3528 Nucleic Acids Research, Vol. 20, No. 13
A novel method for the isolation of tissuespecific genes Ignacio R.Rodriguez and Gerald J.Chader National Eye Institute, NIH, Building 6, Room 307, Bethesda, MD 20892, USA Submitted February 10, 1992 Subtraction cloning has become a popular method for isolating tissue specific genes (1, 2). One of the most common problems with subtraction techniques in that specific mRNAs or cDNAs from the target tissue are subtracted away by common sequences (e.g. gene families) or repetitive elements (e.g. Alu repetitive elements) leaving little or no product to clone or use as a probe. We have devised a new subtraction method for the generation of tissue-specific probes which avoids these problems. The method depicted in Figure 1 consists of synthesizing singlestranded antisense cDNA from the target tissue (i.e. retina) on magnetic beads (Dynabeads® oligo dT, Dynal Inc.), destroying the mRNA, then random priming the immobilized target cDNA to generate short labeled sense fragmnts. The synthesis of cDNA on Dynabeads® has previously been described (3). However, we provide here an improved method for synthesising cDNA on these beads. These labeled fragments are then eluted from the beads and mixed with an excess of similarly synthesized cDNA (on a different set of beads) from the driver tissue(s) (e.g. liver, etc.) and the common fragments allowed to anneal. Those fragments which do not anneal are left in the supernatant while those common to the two tissues are left bound to the beads containing the driver tissue cDNA. These specific fragments can be used to screen cDNA, genomic or chromosome-specific libraries. Since our interests are in retina-specific genes, we synthesized cDNA on the magnetic beads from total human adult retinal RNA (target) as well as from human adult liver and muscle (drivers). An amount of beads containing 100 ng of retinal cDNA was copied and labeled using 200 ACi of 32P dCTP by random priming. After the random priming reaction was completed, 2 Ag of tRNA were added as carrier. The random priming mix with the unincorporated label was removed from the beads, and 32P-sscDNA was eluted by the addition of 100 Al of 0.15 N NaOH for 5 min at room temperature. The 32P-sscDNA was neutrilized by the addition of 100 1 of 10 x SSC to make a final concentration of 200 Al of 5 x SSC and an additional 2 Ag of tRNA added as carrier. The 32P-sscDNA fragments in 5 x SSC were added to another set of beads containing 200 ng of liver cDNA and allowed to denature at 940C for 2 min and to anneal for 1 hr at 550C. The 32P-sscDNA which fails to anneal to the beads containing the liver cDNA was used to screen a human adult retina cDNA library. A more specific probe can be generated by sequentially subtracting the 32P-sscDNA on beads containing cDNA from other tissues. The secondary screening of-the clones was performed with a probe subtracted from both liver and muscle cDNA. After re-screening we were able to isolate 15-20 clones for every 300,000 clones screened. The sequence analysis of these clones is still in progress, however,
several retina-specific cDNAs such as the green visual pigment, rhodopsin and interphotoroceptor retinoid-binding protein have been found.
REFERENCES 1. Swaroop,A., et al. (1991) Nucleic Acids Res. 19, 1954. 2. Hara,E., et al. (1991) Nucleic Acids Res. 19, 7097-7104. 3. Raineri,I., Moroni,C. and Senn,H.P. (1991) Nucleic Acids Res. 19, 4010. Annee mRNA to Dynabeads
(AvnaoUer, a) Destroy mRNA with O.1N NaOH (Uver Driver)
Destroy mRNA wth RNAseH (Reire or Target) Random prime cDNA Pp dCTP
I
LIVER cDNA ON DYNABEADS
Rndr cDNA_ Ree random prime fragen 0.1N NaOH
Equilibrate in 5X SSC
Add random pimed rag to Uver cDNA iar WMA rANA LJUV
Alow cornmon f mn ts to anneal
Screen cDNA or Genomic Ubraries Figure 1. Schematic protocol for the preparation of a tissue-specific probe. The oligo dT Dynabeads® and the total RNA were equilibrated with 10 mM Tris-Cl pH 7.0, 1 mM EDTA, 1 M KCI. The RNA 30 1I, (30 jtg, 1 gg/Al) was mixed with the equilibrated Dynabeads® (0.5 mg) and heated to 700C for 2 minutes. The poly A RNA was allowed to anneal to the beads for 30 min at 30°C and the excess RNA removed. The beads with the attached poly A mRNA were then suspended in 2.5 A1 buffer A (200 mM Tris-CJ pH 8.3, 1.0 M KCl), 2.5 1l buffer B (30 mM MgCl2, 15 mM MnCl2), 20 1l 10 mM dNTPs (2.5 mM each), 1 A1 of 32P-labeled dCTP (5 uCi, -3000 Ci/mmol, Amersham, Arlington Hts, IL), 1 AL RNAsine (Promega, Madison, WI), 2 1l SuperScript® RT (BRL, Gaithersburg, MD), 5 AL Retrotherme RT (1 Unit/Al, Epicentre Technologies, Madison, WI) and 16 1d of H20 to make a final volume of 50 Al. The reaction mixture was incubated at 40°C for 30 min, then at 70°C for 1 hr under mineral oil. The cDNA was quantitated by measuring the amount of 32p incorporated into the beads.
A novel method for the isolation of tissuespecific genes Ignacio R.Rodriguez and Gerald J.Chader National Eye Institute, NIH, Building 6, Room 307, Bethesda, MD 20892, USA Submitted February 10, 1992 Subtraction cloning has become a popular method for isolating tissue specific genes (1, 2). One of the most common problems with subtraction techniques in that specific mRNAs or cDNAs from the target tissue are subtracted away by common sequences (e.g. gene families) or repetitive elements (e.g. Alu repetitive elements) leaving little or no product to clone or use as a probe. We have devised a new subtraction method for the generation of tissue-specific probes which avoids these problems. The method depicted in Figure 1 consists of synthesizing singlestranded antisense cDNA from the target tissue (i.e. retina) on magnetic beads (Dynabeads® oligo dT, Dynal Inc.), destroying the mRNA, then random priming the immobilized target cDNA to generate short labeled sense fragmnts. The synthesis of cDNA on Dynabeads® has previously been described (3). However, we provide here an improved method for synthesising cDNA on these beads. These labeled fragments are then eluted from the beads and mixed with an excess of similarly synthesized cDNA (on a different set of beads) from the driver tissue(s) (e.g. liver, etc.) and the common fragments allowed to anneal. Those fragments which do not anneal are left in the supernatant while those common to the two tissues are left bound to the beads containing the driver tissue cDNA. These specific fragments can be used to screen cDNA, genomic or chromosome-specific libraries. Since our interests are in retina-specific genes, we synthesized cDNA on the magnetic beads from total human adult retinal RNA (target) as well as from human adult liver and muscle (drivers). An amount of beads containing 100 ng of retinal cDNA was copied and labeled using 200 ACi of 32P dCTP by random priming. After the random priming reaction was completed, 2 Ag of tRNA were added as carrier. The random priming mix with the unincorporated label was removed from the beads, and 32P-sscDNA was eluted by the addition of 100 Al of 0.15 N NaOH for 5 min at room temperature. The 32P-sscDNA was neutrilized by the addition of 100 1 of 10 x SSC to make a final concentration of 200 Al of 5 x SSC and an additional 2 Ag of tRNA added as carrier. The 32P-sscDNA fragments in 5 x SSC were added to another set of beads containing 200 ng of liver cDNA and allowed to denature at 940C for 2 min and to anneal for 1 hr at 550C. The 32P-sscDNA which fails to anneal to the beads containing the liver cDNA was used to screen a human adult retina cDNA library. A more specific probe can be generated by sequentially subtracting the 32P-sscDNA on beads containing cDNA from other tissues. The secondary screening of-the clones was performed with a probe subtracted from both liver and muscle cDNA. After re-screening we were able to isolate 15-20 clones for every 300,000 clones screened. The sequence analysis of these clones is still in progress, however,
several retina-specific cDNAs such as the green visual pigment, rhodopsin and interphotoroceptor retinoid-binding protein have been found.
REFERENCES 1. Swaroop,A., et al. (1991) Nucleic Acids Res. 19, 1954. 2. Hara,E., et al. (1991) Nucleic Acids Res. 19, 7097-7104. 3. Raineri,I., Moroni,C. and Senn,H.P. (1991) Nucleic Acids Res. 19, 4010. Annee mRNA to Dynabeads
(AvnaoUer, a) Destroy mRNA with O.1N NaOH (Uver Driver)
Destroy mRNA wth RNAseH (Reire or Target) Random prime cDNA Pp dCTP
I
LIVER cDNA ON DYNABEADS
Rndr cDNA_ Ree random prime fragen 0.1N NaOH
Equilibrate in 5X SSC
Add random pimed rag to Uver cDNA iar WMA rANA LJUV
Alow cornmon f mn ts to anneal
Screen cDNA or Genomic Ubraries Figure 1. Schematic protocol for the preparation of a tissue-specific probe. The oligo dT Dynabeads® and the total RNA were equilibrated with 10 mM Tris-Cl pH 7.0, 1 mM EDTA, 1 M KCI. The RNA 30 1I, (30 jtg, 1 gg/Al) was mixed with the equilibrated Dynabeads® (0.5 mg) and heated to 700C for 2 minutes. The poly A RNA was allowed to anneal to the beads for 30 min at 30°C and the excess RNA removed. The beads with the attached poly A mRNA were then suspended in 2.5 A1 buffer A (200 mM Tris-CJ pH 8.3, 1.0 M KCl), 2.5 1l buffer B (30 mM MgCl2, 15 mM MnCl2), 20 1l 10 mM dNTPs (2.5 mM each), 1 A1 of 32P-labeled dCTP (5 uCi, -3000 Ci/mmol, Amersham, Arlington Hts, IL), 1 AL RNAsine (Promega, Madison, WI), 2 1l SuperScript® RT (BRL, Gaithersburg, MD), 5 AL Retrotherme RT (1 Unit/Al, Epicentre Technologies, Madison, WI) and 16 1d of H20 to make a final volume of 50 Al. The reaction mixture was incubated at 40°C for 30 min, then at 70°C for 1 hr under mineral oil. The cDNA was quantitated by measuring the amount of 32p incorporated into the beads.
