. To prepare the cut vector, digest the vector DNA with NotI to completion. Monitor the digestion on an agarose gel. Digest the NotI cut vector with SalI and gel-purify the linear DNA. Extract the cut DNA from gel slices with a QIAEX gel extraction kit. Because the NotI and SalI sites are close to each other in the polylinker region of the vector, it is difficult to determine whether the second digestion is complete.
80
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To monitor the completeness of second digestion, we have end-labeled 3% of NotI-digested DNA with 3Zp and then digested the DNA with the second enzyme, SalI. The extent of SalI digestion can be checked by digesting with an enzyme that cuts the vector backbone. Two 32p-labeled bands are expected if the second digestion has been complete. A partial SalI digestion should give three 32p bands. Alternatively, transformation efficiency of ligated SalI-digested vector can be used to assess whether the second digestion is complete. Low transformation efficiency is expected if the second digestion has occurred successfully. As described in the previous section for preparing cut vector, ligating the NotI- and SalI-digested vector, followed by gel purifying the linear unligated DNA, can be used to purify double digested vector DNA away from single restricted vector. Total RNA Extraction and mRNA Purification Construction of a good cDNA library begins with the preparation of highquality mRNA. Generally, 5/xg of mRNA is sufficient to construct a cDNA library containing about 106 clones in E. coll. Since only 1.3 to 1.4% of a total RNA population in yeast is mRNA, it is necessary to start with at least several milligrams of total RNA. To avoid RNase contamination, it is important to use sterile disposable plasticware and diethyl pyrocarbonate (DEPC)-treated H20 throughout RNA purification. The hot acidic phenol method 17 works well for the extraction of total RNA from yeast. For a scaled-up preparation, about 101° (500 ml culture) S. cerevisiae cells in mid-exponential phase are collected in 50-ml polypropylene tubes, washed once with ice-cold water. Resuspend cells in 12 ml TES (10 mM Tris-Cl, pH 7.5, 10 mM EDTA, 0.5% SDS). Add 12 ml acid phenol (unbuffered liquefied phenol from Sigma, St. Louis, MO) to the tube and vortex vigorously for 10 sec. Incubate for 30 to 60 min at 65 ° with occasional vortexing. Centrifuge and transfer the aqueous (top) phase to a clean 50-ml tube, add 12 ml acid phenol, vortex vigorously, and centrifuge 10 min at 3000 rpm in a swing-bucket rotor in a SorvaU table top centrifuge. Transfer aqueous phase to a new tube, add 1/10 volume of 3 M sodium acetate, pH 5.3, and 2 volumes of ice-cold 100% ethanol, and precipitate. Centrifuge at top speed for 30 min at 4 °. Wash the RNA pellet with 70% ice-cold ethanol. Centrifuge, air-dry the RNA pellet, and resuspend the pellet in 0.5 ml H20. Fully dissolve RNA before measuring OD260. The yield should be around 5 mg. mRNA is isolated from total RNA by oligo(dT) cellulose column chromatography) 8 For 5 mg RNA, use 0.75 ml of oligo(dT)-cellulose in a column. About 17F. M. Ausubel, R. Brent, R. E. Kingston, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (eds.), in "Current Protocols in Molecular Biology," Vol. 1. John Wiley & Sons, New York, 1987. 18 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.
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1/10 of the RNA is expected to be recovered from the first round of purification. Then, the oligo(dT) column is regenerated with 0.1 M NaC1 and equilibrated. The eluted poly(A) mRNA can be loaded onto the oligo(dT) column directly without the need for precipitation for the second round of purification by adjusting the NaC1 concentration of the eluted mRNA to 0.5 M. About 50% of the applied RNA is expected to be recovered in the elution. After three cycles of purification through the oligo(dT) column, most of the tRNA and rRNA should be removed, and the material should be highly enriched in mRNA. Quality of total RNA and purified mRNA should be evaluated by a RNA gel. Because SDS is in the loading buffer of the oligo(dT) column and can interfere with the cDNA synthesis, it is necessary to precipitate and wash the mRNA twice after the column elution to reduce the content of SDS in the mRNA.
cDNA Synthesis and Fractionation For cDNA synthesis and fractionation, we recommend using the SuperScript Plasmid System from Invitrogen Corporation. The system contains all necessary reagents sufficient for three experiments, each converting up to 5 #g of mRNA to fractionated ready-to-ligate cDNA. The instruction manual from Invitrogen, Carlsbad, CA, is very explicit, and if followed closely, one should succeed in making a cDNA library without too much trouble. The important steps for preparing cDNA inserts are highlighted below. First-Strand Synthesis. First strand cDNA synthesis from mRNA is achieved with SuperScript II RT (reverse transcriptase) in the following 20/zl reaction. The engineered RT enzyme SuperScript II does not contain RNase H activity, but still exhibits an improved polymerase activity. 1. Add 2/zl of Nod primer-adapter to 1 to 5/zg of mRNA; dilute as needed to make the final reaction 20/zl (including RT and 8-#1 reagents that will be added in step 2). 2. Heat to 70 ° for 10 min and quick-chill on ice. Spin briefly and add: 4/zl of 5 x First strand buffer 2 #1 of 0.1 M D T T 1 #1 of 10 mM dNTP mix 1/zl of [ot-32p]dCTP (1/zCi//zl) 3. Incubate at 37 ° for 2 min to equilibrate the temperature. Add SuperScript II RT (use 1/zl of RT for each/zg of mRNA used in the reaction). 4. Incubate at 37 ° for 1 hr. Place the tube on ice to terminate the reaction. 5. To determine the yield of first strand synthesis, remove 2 tzl from the reaction and add it to 43 tzl of 20 mM EDTA (pH 7.5) and 5/zl of 1 /zg//zl yeast tRNA. From the diluted sample, spot duplicate 10-/zl aliquots onto glassfiber filters. Dry one to determine the specific activity (SA) of the dCTP
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reaction. Wash the other filter three times, 5 min each, in a beaker containing 50 ml of ice-cold 10% TCA with 1% sodium pyrophosphate, and once in 50 ml of 95% ethanol. Dry the filter to determine the yield of first strand cDNA. The specific activity (SA) is determined by dividing the counts per rain from the 10-#1 aliquot from the unwashed filter with the quantity (in pmol) of the same nucleotide in the 10-/zl aliquot: SA(cpm/pmol dCTP) =
cpm/10/.tl 200 pmol dCTP/10/zm
Once the SA is determined, the amount of cDNA synthesized in the first-strand reaction can be calculated from the amount of acid-precipitable radioactivity. Amount of cDNA(/zg) = (acid-precipitablecpm) x (50/zl/10/zl) x (20 ~1/2/zl) x (4pmol dNTP/pmol dCTP) (SA cpm/pmol dCTP) x (3030 pmol dNTP//xg cDNA) "3030" is the amount of nucleotide equivalent to 10/zg of single-strand DNA. The yield of first-strand synthesis can be calculated by dividing the amount of cDNA synthesized by the amount of starting mRNA. We have had around 38% yield from 3.1 /zg of yeast mRNA. A lower yield of first-strand synthesis does not necessarily indicate that a library cannot be made, since each ligation reaction only requires 10 ng of cDNA. It is critical, however, that the size distribution of the cDNA products be similar to that of the mRNA. Shorter cDNA products may indicate the danger of RNase contamination. Second-Strand Synthesis
1. On ice, add the following reagents to the remaining 18-#1 first-strand reaction: 93/zl of H20 30/zl of 5 x Second strand buffer 3/zl of 10 mM dNTP mix 1 izl E. coli DNA ligase (10 U//xl) 4 Ixl E. coli DNA polymerase I (10 U//xl) 1 Ixl E. coli RNase H (2 U//xl) Final volume is 150/~1. 2. Incubate for 2 hr at 16°. 3. Add 2/zl (10 units) of T4 DNA polymerase, continue incubating at 16° for 5 min. 4. Place the reaction on ice, and add 10/zl of 0.5 M EDTA.
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83
5. Extract with 150/zl phenol: chloroform: isoamyl alcohol (25 : 24 : 1). Precipitate the cDNA with 70 /xl of 7.5-m ammonium acetate and 0.5 ml ethanol (-20°). Wash with 0.5 ml of 70% ethanol (-20°). Air-dry at 37 ° for 10 min. 6. To determine the quality of synthesized cDNA, run some (5 /zl leftover from the aqueous phase of each phenol extraction is enough) second-strand reactions on an agarose gel and dry the gel. Visualize by exposing the gel to an X-ray film. Compare the range of cDNA smear to that of the starting mRNA to determine the quality of cDNA.
SalI Adapter Addition and NotI Digestion 1. Set up a 50-#1 ligation reaction to add SalI adapter to the cDNA from step 5 of second-strand synthesis. cDNA 25/zl H20 10 #1 5× T4 DNA Ligase buffer 10/zl SalI Adapters 5/zl T4 DNA Ligase 2. Incubate at 16° overnight. 3. Extract with phenol : chloroform :isoamyl alcohol. Precipitate, wash, and air-dry DNA. 4. Set up a 50-/,1 Nod digestion. 5. Incubate for 2 hr at 37 °. 6. Extract with phenol : chloroform : isoamyl alcohol. Precipitate, wash, and air-dry DNA.
Fractionation by Column Chromatography. To ensure the quality of your cDNA library, it is crucial to remove all residual SalI adapters and the NotI fragments released by restriction digestion with NotI. This is accomplished by filtrating the cDNA through a size fractionation column provided by the SuperPlasmid System. 1. Dissolve the NotI digested cDNA in 100 #1 TEN buffer (10 mM Tris-HC1 pH 7.5, 0.1 mM EDTA, 25 mM NaC1). 2. Wash a cDNA fractionation column (provided in the Invitrogen SuperScript Plasmid System) with 0.8 ml TEN buffer four times; let the column drain completely for each wash. 3. Load the 100/zl cDNA to the column; collect flow-through into tube 1. 4. Add 100 #1 TEN buffer to the column and collect into tube 2. Let it drain completely.
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5. From the second 100/zl TEN wash, collect single drop fractions (-'~35/zl) into each microcentrifuge tube. Continue adding 100-/zl aliquots of TEN until a total of 18 drops have been collected into tubes 3 through 20, one drop per tube. 6. Measure the volume in each tube with a fresh tip. Identify the fraction whose cumulative elution volume (sum from tube 1 to this fraction) is closest to but not exceeding 550/zl. Discard all tubes after this fraction. If fractions beyond 550 ~1 are used, the library will contain "empty" clones, which come from ligation of the short NotI-primer-adapter-SalI fragment released from the NotI digestion. 7. Place the remaining tubes in a scintillation counter and obtain Cerenkov counts for each tube in the tritium channel without using scintillation fluid. Counts should increase after reaching 400 to 450/zl of elution volume. Calculate the amount of cDNA in each fraction by using the following equation with the SA determined for the first strand synthesis: Amount of ds cDNA (ng) (Cerenkov cpm) x 2 x (4pmol dNTP/pmol dCTP) x (1000ng//zg ds cDNA) SA(cpm/pmol dCTP) × (1515 pmol dNTP//zg ds cDNA) .
Determine the cDNA concentration for each fraction by dividing the amount of cDNA in each fraction with the volume. If the cDNA concentration is higher than 0.71 ng//zl, 10 ng cDNA from that fraction can be used directly for ligation with the vector without precipitation. Otherwise, the cDNA has to be concentrated. To maximize the average insert size in the cDNA library, cDNA from the earliest fraction should be used for ligation. Often, the earliest fraction does not have enough cDNA and is not concentrated enough to be used directly. In this case, fractions may have to be pooled together and precipitated to obtain the 10 ng of cDNA needed for ligation.
Ligation of cDNA to Vector and Transformation Set up ligation reactions with 10 ng of cDNA and 50 ng of SalI- and NotI-cut vector in 20/zl reactions, as following: 14/xl of 10 ng cDNA in TEN buffer 1/_tl of Notl-SalI cut vector DNA (50 ng//zl) 4 ~1 of 5 x T4 DNA ligase buffer 1/zl of T4 DNA ligase (1 unit//zl ) Incubate for 3 hr at room temperature.
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Ligation reactions from each column fraction should be kept separately as different pools of library. A negative control ligation with the same amount of cutvector DNA, but without cDNA, should be used. In addition, we suggest setting up a control ligation reaction using cDNA from one of the later fractions (less valuable) with the Nod- and SalI-cut pSPORT vector DNA provided in the SuperScript Plasmid System. The transformation efficiency of this reaction will inform you whether a ligation problem is caused by cDNA or by the cut vector. In order to generate a sufficiently large eDNA library, it is important to use the most competent E. coli cells in transformation. Electroporation usually offers higher transformation efficiency. With a successful ligation, we typically obtain around 10 6 transformants per ligation from electroporation of ElectroMAX DH10B cells from Invitrogen (transformation efficiency at 109-101° transformants/#g of supercoiled plasmid). If the transformation efficiency is 100-fold lower, the library size will be on the borderline for genes of low abundance. Transformation efficiency is also a good indication of whether the ligation reaction has worked or not. We expect that the ligation with cDNAs should give 50-100 times more transformants than the negative control without cDNAs. If not, there may be problems with ligation reactions, which can come from either cDNA or the cut vector. If the ligation reaction with the pSPORT vector gives 100-fold higher transformation efficiency than the ligation with the cut vector, the vector preparation may have a problem. If the transformation efficiencies of both ligations are low (<105 transformants/ligation, assuming that supercoiled plasmid yields >109 transformants//zg) the ligation reactions may not be proceeding properly and cDNA is likely the cause of the problem. Since synthesized cDNA has been visualized on an agarose gel and length of cDNA should not affect transformation efficiency, the first and second strand syntheses are not likely to cause the problem. On the other hand, problems in SalI adapter addition and NotI digestion will not be easily detected during the process and will affect the ligation efficiency dramatically. To dissect which of the two steps has gone wrong, the ligation reaction can be checked by running the remaining ligation reaction on an agarose gel next to the cut vector DNA. Successfully ligated DNA should show an upward smear from the position of the cut vector. However, the same upward smear will be seen if either NotI or SalI end of the inserts has ligated to the vector. To pinpoint the cause of a failed ligation, we have digested the ligation reaction with either SalI or NotI and run the digestions on an agarose gel next to the ligated DNA and cut vector DNA. If NotI digestion, for example, can convert the upward smear to a band same size as the cut vector, the SalI end must have not ligated properly. Alternative, the adapter addition step can be monitored by nondenaturing polyacrylamide gel electrophoresis during the process before the final ligation step. If 10/zl of the adapter ligation reaction is run on a 12% acrylamide gel and the DNA is visualized by ethidium bromide staining, the
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ligation of adapters to each other, mostly at their blunt ends but occasionally at the sticky ends, will be evidenced by a ladder of fragments of 16, 32, 60, 88 bp, etc. Although this does not verify that the SalI adaptors have ligated to the cDNA, it does show that the ligation reaction is successful. To examine Nod digestion, 4/zl of NotI digestion can be run on the same 12% acrylamide gel together with SalI ligation reaction. After visualizing the SalI adapter ligation ladder with ethidium bromide, the gel can be dried and exposed to X-ray film to visualize a NotI-SalI fragment of 40 bp, which should be between dimers and quadruples of the SalI adapters.
Determining Quality of Library A good cDNA library has three key characteristics that set it apart from the mediocre counterparts: (1) it should be large enough to contain genes of all genes expressed, including the ones expressed at low abundance, (2) it should contain full-length cDNA inserts, and (3) over 90% of its clones should contain cDNA inserts at the expected orientation. Either a complementation in a yeast mutant strain or a colony hybridization screen with the library could be performed to determine whether the library contains cDNAs for mRNAs of low abundance. The last two stipulations can be assessed by restriction digestion of some randomly picked clones with SalI and NotI and by sequencing each insert with a primer from the promoter in the vector. Acknowledgment I thank Craig M. Thompson for sharing his protocol and expertise in making a yeast genomic library. I thank Gerald R. Fink and Anthony Bretscher for providing laboratory space and support during this work.