Nucleic Acids Research, Vol. 18, No. 24 7459
DNA fingerprinting with 35S nucleotides Brenda S.Johnson* and John P.H.Th'ng1 Department of Zoology, University of California, Davis, CA 95616-8755 and 1Department of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616-8635, USA Submitted November 2, 1990 Recent forensic investigations and studies of genetic relatedness in diverse organisms have employed DNA fingerprinting techniques that nearly exclusively use probes labelled with Phosphorus-32 (32p). 32p is generally preferred for this purpose because it is a strong fl-emitter that produces an intense signal after a relatively short autoradiographic exposure. This characteristic, however, is not without shortcomings. Even brief exposures to the high-energy of 32p, particularly with commonly-used intensifying screens, can cause the resulting unavoidably fuzzy bands to bleed into one another, which may obscure true band number and location. Accuracy may thus be compromised in the interest of obtaining the results quickly. In addition, incorporation of 32p causes inevitable degradation of the probe DNA due to strand scission, limiting the potential storage time for 32P-labelled probes to no more than 1 week. Use of 32P also requires more extensive and costly safety precautions, such as shielding and remote manipulation, than do radionuclides of lower energy. We investigated the potential of Sulfur-35 (35S) nucleotides to circumvent these drawbacks. As 35S is an order of magnitude less energetic than 32p (0ma of 167 keV compared to 1.7 MeV), it is relatively safer and is easier to use routinely. 35S has a halflife 6 times that of 32p (87.4 compared to 14.3 days), and it does not cause as much degradation of probe DNA. This greater stability contributes to a longer probe storage life and increased radionuclide economy, as a stock vial can be drawn from much longer. Genomic avian DNA was isolated by standard methods of phenol:chloroform extraction and ethanol precipitation (1, 2). Five jig were digested with HinfI and fractionated on a 0.6% agarose gel run at 1.2 V/cm for 40 hours. Southern transfer to a nylon membrane (Nytran), hybridization, and washes were performed according to the protocol described by Westneat et al. (3). We labelled minisatellite probe pV47-2 (4) by random primer extension, using either 50 AtCi [a-32P] dCTP (> 3000 Ci/mMol) or 50 ,.tCi each of [a-35S] dCTP and dATP (> 1000 Ci/mMol) (Amersham); and we hybridized the membrane with 1 -2 x 106 cpm/ml solution. 32P blots were enveloped in Saran Wrap and exposed to Kodak X-Omat AR film for up to 3 days at 80°C with 1 screen, or longer than 6 days at room -
temperature. 35S blots can be thoroughly air-dried and laid directly on film if exposure is limited to no more than 3 days. However, this method can cause the probe to bind permanently
*
To whom correspondence should be addressed
kb --V.
.
*.4iB
MRs_B
9-
A,
. ...
s?s
B
C
Figure 1. Avian DNA fingerprints as detected by pV47-2 probe labelled with 35S (panel A) and 32p (panels B and C).
to some membranes. An alternative to exposing the dry blot is to lay the moist membrane signal-side-up on a piece of filter paper and to wrap these smoothly together with 1.5 micron mylar
sheeting (Ambis Systems, Inc., 3939 Ruffin Rd., San Diego, CA 92123). We exposed the film to the DNA-side of the blot for 3-6 days at room temperature. To reprobe either membrane, signal was stripped by boiling in .01 x SSC/0.5 % SDS for 20 minutes, and the blot was then rehybridized. Figure 1 shows avian DNA fingerprints that were detected, as described above, by 35S nucleotides (panel A) and by intensified and unintensified 32p (panels B and C, respectively). The membrane was first probed with 35S-labelled pV47-2, then stripped and reprobed with 32P-labelled pV47-2. The bands obtained with 35S are generally sharper, and proximate bands can be more easily distinguished. For example, bands ambiguously smeared or not visible when probed with 32p were revealed by 35S to be made up of several distinct markers (see bands delineated by filled arrows in figure). In other cases (unfilled arrows), single markers on the 32P blots were shown by 35S to comprise 2 bands. Although 32P nucleotides are adequate for most DNA fingerprinting needs, 35S may be useful in special cases, such as in the discrimination of closely-migrating fragments. 35S can significantly improve DNA fingerprint resolution because it produces better-defined bands, especially in the low molecular weight range where bands tend to lie closer together. This may be especially important in the case of high-concentration agarose gels that are run relatively quickly.
7460 Nucleic Acids Research, Vol. 18, No. 24
ACKNOWLEDGEMENTS This work was supported in part by ACS grant NP473 to E.M.Bradbury. Donna Gadbois recommended and provided the mylar sheeting.
REFERENCES 1. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 2. Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., SmTith,J.A. and Struhl,K. (1987) Current Protocols in Molecular Biology. Green Publishing Associates and Wiley-Interscience, New York. 3. Westneat,D., Noon,W.A., Reeve,H.K. and Aquadro,C.F. (1988) Nucl. Acids Res. 16, 4161. 4. Longmire,J.L., Kraemer,P.M., Brown,N.C., Hardekopf,L.C. and Deaven,L.L. (1990) Nucl. Acids Res. 18, 1658.
DNA fingerprinting with 35S nucleotides Brenda S.Johnson* and John P.H.Th'ng1 Department of Zoology, University of California, Davis, CA 95616-8755 and 1Department of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616-8635, USA Submitted November 2, 1990 Recent forensic investigations and studies of genetic relatedness in diverse organisms have employed DNA fingerprinting techniques that nearly exclusively use probes labelled with Phosphorus-32 (32p). 32p is generally preferred for this purpose because it is a strong fl-emitter that produces an intense signal after a relatively short autoradiographic exposure. This characteristic, however, is not without shortcomings. Even brief exposures to the high-energy of 32p, particularly with commonly-used intensifying screens, can cause the resulting unavoidably fuzzy bands to bleed into one another, which may obscure true band number and location. Accuracy may thus be compromised in the interest of obtaining the results quickly. In addition, incorporation of 32p causes inevitable degradation of the probe DNA due to strand scission, limiting the potential storage time for 32P-labelled probes to no more than 1 week. Use of 32P also requires more extensive and costly safety precautions, such as shielding and remote manipulation, than do radionuclides of lower energy. We investigated the potential of Sulfur-35 (35S) nucleotides to circumvent these drawbacks. As 35S is an order of magnitude less energetic than 32p (0ma of 167 keV compared to 1.7 MeV), it is relatively safer and is easier to use routinely. 35S has a halflife 6 times that of 32p (87.4 compared to 14.3 days), and it does not cause as much degradation of probe DNA. This greater stability contributes to a longer probe storage life and increased radionuclide economy, as a stock vial can be drawn from much longer. Genomic avian DNA was isolated by standard methods of phenol:chloroform extraction and ethanol precipitation (1, 2). Five jig were digested with HinfI and fractionated on a 0.6% agarose gel run at 1.2 V/cm for 40 hours. Southern transfer to a nylon membrane (Nytran), hybridization, and washes were performed according to the protocol described by Westneat et al. (3). We labelled minisatellite probe pV47-2 (4) by random primer extension, using either 50 AtCi [a-32P] dCTP (> 3000 Ci/mMol) or 50 ,.tCi each of [a-35S] dCTP and dATP (> 1000 Ci/mMol) (Amersham); and we hybridized the membrane with 1 -2 x 106 cpm/ml solution. 32P blots were enveloped in Saran Wrap and exposed to Kodak X-Omat AR film for up to 3 days at 80°C with 1 screen, or longer than 6 days at room -
temperature. 35S blots can be thoroughly air-dried and laid directly on film if exposure is limited to no more than 3 days. However, this method can cause the probe to bind permanently
*
To whom correspondence should be addressed
kb --V.
.
*.4iB
MRs_B
9-
A,
. ...
s?s
B
C
Figure 1. Avian DNA fingerprints as detected by pV47-2 probe labelled with 35S (panel A) and 32p (panels B and C).
to some membranes. An alternative to exposing the dry blot is to lay the moist membrane signal-side-up on a piece of filter paper and to wrap these smoothly together with 1.5 micron mylar
sheeting (Ambis Systems, Inc., 3939 Ruffin Rd., San Diego, CA 92123). We exposed the film to the DNA-side of the blot for 3-6 days at room temperature. To reprobe either membrane, signal was stripped by boiling in .01 x SSC/0.5 % SDS for 20 minutes, and the blot was then rehybridized. Figure 1 shows avian DNA fingerprints that were detected, as described above, by 35S nucleotides (panel A) and by intensified and unintensified 32p (panels B and C, respectively). The membrane was first probed with 35S-labelled pV47-2, then stripped and reprobed with 32P-labelled pV47-2. The bands obtained with 35S are generally sharper, and proximate bands can be more easily distinguished. For example, bands ambiguously smeared or not visible when probed with 32p were revealed by 35S to be made up of several distinct markers (see bands delineated by filled arrows in figure). In other cases (unfilled arrows), single markers on the 32P blots were shown by 35S to comprise 2 bands. Although 32P nucleotides are adequate for most DNA fingerprinting needs, 35S may be useful in special cases, such as in the discrimination of closely-migrating fragments. 35S can significantly improve DNA fingerprint resolution because it produces better-defined bands, especially in the low molecular weight range where bands tend to lie closer together. This may be especially important in the case of high-concentration agarose gels that are run relatively quickly.
7460 Nucleic Acids Research, Vol. 18, No. 24
ACKNOWLEDGEMENTS This work was supported in part by ACS grant NP473 to E.M.Bradbury. Donna Gadbois recommended and provided the mylar sheeting.
REFERENCES 1. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 2. Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., SmTith,J.A. and Struhl,K. (1987) Current Protocols in Molecular Biology. Green Publishing Associates and Wiley-Interscience, New York. 3. Westneat,D., Noon,W.A., Reeve,H.K. and Aquadro,C.F. (1988) Nucl. Acids Res. 16, 4161. 4. Longmire,J.L., Kraemer,P.M., Brown,N.C., Hardekopf,L.C. and Deaven,L.L. (1990) Nucl. Acids Res. 18, 1658.
