Biochem. J. (1990) 266, 615-617 (Printed in Great Britain)
615
Genetic characterization of an alloalbumin, albumin Kashmir, using gene amplification and allele-specific oligonucleotides Demetris SAVVA,*t Andra's L. TARNOKY* and Michael F. VICKERSt *Department of Biochemistry & Physiology, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 2AJ, and tDepartment of Clinical Biochemistry, Royal Berkshire Hospital, Reading RGl SAN, U.K.
The molecular basis for albumin Kashmir was studied using the polymerase chain reaction to amplify a DNA fragment containing codon 501 in exon 12 of the human albumin gene. Southern blots of the amplified DNA were hybridized to oligonucleotide probes specific either for the normal allele of albumin or for albumin Kashmir. The results provide strong evidence that codon 501 in albumin Kashmir is AAG (lysine) instead of GAG (glutamic acid), thus confirming the protein sequences reported. This approach was used to characterize a bisalbuminaemic individual as a carrier for albumin Kashmir. Similar strategies may be devised to study the molecular basis and to identify carriers of other alloalbumins.
INTRODUCTION
The variant albumin Kashmir described in 1969 [1] was the first of several reports probably describing the same widely distributed variant in families of Asian descent. The serum of the propositus, a heterozygote, contained the expected near-equal proportions of the
wild-type and the variant albumins; only the wild-type albumin showed the normal binding to HABA [2-(4'hydroxyazobenzene)benzoic acid], whereas both bound Bromocresol Green. Huss et al. [2] have recently determined the substitution in three alloalbumins, namely Vancouver, Birmingham and Adana, as glutamic acid-501 to lysine, and assumed identity with albumin Kashmir, which they did not examine. Their assumption was reasonable on ethnic grounds and was one made by Tarnoky for albumin Birmingham [3]. When blood from the members of the original Kashmiri family became available, we decided to extend the work of Huss et al. [2] to the genetic characterization of this variant. The application of recombinant DNA procedures to the study of albumin has resulted in the isolation of the cDNA for human albumin [4,5]; it has also allowed the characterization of the genomic DNA and the localization of the gene on chromosome 4 [6]. Recent years have seen the development of gene amplification procedures and in particular the polymerase chain reaction (PCR), which uses a thermostable DNA polymerase for the amplification reaction [7]. In this paper we describe the use of PCR to amplify part of the human albumin gene, and the use of allele-specific oligonucleotides (ASOs) to identify bisalbuminaemic individuals possessing the Kashmir variant. Similar methods have been used in other laboratories for, amongst others, the prenatal diagnosis of sickle-cell anaemia [8], the detection of mutations leading to aclantitrypsin deficiency [9] and HLA histocompatibility antigen typing [10].
MATERIALS AND METHODS Materials and electrophoresis Blood samples were collected from daughters A and B of the propositus, from an unrelated Asian patient C with bisalbuminaemia and from his two children D and E. The blood samples were allowed to clot and were then centrifuged for 5 min at 3000 g. Serum samples were applied on to cellulose acetate membranes (Sartorius Ltd., Epsom, Surrey, U.K.) using a multiapplicator (Shandon Southern, Runcorn, Cheshire, U.K.). Proteins were separated by electrophoresis in barbitone acetate buffer (pH 8.6, I 0.1) at 80 V for 75 min, stained with 0.2 % (w/v) Ponceau S in 3 % (w/v) trichloroacetic acid, washed with 7 % (v/v) acetic acid and scanned. Isolation of DNA Crude DNA preparations were obtained from heparinized blood or, in some cases, from homogenization of blood clots obtained as described above. Samples (0.5 ml) were centrifuged for 5 min in an Eppendorf Microfuge, and cell pellets were resuspended in 1 ml of TE buffer (10 mM-Tris/HCl (pH 7.5)/i mMNa2EDTA) and recentrifuged. The cell pellets were then resuspended in 100 ,tl of water, incubated for 20 min in a boiling water bath and centrifuged in an Eppendorf Microfuge for 15 min. The supernatants were stored at -20 °C and used as sources of DNA. Amplification of albumin DNA using the PCR The sequences of the two oligonucleotides (oligos; 20-mers) were based on the published sequence for the human albumin gene [6], with one of these (DS40) being of the same sense as this sequence and the other (DS41) being of the opposite sense; both sequences are located in exon 12 of the gene. Oligo DS40 corresponds to nucleotides 13805-13824 of the albumin gene (5'TTGTCCTGAACCAGTTATGT-3') and oligo DS41 (5'-GATTTGTCTCTCCTTCTCAG-3') to nucleotides
Abbreviations used: PCR, polymerase chain reaction; ASO, allele-specific oligonucleotide; oligo, oligonucleotide. $ To whom correspondence should be addressed.
Vol. 266
D. Savva, A. L. Tarnoky and M. F. Vickers
616
14010-13991. These oligos should therefore result in the amplification of a 206 bp fragment of DNA. DNA amplification experiments were performed in a final volume of 50 ,1 using Amplitaq (Perkin-Elmer, Beaconsfield, Bucks., U.K.), a thermostable recombinant Taq DNA polymerase. In addition to the DNA to be amplified (15 ,u of crude DNA preparation), each reaction mixture also contained 10 mM-Tris/HCl, pH 8.3, 50 mM-KCl, 1.5 mM-MgCl2, 0.01 00 (w/v) gelatin, 0.2 mM of each deoxynucleotide triphosphate, 0.2 4uM-oligo DS40, 0.2 jtM-oligo DS41 and 2.5 units of DNA polymerase; mineral oil (50 ,1) was added to each reaction to prevent evaporation. The DNA in each reaction was initially ' melted ' by incubating at 95 °C for 5 min before 30 cycles of amplification were performed using a programmable Dri-Block (Techne, Cambridge, U.K.). Each amplification cycle consisted of incubations at 95 °C for 1 min, 50 °C for 1 min and 74 °C for 1.5 min. At the end of the 30 cycles, the reaction mixtures were incubated at 25 °C for 5 min. Southern hybridizations Hybridizations were performed using two ASOs with their sequences based on the same sense as the sequence of the human albumin gene [6] and corresponding to bases 13935-13951: oligo DS42 (5'-TTCCCAAAGAGTTTAAT-3') was specific for the normal allele of the gene (which codes for glutamic acid at position 501) and oligo DS43 (same as DS42 but with A instead of G at nucleotide 9) was specific for the Kashmir variant [2], assuming the minimum number of mutations. Both DS42 and DS43 were radiolabelled using [y-32P]ATP (3000 Ci/mmol) and T4 polynucleotide kinase (both from Amersham International, Amersham, Bucks., U.K.) to a specific radioactivity of about 3 x 108 d.p.m./,ug of oligo. Electrophoresis of DNA using 10 ,1 aliquots from each PCR was performed as described previously [11] on 1.4 0'; (w/v) agarose gels. Southern blotting on to Hybond-N membranes was carried out as recommended by the supplier of the membrane (Amersham International). Hybridizations were performed at 29 °C using 100 ng of radiolabelled oligo (DS42 or DS43) in 2 x SSC (SSC is 0.15 M-NaCl/0.01 5 M-trisodium citrate, pH 7.0) containing 0.5 0 (w/v) dried skimmed milk. The hybridization temperature was calculated according to Wallace & Miyada [12] so as to allow the formation of perfect hybrids only. Membranes were washed twice (10 min each) in 2 x SSC at room temperature, air-dried and exposed to Fuji-RX X-ray film. Following the development of the X-ray film, the membranes were washed for 10 min in 0.1 x SSC at 65 °C to remove the bound probe and then re-used in hybridizations with the alternative probe. RESULTS AND DISCUSSION Electrophoretic separation of the serum proteins (Fig. 1) shows two albumin bands in individual A but not in B, although both were members of the authentic albumin Kashmir family. The results also show two similar bands in patient C, whose daughters D and E, however, do not carry the trait. Huss et al. [2] located the change in the albumins presumed to be Kashmir at position 501, which changed from glutamic acid to lysine. From the sequence described
B
A ..:
....
...
D
C ::::.::
::
E
..:: :.
Fig. 1. Cellulose acetate electrophoresis of normal and alloalbuminaemic sera Serum samples from each individual were electrophoresed on cellulose acetate membranes as described in the Materials and methods section. Letters above each lane identify the individuals studied.
by Minghetti et al. [6], the codon for glutamic acid-501 in normal albumin is GAG. There are two possible codons for lysine (AAG and AAA), but assuming that the change from glutamic acid to lysine was due to only one mutational event, the change in the DNA would have been from GAG to AAG. In order to confirm this assumption in individual A, as well as to confirm the diagnosis for the bisalbuminaemic individual C, the PCR has been used to amplify a 206 bp fragment of human DNA located totally within exon 12 of the albumin gene. This amplified fragment was then used in hybridizations with oligos specific either for the normal allele or for that coding for the Kashmir variant. The PCR was performed using the crude DNA preparations obtained from the blood or blood clots of individuals A-E and the amplification products were analysed by electrophoresis on agarose gels. The results (not shown) indicated that in all cases the amplification product was a DNA fragment of about 200 bp, which is in close agreement with the expected size (206 bp). Although blood clots (from subjects A and B) resulted in less amplification than whole blood, the amount of DNA obtained was sufficient for subsequent hybridization. Southern hybridization of the PCR amplification products (Fig. 2) with the radiolabelled oligo corresponding to the normal allele of albumin (DS42) gave a positive result with all five individuals, with the strength of the hybridization signal reflecting the amount of PCR product present in each sample. Furthermore, the mutant allele for albumin Kashmir (DS43) hybridized to two PCR products only (those for individuals A and C). This suggests that the hybridization conditions employed were sufficiently stringent to prevent the formation of hybrids containing even a single mismatch. Computer analysis of the nucleotide sequence of the amplified DNA using the WORDSEARCH program in the sequence analysis package of the University of Wisconsin (available at the Daresbury Laboratory) showed (1) that the only perfect match between this and oligo DS42 was at the expected position (bases 13935-13951 [6]), (2) that DS43 showed 1990
Albumin Kashmir: diagnosis using DNA hybridization
(a)
C
D
E
A
B
(bp)
617 as suggested by his ethnic background and electrophoretic pattern. A strategy similar to that adopted here may also be applied to the molecular characterization of other alloalbumins. It will of course be necessary to alter the sequences of the oligos used depending on the particular albumin variant under investigation; information to allow the design of appropriate oligos is available, since the albumin gene has been characterized [6] and amino acid substitutions in a number of albumin mutants are known [14]. Of particular interest will be an examination of the DNA changes resulting in albumins Vancouver, Birmingham and Adana, which contain the same change at position 501 as that for albumin Kashmir. For example, it will be interesting to establish whether the codon change resulting in these variants was the same as that for albumin Kashmir (GAG to AAG).
Thanks are due to Miss Jennifer Morris for expert technical assistance during the course of this work.
REFERENCES
Fig. 2. Analysis of the PCR by Southern hybridization. The PCR amplification products were electrophoresed on agarose gels, blotted on to Hybond-N membranes and hybridized with the two allele-specific oligonucleotide probes for normal albumin (DS42; a) and for albumin Kashmir (DS43; b) as described in the Materials and methods section. Letters above each lane identify the individuals studied. The positions of the molecular mass markers (in bp) are indicated by arrows.
only one mismatch with this sequence at base 13943 [6], and (3) that there were no other sequences with any similarity to either oligo. Fig. 2 shows some additional bands in certain tracks (C, D and E with DS42 and C with DS43); these are due to the formation of primer artefacts and incomplete strands in PCRs of more than 20 cycles [13]. Huss et al. [2] assumed that their sera contained the same mutation as that in albumin Kashmir. Our results, obtained with DNA from the original family, substantiate this and are evidence for the assumption (made when designing oligo DS43) that the mutation resulting in the albumin Kashmir variant contained a change of codon 501 from GAG to AAG. They also confirm that -individual C was a carrier for the albumin Kashmir allele, Received 12 October 1989/29 November 1989; accepted 5 January 1990
Vol. 266
1. Tarnoky, A. L. & Dowding, B. (1969) Clin. Chim. Acta 26, 455-458 2. Huss, K., Madison, J., Ishioka, N., Takahashi, N., Arai, K. & Putnam, F. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6692-6696 3. Tarnoky, A. L. (1980) Adv. Clin. Chem. 21, 101-146 4. Lawn, R. M., Adelman, J., Bock, S. C., Franke, A. E., Houck, C. M., Najarian, R. C., Seeburg, P. H. & Wion, K. L. (1981) Nucleic Acids Res. 9, 6103-6114 5. Dugaiczyk, A., Law, S. W. & Dennison, 0. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 71-75 6. Minghetti, P. P., Ruffner, D. E., Kuang, W.-J., Dennison, 0. E., Hawkins, J. W., Beattie, W. G. & Dugaiczyk, A. (1986) J. Biol. Chem. 261, 6747-6757 7. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. & Arnheim, N. (1985) Science 230, 1350-1354 8. Kazazian, H. H. (1989) in Polymerase Chain Reaction (Erlich, H. A., Gibbs, R. & Kazazian, H. H., eds.), pp. 47-55, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 9. Kidd, V. J., Wallace, R. B., Itakura, K. & Woo, S. L. C. (1983) Nature (London) 304, 230-234 10. Saiki, R. K., Walsh, P. S., Levenson, C. H. & Erlich, H. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6230-6234 11. Savva, D. & Mandelstam, J. (1984) J. Gen. Microbiol. 130, 2137-2145 12. Wallace, R. B. & Miyada, C. G. (1987) Methods Enzymol. 152, 432-442 13. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science 239, 487-491 14. Takahashi, N., Takahashi, Y., Blumberg, B. S. & Putnam, F. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4413-4417
615
Genetic characterization of an alloalbumin, albumin Kashmir, using gene amplification and allele-specific oligonucleotides Demetris SAVVA,*t Andra's L. TARNOKY* and Michael F. VICKERSt *Department of Biochemistry & Physiology, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 2AJ, and tDepartment of Clinical Biochemistry, Royal Berkshire Hospital, Reading RGl SAN, U.K.
The molecular basis for albumin Kashmir was studied using the polymerase chain reaction to amplify a DNA fragment containing codon 501 in exon 12 of the human albumin gene. Southern blots of the amplified DNA were hybridized to oligonucleotide probes specific either for the normal allele of albumin or for albumin Kashmir. The results provide strong evidence that codon 501 in albumin Kashmir is AAG (lysine) instead of GAG (glutamic acid), thus confirming the protein sequences reported. This approach was used to characterize a bisalbuminaemic individual as a carrier for albumin Kashmir. Similar strategies may be devised to study the molecular basis and to identify carriers of other alloalbumins.
INTRODUCTION
The variant albumin Kashmir described in 1969 [1] was the first of several reports probably describing the same widely distributed variant in families of Asian descent. The serum of the propositus, a heterozygote, contained the expected near-equal proportions of the
wild-type and the variant albumins; only the wild-type albumin showed the normal binding to HABA [2-(4'hydroxyazobenzene)benzoic acid], whereas both bound Bromocresol Green. Huss et al. [2] have recently determined the substitution in three alloalbumins, namely Vancouver, Birmingham and Adana, as glutamic acid-501 to lysine, and assumed identity with albumin Kashmir, which they did not examine. Their assumption was reasonable on ethnic grounds and was one made by Tarnoky for albumin Birmingham [3]. When blood from the members of the original Kashmiri family became available, we decided to extend the work of Huss et al. [2] to the genetic characterization of this variant. The application of recombinant DNA procedures to the study of albumin has resulted in the isolation of the cDNA for human albumin [4,5]; it has also allowed the characterization of the genomic DNA and the localization of the gene on chromosome 4 [6]. Recent years have seen the development of gene amplification procedures and in particular the polymerase chain reaction (PCR), which uses a thermostable DNA polymerase for the amplification reaction [7]. In this paper we describe the use of PCR to amplify part of the human albumin gene, and the use of allele-specific oligonucleotides (ASOs) to identify bisalbuminaemic individuals possessing the Kashmir variant. Similar methods have been used in other laboratories for, amongst others, the prenatal diagnosis of sickle-cell anaemia [8], the detection of mutations leading to aclantitrypsin deficiency [9] and HLA histocompatibility antigen typing [10].
MATERIALS AND METHODS Materials and electrophoresis Blood samples were collected from daughters A and B of the propositus, from an unrelated Asian patient C with bisalbuminaemia and from his two children D and E. The blood samples were allowed to clot and were then centrifuged for 5 min at 3000 g. Serum samples were applied on to cellulose acetate membranes (Sartorius Ltd., Epsom, Surrey, U.K.) using a multiapplicator (Shandon Southern, Runcorn, Cheshire, U.K.). Proteins were separated by electrophoresis in barbitone acetate buffer (pH 8.6, I 0.1) at 80 V for 75 min, stained with 0.2 % (w/v) Ponceau S in 3 % (w/v) trichloroacetic acid, washed with 7 % (v/v) acetic acid and scanned. Isolation of DNA Crude DNA preparations were obtained from heparinized blood or, in some cases, from homogenization of blood clots obtained as described above. Samples (0.5 ml) were centrifuged for 5 min in an Eppendorf Microfuge, and cell pellets were resuspended in 1 ml of TE buffer (10 mM-Tris/HCl (pH 7.5)/i mMNa2EDTA) and recentrifuged. The cell pellets were then resuspended in 100 ,tl of water, incubated for 20 min in a boiling water bath and centrifuged in an Eppendorf Microfuge for 15 min. The supernatants were stored at -20 °C and used as sources of DNA. Amplification of albumin DNA using the PCR The sequences of the two oligonucleotides (oligos; 20-mers) were based on the published sequence for the human albumin gene [6], with one of these (DS40) being of the same sense as this sequence and the other (DS41) being of the opposite sense; both sequences are located in exon 12 of the gene. Oligo DS40 corresponds to nucleotides 13805-13824 of the albumin gene (5'TTGTCCTGAACCAGTTATGT-3') and oligo DS41 (5'-GATTTGTCTCTCCTTCTCAG-3') to nucleotides
Abbreviations used: PCR, polymerase chain reaction; ASO, allele-specific oligonucleotide; oligo, oligonucleotide. $ To whom correspondence should be addressed.
Vol. 266
D. Savva, A. L. Tarnoky and M. F. Vickers
616
14010-13991. These oligos should therefore result in the amplification of a 206 bp fragment of DNA. DNA amplification experiments were performed in a final volume of 50 ,1 using Amplitaq (Perkin-Elmer, Beaconsfield, Bucks., U.K.), a thermostable recombinant Taq DNA polymerase. In addition to the DNA to be amplified (15 ,u of crude DNA preparation), each reaction mixture also contained 10 mM-Tris/HCl, pH 8.3, 50 mM-KCl, 1.5 mM-MgCl2, 0.01 00 (w/v) gelatin, 0.2 mM of each deoxynucleotide triphosphate, 0.2 4uM-oligo DS40, 0.2 jtM-oligo DS41 and 2.5 units of DNA polymerase; mineral oil (50 ,1) was added to each reaction to prevent evaporation. The DNA in each reaction was initially ' melted ' by incubating at 95 °C for 5 min before 30 cycles of amplification were performed using a programmable Dri-Block (Techne, Cambridge, U.K.). Each amplification cycle consisted of incubations at 95 °C for 1 min, 50 °C for 1 min and 74 °C for 1.5 min. At the end of the 30 cycles, the reaction mixtures were incubated at 25 °C for 5 min. Southern hybridizations Hybridizations were performed using two ASOs with their sequences based on the same sense as the sequence of the human albumin gene [6] and corresponding to bases 13935-13951: oligo DS42 (5'-TTCCCAAAGAGTTTAAT-3') was specific for the normal allele of the gene (which codes for glutamic acid at position 501) and oligo DS43 (same as DS42 but with A instead of G at nucleotide 9) was specific for the Kashmir variant [2], assuming the minimum number of mutations. Both DS42 and DS43 were radiolabelled using [y-32P]ATP (3000 Ci/mmol) and T4 polynucleotide kinase (both from Amersham International, Amersham, Bucks., U.K.) to a specific radioactivity of about 3 x 108 d.p.m./,ug of oligo. Electrophoresis of DNA using 10 ,1 aliquots from each PCR was performed as described previously [11] on 1.4 0'; (w/v) agarose gels. Southern blotting on to Hybond-N membranes was carried out as recommended by the supplier of the membrane (Amersham International). Hybridizations were performed at 29 °C using 100 ng of radiolabelled oligo (DS42 or DS43) in 2 x SSC (SSC is 0.15 M-NaCl/0.01 5 M-trisodium citrate, pH 7.0) containing 0.5 0 (w/v) dried skimmed milk. The hybridization temperature was calculated according to Wallace & Miyada [12] so as to allow the formation of perfect hybrids only. Membranes were washed twice (10 min each) in 2 x SSC at room temperature, air-dried and exposed to Fuji-RX X-ray film. Following the development of the X-ray film, the membranes were washed for 10 min in 0.1 x SSC at 65 °C to remove the bound probe and then re-used in hybridizations with the alternative probe. RESULTS AND DISCUSSION Electrophoretic separation of the serum proteins (Fig. 1) shows two albumin bands in individual A but not in B, although both were members of the authentic albumin Kashmir family. The results also show two similar bands in patient C, whose daughters D and E, however, do not carry the trait. Huss et al. [2] located the change in the albumins presumed to be Kashmir at position 501, which changed from glutamic acid to lysine. From the sequence described
B
A ..:
....
...
D
C ::::.::
::
E
..:: :.
Fig. 1. Cellulose acetate electrophoresis of normal and alloalbuminaemic sera Serum samples from each individual were electrophoresed on cellulose acetate membranes as described in the Materials and methods section. Letters above each lane identify the individuals studied.
by Minghetti et al. [6], the codon for glutamic acid-501 in normal albumin is GAG. There are two possible codons for lysine (AAG and AAA), but assuming that the change from glutamic acid to lysine was due to only one mutational event, the change in the DNA would have been from GAG to AAG. In order to confirm this assumption in individual A, as well as to confirm the diagnosis for the bisalbuminaemic individual C, the PCR has been used to amplify a 206 bp fragment of human DNA located totally within exon 12 of the albumin gene. This amplified fragment was then used in hybridizations with oligos specific either for the normal allele or for that coding for the Kashmir variant. The PCR was performed using the crude DNA preparations obtained from the blood or blood clots of individuals A-E and the amplification products were analysed by electrophoresis on agarose gels. The results (not shown) indicated that in all cases the amplification product was a DNA fragment of about 200 bp, which is in close agreement with the expected size (206 bp). Although blood clots (from subjects A and B) resulted in less amplification than whole blood, the amount of DNA obtained was sufficient for subsequent hybridization. Southern hybridization of the PCR amplification products (Fig. 2) with the radiolabelled oligo corresponding to the normal allele of albumin (DS42) gave a positive result with all five individuals, with the strength of the hybridization signal reflecting the amount of PCR product present in each sample. Furthermore, the mutant allele for albumin Kashmir (DS43) hybridized to two PCR products only (those for individuals A and C). This suggests that the hybridization conditions employed were sufficiently stringent to prevent the formation of hybrids containing even a single mismatch. Computer analysis of the nucleotide sequence of the amplified DNA using the WORDSEARCH program in the sequence analysis package of the University of Wisconsin (available at the Daresbury Laboratory) showed (1) that the only perfect match between this and oligo DS42 was at the expected position (bases 13935-13951 [6]), (2) that DS43 showed 1990
Albumin Kashmir: diagnosis using DNA hybridization
(a)
C
D
E
A
B
(bp)
617 as suggested by his ethnic background and electrophoretic pattern. A strategy similar to that adopted here may also be applied to the molecular characterization of other alloalbumins. It will of course be necessary to alter the sequences of the oligos used depending on the particular albumin variant under investigation; information to allow the design of appropriate oligos is available, since the albumin gene has been characterized [6] and amino acid substitutions in a number of albumin mutants are known [14]. Of particular interest will be an examination of the DNA changes resulting in albumins Vancouver, Birmingham and Adana, which contain the same change at position 501 as that for albumin Kashmir. For example, it will be interesting to establish whether the codon change resulting in these variants was the same as that for albumin Kashmir (GAG to AAG).
Thanks are due to Miss Jennifer Morris for expert technical assistance during the course of this work.
REFERENCES
Fig. 2. Analysis of the PCR by Southern hybridization. The PCR amplification products were electrophoresed on agarose gels, blotted on to Hybond-N membranes and hybridized with the two allele-specific oligonucleotide probes for normal albumin (DS42; a) and for albumin Kashmir (DS43; b) as described in the Materials and methods section. Letters above each lane identify the individuals studied. The positions of the molecular mass markers (in bp) are indicated by arrows.
only one mismatch with this sequence at base 13943 [6], and (3) that there were no other sequences with any similarity to either oligo. Fig. 2 shows some additional bands in certain tracks (C, D and E with DS42 and C with DS43); these are due to the formation of primer artefacts and incomplete strands in PCRs of more than 20 cycles [13]. Huss et al. [2] assumed that their sera contained the same mutation as that in albumin Kashmir. Our results, obtained with DNA from the original family, substantiate this and are evidence for the assumption (made when designing oligo DS43) that the mutation resulting in the albumin Kashmir variant contained a change of codon 501 from GAG to AAG. They also confirm that -individual C was a carrier for the albumin Kashmir allele, Received 12 October 1989/29 November 1989; accepted 5 January 1990
Vol. 266
1. Tarnoky, A. L. & Dowding, B. (1969) Clin. Chim. Acta 26, 455-458 2. Huss, K., Madison, J., Ishioka, N., Takahashi, N., Arai, K. & Putnam, F. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6692-6696 3. Tarnoky, A. L. (1980) Adv. Clin. Chem. 21, 101-146 4. Lawn, R. M., Adelman, J., Bock, S. C., Franke, A. E., Houck, C. M., Najarian, R. C., Seeburg, P. H. & Wion, K. L. (1981) Nucleic Acids Res. 9, 6103-6114 5. Dugaiczyk, A., Law, S. W. & Dennison, 0. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 71-75 6. Minghetti, P. P., Ruffner, D. E., Kuang, W.-J., Dennison, 0. E., Hawkins, J. W., Beattie, W. G. & Dugaiczyk, A. (1986) J. Biol. Chem. 261, 6747-6757 7. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. & Arnheim, N. (1985) Science 230, 1350-1354 8. Kazazian, H. H. (1989) in Polymerase Chain Reaction (Erlich, H. A., Gibbs, R. & Kazazian, H. H., eds.), pp. 47-55, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 9. Kidd, V. J., Wallace, R. B., Itakura, K. & Woo, S. L. C. (1983) Nature (London) 304, 230-234 10. Saiki, R. K., Walsh, P. S., Levenson, C. H. & Erlich, H. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6230-6234 11. Savva, D. & Mandelstam, J. (1984) J. Gen. Microbiol. 130, 2137-2145 12. Wallace, R. B. & Miyada, C. G. (1987) Methods Enzymol. 152, 432-442 13. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science 239, 487-491 14. Takahashi, N., Takahashi, Y., Blumberg, B. S. & Putnam, F. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4413-4417
