BEECHNICAL
BIPS
Rapid hybridO~tion analysis to locate mutant genes in genomic DNA Hybridization to detect specific sequences in DNA from various sources has become a routine procedure. Here l describe a fast and simple protocol to detect single point mutations in genomic yeast DNA. Digested and electrophoresed DNA is hybridized with a 32P-labelled otigonucleotide containing the expected mutant sequence. Washing the hybridized agarose gel at different stringencies allows a mutant or the original wild-type sequence to be detected. The oligonucteotides used (23-29 nucleotides) contain one or two mismatches, located approximately in the middle of the sequence. Stringent washing conditions varied between 56°(2 and 71°(2, depending
(a) 50°C
(b) 58°C Kb
~W
11.0 5.2-
agarose gel. The separated DNA was denatured by soaking the gel for 20 rain in 0.4 MNaOH. 0.8 M NaC1. To neutralize, the gel 4 1 2 3 4 was soaked for 20 min in 0.5 i Tris-HCl (pH 7.4), t.5 M NaCt and dried in a gel dryer for 1 h at 60°(2. Hybridization with a FIGR 32p-end-labelled oligonucleotide (specific activity 2×106 c.p.m. pmo1-1) was performed in a mixture of 5xSSC. 20 ggml-1 Autoradiographs of an agarose gel hybridization analysis yeast tRNA and 0.1% SDS (w/v) for 2 h at 42°C. The gel was with a 32P-end-labeUed oligonucleotide. Genomic yeast washed twice at 23°C. 37°C and at 50°C in 5xSSC. each time DNAs, originating from haploid strains of one tetrad, were for five minutes and autoradiographed for 4-15 h. Under these digested with the restriction enzyme PstI and separated on conditions, oligonucleotides containing one or two mismatches a 0.9°,6 agarose get. Hybridization. washing and autostill hybridize to the complementary genomic sequence. radiography (5 h exposure time) was performed as described When the washing temperature is raised to stringent in the text. The sizes of the hybridization signals represent conditions, these hybridization signals disappear and only an integrated mutated actin-LEU2 gene fragment (11 kb) or those produced by complementary homologous sequences the wild-type actin gene (5.2 kb) as described 2. remain stable. Figure 1 shows autoradiographs from a hybridization analysis with genomic yeast DNAs TABLE1. Oligonudeotides used to identify point mutations in the yeast actin gene originating from four haploid strains of one tetrad. The oligonucleotide Mutation Oligonucleotide sequence Stringent used (C374A. see Table 1) contained temperature (°C) a G and a C at positions 12 and 13. which will mismatch with a G and a T A at the corresponding genomic location D2V 5'-GAACATACCAGAAACCATI'GTI'AATTC-3' 68 of the yeast actin gene 1. GA DNAs from lanes 1 and 3 contain E4V 5'-CCAAAGCAGCAACAACTAAACATATAATATAGC-3' 70 a wild-type actin gene. A hybridization signal is readily detected at nonTG stringent washing conditions (Fig. la, D l l Q 5'-CATACCAGAACCGTITrGAATAACCAAAGC-3' 67 5.2 kb bands), but can no longer be A discerned at higher stringency (58°C, 5'-CTACTCAAGATCATCATCAAGTAGT-3' 60 K191M Fig. lb). Two DNAs (lanes 2 and 4) contain a mutant actin gene. It is inGT serted, together with a LEU2 gene, at C374A 5*-GAGATI'AGAAAGCCTrGTGGTGAA-3' 58 the chromosomal actin locus, to replace the wild-type gene2. Their hy- The mismatched bases are shown above the correspondin~ wild-type sequences bridization signals with the mutant More detailed information about the codon exchanges is given in Ref. 2. oligonudeotide probe remain stable at the stringent washing temperature (Fig. lb, I1 kb bands). To prove the validity of this method the mutant actin gene was retrieved by plasmid eviction3 and sequenced. The assay has been used to identify t6 different point mutations in the yeast actin gene 2. Table 1 shows the sequence and stringent temperatures for some selected oligonucleotides. This rapid protocol has two advantages over blotting procedures: (1) the whole analysis, including autoradiography, takes less than 24 h; (2) direct gel hybridization avoids loss of DNA during membrane transfer, thereby increasing the sensitivity of the assay. REFERENCES
1 Gallwitz. D. and Sums, I. (1980) Proc. NatlAcad. Sci. USA 77, 2546-2550 2 Johannes, F.J. and GaUwitz. D. (1991) EMBOJ. 10, 3951-3958 3 Winston, F., Chumley, F. and Fink, G.R. (1983) Meth. Enzymol. 101, 211-228
Contributed by Franz-Josef Johannes, Institute of Cell Biology and Immunology, University of Stuttgart, Pfaffenwaldring 57, 7000 Stuttgart 80, FRG. TIG FEBRUARY1992 VOt.. 8 NO. 2
-t8
BIPS
Rapid hybridO~tion analysis to locate mutant genes in genomic DNA Hybridization to detect specific sequences in DNA from various sources has become a routine procedure. Here l describe a fast and simple protocol to detect single point mutations in genomic yeast DNA. Digested and electrophoresed DNA is hybridized with a 32P-labelled otigonucleotide containing the expected mutant sequence. Washing the hybridized agarose gel at different stringencies allows a mutant or the original wild-type sequence to be detected. The oligonucteotides used (23-29 nucleotides) contain one or two mismatches, located approximately in the middle of the sequence. Stringent washing conditions varied between 56°(2 and 71°(2, depending
(a) 50°C
(b) 58°C Kb
~W
11.0 5.2-
agarose gel. The separated DNA was denatured by soaking the gel for 20 rain in 0.4 MNaOH. 0.8 M NaC1. To neutralize, the gel 4 1 2 3 4 was soaked for 20 min in 0.5 i Tris-HCl (pH 7.4), t.5 M NaCt and dried in a gel dryer for 1 h at 60°(2. Hybridization with a FIGR 32p-end-labelled oligonucleotide (specific activity 2×106 c.p.m. pmo1-1) was performed in a mixture of 5xSSC. 20 ggml-1 Autoradiographs of an agarose gel hybridization analysis yeast tRNA and 0.1% SDS (w/v) for 2 h at 42°C. The gel was with a 32P-end-labeUed oligonucleotide. Genomic yeast washed twice at 23°C. 37°C and at 50°C in 5xSSC. each time DNAs, originating from haploid strains of one tetrad, were for five minutes and autoradiographed for 4-15 h. Under these digested with the restriction enzyme PstI and separated on conditions, oligonucleotides containing one or two mismatches a 0.9°,6 agarose get. Hybridization. washing and autostill hybridize to the complementary genomic sequence. radiography (5 h exposure time) was performed as described When the washing temperature is raised to stringent in the text. The sizes of the hybridization signals represent conditions, these hybridization signals disappear and only an integrated mutated actin-LEU2 gene fragment (11 kb) or those produced by complementary homologous sequences the wild-type actin gene (5.2 kb) as described 2. remain stable. Figure 1 shows autoradiographs from a hybridization analysis with genomic yeast DNAs TABLE1. Oligonudeotides used to identify point mutations in the yeast actin gene originating from four haploid strains of one tetrad. The oligonucleotide Mutation Oligonucleotide sequence Stringent used (C374A. see Table 1) contained temperature (°C) a G and a C at positions 12 and 13. which will mismatch with a G and a T A at the corresponding genomic location D2V 5'-GAACATACCAGAAACCATI'GTI'AATTC-3' 68 of the yeast actin gene 1. GA DNAs from lanes 1 and 3 contain E4V 5'-CCAAAGCAGCAACAACTAAACATATAATATAGC-3' 70 a wild-type actin gene. A hybridization signal is readily detected at nonTG stringent washing conditions (Fig. la, D l l Q 5'-CATACCAGAACCGTITrGAATAACCAAAGC-3' 67 5.2 kb bands), but can no longer be A discerned at higher stringency (58°C, 5'-CTACTCAAGATCATCATCAAGTAGT-3' 60 K191M Fig. lb). Two DNAs (lanes 2 and 4) contain a mutant actin gene. It is inGT serted, together with a LEU2 gene, at C374A 5*-GAGATI'AGAAAGCCTrGTGGTGAA-3' 58 the chromosomal actin locus, to replace the wild-type gene2. Their hy- The mismatched bases are shown above the correspondin~ wild-type sequences bridization signals with the mutant More detailed information about the codon exchanges is given in Ref. 2. oligonudeotide probe remain stable at the stringent washing temperature (Fig. lb, I1 kb bands). To prove the validity of this method the mutant actin gene was retrieved by plasmid eviction3 and sequenced. The assay has been used to identify t6 different point mutations in the yeast actin gene 2. Table 1 shows the sequence and stringent temperatures for some selected oligonucleotides. This rapid protocol has two advantages over blotting procedures: (1) the whole analysis, including autoradiography, takes less than 24 h; (2) direct gel hybridization avoids loss of DNA during membrane transfer, thereby increasing the sensitivity of the assay. REFERENCES
1 Gallwitz. D. and Sums, I. (1980) Proc. NatlAcad. Sci. USA 77, 2546-2550 2 Johannes, F.J. and GaUwitz. D. (1991) EMBOJ. 10, 3951-3958 3 Winston, F., Chumley, F. and Fink, G.R. (1983) Meth. Enzymol. 101, 211-228
Contributed by Franz-Josef Johannes, Institute of Cell Biology and Immunology, University of Stuttgart, Pfaffenwaldring 57, 7000 Stuttgart 80, FRG. TIG FEBRUARY1992 VOt.. 8 NO. 2
-t8
