Reusable cDNA libraries coupled to magnetic beads

NOTES & TIPS Reusable cDNA Libraries Coupled to Magnetic Beads Youn-Ho Lee and Victor D. Vacquier Marine Biology Research Division, 0202, Scripps Institution of Oceanography, University o[ Cali[ornia, San Diego, La JoUa, California 92093 A simple, rapid method has been developed for the construction of cDNA libraries coupled to magnetic beads (Dynal, Inc., Great Neck, NY). This method allows the simple, efficient removal and exchange of reactants between steps. T h e cDNA-magnetic beads may be stored for months and reused. We have used this method with great facility to determine the cDNA sequences of abalone sperm 16 K lysins ( ~ 6 6 0 nt) from congeneric species throughout the world. From tissue sample to single-stranded sequencing templates requires about 20 h. Protocol. (a) Total RNA is isolated from 1 g of tissue (1), and the poly(A) + fraction is purified using the poly(AT) tract system from Promega (Madison, WI). (b) T h e reverse transcription primer (RTpI), p o l y ( T ) - a d a p t o r - b i o t i n (5' biotin-GCCTCGAGTCGACA(T)16 3') is immobilized on Dynal streptavidin magnetic beads. The underlining denotes an XhoI site and the italics a SalI site t h a t can be used for directional cloning. Fifty picomoles of R T P is incubated with 30-~1 beads (10 mg/ml) in 100 #l of 1X binding and washing buffer (BWB, 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1.0 M NaC1) for 30 min at 23°C. T h e beads are collected on the side of a 1.5-ml microcentrifuge tube using the Dynal magnetic stand and washed once with 0.2 ml BWB and twice with 0.2 ml RNase-free water. One microgram of poly(A) + RNA is heated 10 min to 70°C in 12-#1 water in the tube containing the R T P immobilized on Dynal beads. T h e tube is cooled in ice and the following reactants are added: 4 #l of 5x reverse transcriptase buffer (Promega), 0.5 #l RNasin (20 units), 2 ~1 d N T P s at 10 mM each, and 1.5 #1 AMV reAbbreviations used: RTP, reverse transcription primer; BWB, binding and washing buffer; RNasin, ribonuclease inhibitor; AMV, avian myeloblastosis virus; 3'GSBP, 3' gene-specific biotinylated primer; 5'ACP, 5' adaptor d(C),4 primer; 5'GSP, 5' gene-specific primer; 3'TBP, 3' poly(T) biotinylated primer; Pfu, Pyrococcus[urioSUB.

ANALYTICAL BIOCHEMISTRY 206, 206-207 (1992) 0003-2697/92 $5.00 Copyright © 1992 by AcademicPress, Inc. All rights of reproductionin any form reserved.

verse transcriptase (15 units). Synthesis is performed for 1 h at 42°C and stopped by addition of 100 #l of 0.1 N NaOH, followed by 10 min at 23°C. N a O H t r e a t m e n t denatures the m R N A - c D N A hybrids and hydrolyzes the mRNA. The first strand cDNA synthesized on the magnetic beads is purified using the magnetic stand to capture the beads, followed by one wash with 100 ~1 BWB and two washes with 100 ~l water (Fig. 1). (c) For tailing the first strand cDNA coupled to Dynal beads, 6 ~l of 5 x tailing buffer (Promega), 3 #1 of 10 mM dGTP, 0.7 ul terminal deoxynucleotide transferase (Promega, 20 units), and 20.3 ~l water (total volume 30 #1) are incubated 1 h at 37°C. T h e oligo (dG)-tailed cDNA is purified by washing in BWB using the magnetic stand to capture the beads. T h e cDNA coupled to the beads is stored at 4°C in 50 #l T E (10 mM Tris-HCl, pH 7.6, 1 mM EDTA) with 0.02% azide. (d) Two separate P C R reactions are performed. For 5' end amplification, 5 #l poly(dG)-tailed cDNA-magnetic beads is washed once with 100 #1 BWB and twice with 100 #1 water. Eighty-nine microliters, consisting of 10 #l of 10X PCR buffer (Stratagene, La Jolla, CA), 50 pmol of each primer, a 3' gene-specific biotinylated p r i m e r (3'GSBP), and a 5' adaptor d(C)14 primer (5'ACP), is mixed with the washed beads. For 3' end amplification, an identical reaction is assembled with 50 pmol each of a 5' gene-specific primer (5'GSP) and a 3' poly(T) biotinylated primer (3'TBP). T h e two reactions are heated to 95°C for 5 min and cooled to 85°C, and 1 ~1 Pfu DNA polymerase (2.5 units, Stratagene) and 10 #l d N T P s at 2 mM each are added. The two reaction mixtures are overlaid with 70 #l mineral oil and annealed at 50°C for 2 min and extended at 74°C for 5 min. Thirtyfive cycles of PCR are performed: 94°C for 1.5 min, 50°C for 2 min, and 74°C for 3 min, followed by a final extension at 72°C for 10 min (Fig. 1). (e) T h e two PCR products (5' and 3' end amplicons) are separated from the cDNA-magnetic beads using the magnetic stand. Since the streptavidin binding sites on the beads are saturated with excess biotinylated primers, the PCR products can be removed in the supernatant. The biotinylated cDNA strand is sequenced by a standard solid-phase sequencing protocol (2), and the complementary, nonbiotinylated strand by a protocol developed in our laboratory (3). (f) T h e dG-tailed cDNA-bead template can be recovered and reused for at least five rounds of PCR. Sequences of the sperm lysin cDNA on the first and the 206

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1. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156159. 2. Hultman, T., Stahl, S., Hornes, E., and Uhlen, M. (1989) Nucleic Acids Res. 17, 4937-4946. 3. Lee, Y.-H., and Vacquier, V. D. (1993) BioTechniques, in press.

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Acknowledgments. This work was supported by NIH Grant HD 12986 to V. D. Vacquier and by a Korean Government Overseas Scholarship to Y.-H. Lee. REFERENCES

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amplification of a specific gene. The first strand cDNA is synthesized on the bead using a poly(T)-adaptor-biotin primer (RTP) coupled to the streptavidin bead. The cDNA is purified using a magnetic stand and tailed with dGTP. The oligo (dG)-tailed cDNA is stored at 4°C in TE with 0.02% azide. This cDNA library is then used in two separate PCR reactions to amplify both the 5' and 3' regions of a specific cDNA using gene-specificprimers. After PCR the amplicons are removed in the supernatant for sequencing, and the original bead-coupled cDNA is recovered, washed with NaOH, BWB, and water, and stored in TE at 4°C. fifth r o u n d s were identical. After P C R , the reaction m i x t u r e u n d e r the mineral oil is t r a n s f e r r e d to a new 1.5-ml tube. T h e beads with coupled t e m p l a t e c D N A are c a p t u r e d on the side of the tube using the magnetic stand, a n d the s u p e r n a t a n t c o n t a i n i n g amplified doub l e - s t r a n d e d c D N A s t r a n s f e r r e d to a new tube for seq u e n c i n g (Fig. 1). T h e c a p t u r e d beads are mixed with 30 #1 of 0.1 N N a O H , i n c u b a t e d for 10 min at 23°C, recapt u r e d using the m a g n e t , w a s h e d once with 100 #l B W B a n d twice with 100 #1 water, a n d stored in T E (pH 7.6) with 0.02% azide. Conclusion. T h i s m e t h o d requires fewer steps t h a n a n y o t h e r for c o n v e r t i n g m R N A into c D N A . It utilizes m a g n e t i c r e t e n t i o n for fast, efficient w a s h i n g of t e m plates. This m e t h o d could also be applied to genomic D N A library c o n s t r u c t i o n . In principle, all genomic D N A s can be copied using a p p r o p r i a t e p r i m e r s coupled to magnetic beads.

R a d i o i o d i n a t e d insulin is used in m a n y applications including r a d i o r e c e p t o r a n d r a d i o i m m u n o a s s a y s . T h e validity o f these applications often d e m a n d s t h a t the binding a n d biological p r o p e r t i e s of the radioiodinated insulin be essentially indistinguishable f r o m the native h o r m o n e . A 1 4 - t y r o s y l - m o n o i o d i n a t e d insulin is an iodination p r o d u c t of insulin t h a t best satisfies this criterion (1-4). Various purification p r o c e d u r e s have been applied to the isolation of A14-tyrosyl [125I]iodoinsulin f r o m the o t h e r species of radiolabelled insulin a n d native insulin including H P L C {1,4), i o n - e x c h a n g e chrom a t o g r a p h y (5), a n d p o l y a c r y l a m i d e gel electrophoresis (6). However, all these m e t h o d s have significant limitations. First, t h e y often require extensive h a n d l i n g of the iodination mixtures outside the safe confines of a fumehood. Second, because of the levels of radioactivity involved in an iodination, expensive e q u i p m e n t m u s t be dedicated to the task. Third, H P L C a n d P A G E m e t h o d s are relatively laborious. W e have o v e r c o m e these limitations using a m e t h o d t h a t entails the differential elution of free iodine, native insulin, a n d the various m o n o i o d o a n d diiodoinsulins on disposable cartridges p a c k e d with a C18 reverse-phase c h r o m a t o g r a p h i c support. T h i s m e t h o d is inexpensive a n d e x t r e m e l y simple a n d can be entirely confined to a f u m e h o o d e n v i r o n m e n t , m a k i n g it p a r t i c u l a r l y safe. Materials and methods: Zinc-free insulin. Zinc-free insulin was p r e p a r e d according to the m e t h o d outlined by 1This study was in part supported by grants from the Australian Research Council (A08931905) and Alphapharm/Diabetes Australia Research Trust. 2To whom correspondence and reprint requests should be addressed at School of Biomedical Sciences, Curtin University of Technology, GPO Box U1987, Perth 6001, Western Australia. Fax: 09 351 2342. ANALYTICALBIOCHEMISTRY206, 207-210 (1992) 0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in a n y form reserved.