\./ 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 16 4557
DNA fingerprinting by specific priming of concatenated ol igonucleotides Celia A.May and Jon H.Wetton Department of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK Submitted June 25, 1991 Simple sequences consisting of direct or cryptic tandem repeats of di-, tri- or tetra nucleotide motifs give rise to VNTR regions in a variety of plant and animal species (1, 2, 3). To date the principal detection method for these so-called microsatellites has involved end labelling of short oligonucleotides (9-20 bp) with -y-ATP and subsequent in-gel hybridization (3). By concatenating surplus oligos we have produced probes of higher specific activity with random priming reagents and a32P dCTP which are more routinely used in minisatellite fingerprinting. We have found that this greatly facilitates sequential probing of Southern blots with either micro- or minisatellite sequences with concomitant saving in time and expense. 1 mg each of (TCC)4 and the complementary (GAG)4 oligonucleotides were produced by an Applied Biosystems 381A DNA Synthesizer. The oligos were cleaved from the glass bead matrix by mixing with .880 SG ammonia at room temperature and deprotected by overnight incubation at 55°C. After lyophilization the oligos were resuspended in sterile distilled water and further purified by passage through anion exchange columns (Qiagen) to remove residual ammonium ions which are inhibitory to T4 polynucleotide kinase (4). Equimolar quantities of each were phosphorylated at the 5' end for lhr at 37°C, hybridized together and then ligated at 4°C for 24 hours (as in ref 4). The ligation reaction was repeated until the average length of concatenated oligos fell within the range 0.5 to 1.0 kb as assayed by minigel, at which stage Klenow polymerase was added to fill in the gaps left by hybridization of unevenly matched overhangs. The concatenated oligos were run through a 2% TBE minigel and the fraction between 0.5 and 5.0 kb was electroeluted onto dialysis membrane, cleaned by organic extractions and ethanol precipitated. Concatemers were resuspended at 20 ng ul-I in sterile distilled water. 100 ng of concatemers were heat denatured for 10 mins at 100°C then cooled rapidly to 4°C. The following additions, modified from ref 5, were then made: 5 1.l IM HEPES pH 6.6, 5 Id DTM stock (100 mM each of dATP, dGTP and dTTP in 250 mm Tris HCl, 25 mM MgCl2, 50 mM 2-mercaptoethanol, pH 8.0), 1.4 Al (1 mM Tris, 1 mM EDTA pH 7.5), 2 IL BSA (5 mg mli-), 2.5 ng (TCC)4 primers, 1 tl Klenow fragment (3 units) and 5 ,11 a32P dCTP (3000 Ci mmol'-). The reaction was incubated at room temperature for 4-5 hours and the unincorporated nucleotides removed by Biogel P-60 spun column
chromatography. Southern blots were prehybridized in 4 x SSC, 2% SDS and 1 xBLOTTO (6) at 420C for 6-8 hours, and then 1 x 104 cpm of probe were added per mi of prehybridization solution and the
hybridization carried out overnight. Blots were washed with 4 changes of 4 x SSC, 0.1% SDS at 42°C and autoradiographed overnight with intensifying screens, or for 5 days without, representing a significant reduction in exposure time. Fig. 1 shows 6 HaeIll digests (4 ug) of unrelated House Sparrows (Passer domesticus) run on a 1% TAE agarose gel for 2820V hrs. The average individual possesses 24.8 bands with a mean band sharing between adjacent lanes of 0.182 giving a probability of profile sharing between unrelateds of 4.5 x 10-19.
ACKNOWLEDGEMENT This work was supported by a NERC research studentship (CAM) and SERC research grant (JHW).
REFERENCES 1. Tautz,D. and Renz,M. (1984) Nucl. Acids Res. 12, 4127-4137. 2. Weising,K., Weigand,F., Driesel,A.J., Kahl,G., Zischler,H. and Epplen,J.T. (1989) Nucl. Acids Res. 17, 10128. 3. Zischler,H., Nanda,I., Steeg,C., Schmid,M. and Epplen,J.T. (1989) In: Geldermann,H., Ellendorff,F., Stranzinger,G. (Eds) Proceedings of the Congress on Genome Analysis in Domestic Animals. VCH, Weinheim. 4. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning-A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 5. Feinberg,A.P. and Vogelstein,B. (1983) Anal. Biochem. 132, 6-13. 6. Johnson,D.A., Gautsch,J.W., Sportsman,J.R. and Elder,J.H. (1984) Gene Anal. Techn. 1, 3-8.
-
1t21kb
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40
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2:*1 *~ **iI.w
Nucleic Acids Research, Vol. 19, No. 16 4557
DNA fingerprinting by specific priming of concatenated ol igonucleotides Celia A.May and Jon H.Wetton Department of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK Submitted June 25, 1991 Simple sequences consisting of direct or cryptic tandem repeats of di-, tri- or tetra nucleotide motifs give rise to VNTR regions in a variety of plant and animal species (1, 2, 3). To date the principal detection method for these so-called microsatellites has involved end labelling of short oligonucleotides (9-20 bp) with -y-ATP and subsequent in-gel hybridization (3). By concatenating surplus oligos we have produced probes of higher specific activity with random priming reagents and a32P dCTP which are more routinely used in minisatellite fingerprinting. We have found that this greatly facilitates sequential probing of Southern blots with either micro- or minisatellite sequences with concomitant saving in time and expense. 1 mg each of (TCC)4 and the complementary (GAG)4 oligonucleotides were produced by an Applied Biosystems 381A DNA Synthesizer. The oligos were cleaved from the glass bead matrix by mixing with .880 SG ammonia at room temperature and deprotected by overnight incubation at 55°C. After lyophilization the oligos were resuspended in sterile distilled water and further purified by passage through anion exchange columns (Qiagen) to remove residual ammonium ions which are inhibitory to T4 polynucleotide kinase (4). Equimolar quantities of each were phosphorylated at the 5' end for lhr at 37°C, hybridized together and then ligated at 4°C for 24 hours (as in ref 4). The ligation reaction was repeated until the average length of concatenated oligos fell within the range 0.5 to 1.0 kb as assayed by minigel, at which stage Klenow polymerase was added to fill in the gaps left by hybridization of unevenly matched overhangs. The concatenated oligos were run through a 2% TBE minigel and the fraction between 0.5 and 5.0 kb was electroeluted onto dialysis membrane, cleaned by organic extractions and ethanol precipitated. Concatemers were resuspended at 20 ng ul-I in sterile distilled water. 100 ng of concatemers were heat denatured for 10 mins at 100°C then cooled rapidly to 4°C. The following additions, modified from ref 5, were then made: 5 1.l IM HEPES pH 6.6, 5 Id DTM stock (100 mM each of dATP, dGTP and dTTP in 250 mm Tris HCl, 25 mM MgCl2, 50 mM 2-mercaptoethanol, pH 8.0), 1.4 Al (1 mM Tris, 1 mM EDTA pH 7.5), 2 IL BSA (5 mg mli-), 2.5 ng (TCC)4 primers, 1 tl Klenow fragment (3 units) and 5 ,11 a32P dCTP (3000 Ci mmol'-). The reaction was incubated at room temperature for 4-5 hours and the unincorporated nucleotides removed by Biogel P-60 spun column
chromatography. Southern blots were prehybridized in 4 x SSC, 2% SDS and 1 xBLOTTO (6) at 420C for 6-8 hours, and then 1 x 104 cpm of probe were added per mi of prehybridization solution and the
hybridization carried out overnight. Blots were washed with 4 changes of 4 x SSC, 0.1% SDS at 42°C and autoradiographed overnight with intensifying screens, or for 5 days without, representing a significant reduction in exposure time. Fig. 1 shows 6 HaeIll digests (4 ug) of unrelated House Sparrows (Passer domesticus) run on a 1% TAE agarose gel for 2820V hrs. The average individual possesses 24.8 bands with a mean band sharing between adjacent lanes of 0.182 giving a probability of profile sharing between unrelateds of 4.5 x 10-19.
ACKNOWLEDGEMENT This work was supported by a NERC research studentship (CAM) and SERC research grant (JHW).
REFERENCES 1. Tautz,D. and Renz,M. (1984) Nucl. Acids Res. 12, 4127-4137. 2. Weising,K., Weigand,F., Driesel,A.J., Kahl,G., Zischler,H. and Epplen,J.T. (1989) Nucl. Acids Res. 17, 10128. 3. Zischler,H., Nanda,I., Steeg,C., Schmid,M. and Epplen,J.T. (1989) In: Geldermann,H., Ellendorff,F., Stranzinger,G. (Eds) Proceedings of the Congress on Genome Analysis in Domestic Animals. VCH, Weinheim. 4. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning-A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 5. Feinberg,A.P. and Vogelstein,B. (1983) Anal. Biochem. 132, 6-13. 6. Johnson,D.A., Gautsch,J.W., Sportsman,J.R. and Elder,J.H. (1984) Gene Anal. Techn. 1, 3-8.
-
1t21kb
i$s
40
9g.. ' 00
2:*1 *~ **iI.w
