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DNA typing of HLA-B27 by polymerase chain reaction
Molecular and Cellular Probes (1997) 11, 313–315
Short Communication
DNA typing of HLA-B27 by polymerase chain reaction G. Lucotte1∗ and A. Burckel2 1
Laboratory of Neurogenetics, Maison Blanche Hospital, 51092 Reims and 2 Burckel Laboratory, 27 rue Taine, Paris, France (Received 17 February 1997, Accepted 14 March 1997)
To find a specific method for HLA-B27 typing, we tested an HLA-B27-specific polymerase chain reaction. This method was used for screening 100 randomly selected blood donors, 10 of them being HLA-B27 positive. A flow cytometric method and this PCR method were compared. 1997 Academic Press Limited
KEYWORDS: HLA-B27, PCR method, flow cytometry comparison.
Conventional HLA-B27 typing is based on the detection of HLA antigens at the cell surface, usually by the classical method of complement-dependent cytotoxicity in Teraski plates. For routine purposes (mainly because of the association between HLAB27 and ankylosing spondylitis,1 and several other rheumatic diseases), screening for HLA-B27 is based on flow cytometry. A major disadvantage to serological identification is cross-reactivity of the antibodies used with different HLA antigens. To diminish the potential risk of false-positive results, alternative methods were developed, based on analysis of the genes coding for HLA-B antigens. Some genotyping techniques rely on the combination of specific amplification of the HLA-B gene by the polymerase chain reaction (PCR)2 and hybridization with sequencespecific oligonucleotide probes.3 Allele-specific PCR,4 a more direct method based on specific primer recognition of a sequence in the third exon unique to the HLA-B27 allele, has been described for subtyping the B27 alleles.5 The aim of the present study was to test the feasibility and reliability of the allelespecific PCR assay developed in a routine laboratory
setting, for typing HLA-B27 only. We also wanted to compare the suitability and cost-effectiveness of this PCR method in daily practice with features of the flow cytometric procedure.6 DNA was extracted quickly from 100 ll of each sample of venous blood with the Genefizz blood kit (Eurobio Laboratories, Les Ulis, France), according to the manufacturer’s instructions. This procedure is rapid and permits isolation in less than 15 min of a sufficient quantity of genomic DNA, free of inhibitors, for at least five PCR reactions. The primers used were some of those proposed,5 E91s: 5′-GGGTCTCACACCCTCCAGAAT-3′, and E136as: 5′-CGGCGGTCCAGGAGCT-3′. They amplify codons 91 to 136 of exon 3 from B27 alleles; primer E136as is specific for B alleles, and primer E91s (because of the AT at its 3′ end) is unique to B27 alleles. At least 100 ng of genomic DNA was mixed, in a final volume of 50 ll, with the PCR buffer containing: 50 m KCl, 10 m Tris–HCl pH 8.3, 1.5 m MgCl2, 100 l of each deoxynucleotide triphosphate, 50 p of each primer E91s and E136as, and 1.5 U of Taq polymerase (Amplitaq,
∗ Author to whom all correspondence should be addressed at: Laboratory of Neurogenetics, Neurology Service, Maison Blanche Hospital, 45 rue Cognacq-Jay, 51092 Reims Ce´dex, France.
0890–8508/97/040313+03 $25.00/0/ll970112
1997 Academic Press Limited
G. Lucotte and A. Burckel
B7 B8 B17 B18 B27 B35 B39 B40 B41 B44 B49 B51 B62
Numbers 24 18 3 2 10 11 1 12 5 13 4 3 18
(B57-B58)
(B16) (B60-B61) (B12)
(B15)
B27,B27
B7,B44
B27,B44
M
B18,B44
Perkin-Elmer). The PCR was performed on a 480 model thermocycler (Perkin-Elmer). After an initial denaturation at 94°C for 2 min, 30 cycles were carried out: denaturation at 94°C for 1 min, annealing at 56°C for 1 min (this annealing temperature of 56°C was modified5 to increase the specificity of the PCR reaction), and extension at 72°C for 2 min; the final extension occurred at 72°C for 10 min. A 10 ll aliquot of the amplified products was resolved by 2% agarose minigel electrophoresis at 150 V for 15 min. The gel was stained with ethidium bromide and visualized under 254 u.v. transillumination. Positive samples produced a DNA band of 135 bp. DNA was extracted from blood samples of 100 randomly selected healthy blood donors, fully typed for HLA-B by the microlymphocytoxicity technique (Table 1); 10 of them were HLA-B27 positive and the whole panel was used to confirm the amplification specificity of the PCR assay. The B27-specific PCR
B27,B40
HLA-B alleles
correctly identified all the 10 B27-positive samples of the group of 100, as evidenced by the presence of the 135 bp amplification product (Fig. 1). We have verified that the E91s and E236as primers amplify, as expected,5 all the B27 sub-types (B∗2701 to B∗2706). No false-positive or false-negative results were obtained. To verify amplification reactivity we added a second primer set 5′-GAAGAGCCAAGGACAGGTAC-3′ and 5′-GCTCACTCAGTGTGGCAAAG-3′, which amplifies a 440 bp fragment of the b-globin gene and did not interfere with the 135 bp product; all the samples showed a b-globin-positive amplificate with this set of genomic DNA control primers. To establish a practical and reliable DNA-based procedure to screen samples for HLA-B27, we have modified the specific exon 3 PCR method initially described5 in the following ways: (a) using a rapid method of obtaining DNA from blood samples; (b) using an annealing PCR temperature of 50°C to increase the specificity of the reaction; (c) using an internal control to test amplification reactivity; (d) analysing PCR products by agarose electrophoresis only, because the 135 bp product obtained (even without a further specific hybridization step) is a reliable tool for typing HLA-B27; and (e) using minigel for rapid detection of PCR products in numerous samples. This modified method, which relies on a single-step PCR targeting a sequence of the HLA-B gene unique to the B27 allele, proved to be a simple and reliable method for routine analysis. Another method of typing HLA-B27 by PCR was recently published.7 The sensitivity of our PCR assay was 100%; all 10 B27-positive samples tested were identified correctly. The specificity was tested by characterizing as B27negative the 90 other samples of our panel with
B39,B62
HLA-B classification of 100 samples
B27,B62
Table 1.
B8,B62
314
135
Fig. 1. HLA-B27 typing by allele-specific amplification on minigel with primers E91s and E136a. The expected 135 bp band was detectable only in the B27-positive samples. M=DNA marker.
DNA typing of HLA-B27
different non-B27 alleles, confirming the high specificity of the reaction. This sort of PCR assay offers some advantage over the flow cytometric technique: (a) it is possible to test by PCR about one hundred samples/day (depending on the capacity of the thermocycler used); (b) concerning the time for analysis, 20 samples can be processed conveniently within a maximal time of 4 h; (c) once obtained, genomic DNA can be conserved, in contrast with flow cytometry where samples have to be prepared as soon as they arrive; (d) the PCR procedure requires approximately US$2.5 per sample for material costs, which is comparable to the cost of flow cytometry, but PCR requires no expensive investment, except for the PCR thermocycler; (e) overall, unequivocal results were obtained with our PCR assay; in contrast, 2–5% of the flow cytometry results were, in a first step, uninterpretable because of crossreactivity of the monoclonal antibodies used. Nevertheless the risk of PCR-induced contamination,8 especially sample-to-sample contamination, requires drastic precautions.
315
REFERENCES 1. Schlosstein, L., Terasaki, P. J., Bluestone, R. & Pearson, C. (1973) High association of HLA antigen W27 with ankylosing spondylitis. New England Journal of Medicine 288, 704–6. 2. Saiki, R. K., Scharf, S, Faloona, F. et al. (1985). Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–56. 3. Yoshida, M., Kimura, A., Numano, F. & Susazuki,, T. (1992). Polymerase chain reaction based analysis of polymorphism in the HLA-B gene. Human Immunology 34, 257–66. 4. Wu, D. Y., Ugozzoli, L., Pal, B. K. & Wallace, R. B. (1989) Allele specific enzymatic amplification of bglobin genomic DNA diagnosis of sickle cell anemia. Proceedings of the National Academy of Science, USA 86, 2757–64. 5. Dominguez, O., Coto, E., Martinez-Naves, E., Choo, S. Y. & Lo´pez-Larrea, C. (1992). Molecular typing of HLA-B27 alleles. Immunogenetics 36, 277–82. 6. Albrecht, A. & Mu¨ller, H. A. G. (1987). HLA-B27 typing by use of flow cytofluorometry. Clinical Chemistry 33, 1619–23. 7. Olerup, O. (1994). HLA-B27 typing by a group specific PCR amplification. Tissue Antigens 43, 253–6. 8. Kwok, S. & Higuchi, R. (1989). Avoiding false positives with PCR. Nature 339, 237–8.
Short Communication
DNA typing of HLA-B27 by polymerase chain reaction G. Lucotte1∗ and A. Burckel2 1
Laboratory of Neurogenetics, Maison Blanche Hospital, 51092 Reims and 2 Burckel Laboratory, 27 rue Taine, Paris, France (Received 17 February 1997, Accepted 14 March 1997)
To find a specific method for HLA-B27 typing, we tested an HLA-B27-specific polymerase chain reaction. This method was used for screening 100 randomly selected blood donors, 10 of them being HLA-B27 positive. A flow cytometric method and this PCR method were compared. 1997 Academic Press Limited
KEYWORDS: HLA-B27, PCR method, flow cytometry comparison.
Conventional HLA-B27 typing is based on the detection of HLA antigens at the cell surface, usually by the classical method of complement-dependent cytotoxicity in Teraski plates. For routine purposes (mainly because of the association between HLAB27 and ankylosing spondylitis,1 and several other rheumatic diseases), screening for HLA-B27 is based on flow cytometry. A major disadvantage to serological identification is cross-reactivity of the antibodies used with different HLA antigens. To diminish the potential risk of false-positive results, alternative methods were developed, based on analysis of the genes coding for HLA-B antigens. Some genotyping techniques rely on the combination of specific amplification of the HLA-B gene by the polymerase chain reaction (PCR)2 and hybridization with sequencespecific oligonucleotide probes.3 Allele-specific PCR,4 a more direct method based on specific primer recognition of a sequence in the third exon unique to the HLA-B27 allele, has been described for subtyping the B27 alleles.5 The aim of the present study was to test the feasibility and reliability of the allelespecific PCR assay developed in a routine laboratory
setting, for typing HLA-B27 only. We also wanted to compare the suitability and cost-effectiveness of this PCR method in daily practice with features of the flow cytometric procedure.6 DNA was extracted quickly from 100 ll of each sample of venous blood with the Genefizz blood kit (Eurobio Laboratories, Les Ulis, France), according to the manufacturer’s instructions. This procedure is rapid and permits isolation in less than 15 min of a sufficient quantity of genomic DNA, free of inhibitors, for at least five PCR reactions. The primers used were some of those proposed,5 E91s: 5′-GGGTCTCACACCCTCCAGAAT-3′, and E136as: 5′-CGGCGGTCCAGGAGCT-3′. They amplify codons 91 to 136 of exon 3 from B27 alleles; primer E136as is specific for B alleles, and primer E91s (because of the AT at its 3′ end) is unique to B27 alleles. At least 100 ng of genomic DNA was mixed, in a final volume of 50 ll, with the PCR buffer containing: 50 m KCl, 10 m Tris–HCl pH 8.3, 1.5 m MgCl2, 100 l of each deoxynucleotide triphosphate, 50 p of each primer E91s and E136as, and 1.5 U of Taq polymerase (Amplitaq,
∗ Author to whom all correspondence should be addressed at: Laboratory of Neurogenetics, Neurology Service, Maison Blanche Hospital, 45 rue Cognacq-Jay, 51092 Reims Ce´dex, France.
0890–8508/97/040313+03 $25.00/0/ll970112
1997 Academic Press Limited
G. Lucotte and A. Burckel
B7 B8 B17 B18 B27 B35 B39 B40 B41 B44 B49 B51 B62
Numbers 24 18 3 2 10 11 1 12 5 13 4 3 18
(B57-B58)
(B16) (B60-B61) (B12)
(B15)
B27,B27
B7,B44
B27,B44
M
B18,B44
Perkin-Elmer). The PCR was performed on a 480 model thermocycler (Perkin-Elmer). After an initial denaturation at 94°C for 2 min, 30 cycles were carried out: denaturation at 94°C for 1 min, annealing at 56°C for 1 min (this annealing temperature of 56°C was modified5 to increase the specificity of the PCR reaction), and extension at 72°C for 2 min; the final extension occurred at 72°C for 10 min. A 10 ll aliquot of the amplified products was resolved by 2% agarose minigel electrophoresis at 150 V for 15 min. The gel was stained with ethidium bromide and visualized under 254 u.v. transillumination. Positive samples produced a DNA band of 135 bp. DNA was extracted from blood samples of 100 randomly selected healthy blood donors, fully typed for HLA-B by the microlymphocytoxicity technique (Table 1); 10 of them were HLA-B27 positive and the whole panel was used to confirm the amplification specificity of the PCR assay. The B27-specific PCR
B27,B40
HLA-B alleles
correctly identified all the 10 B27-positive samples of the group of 100, as evidenced by the presence of the 135 bp amplification product (Fig. 1). We have verified that the E91s and E236as primers amplify, as expected,5 all the B27 sub-types (B∗2701 to B∗2706). No false-positive or false-negative results were obtained. To verify amplification reactivity we added a second primer set 5′-GAAGAGCCAAGGACAGGTAC-3′ and 5′-GCTCACTCAGTGTGGCAAAG-3′, which amplifies a 440 bp fragment of the b-globin gene and did not interfere with the 135 bp product; all the samples showed a b-globin-positive amplificate with this set of genomic DNA control primers. To establish a practical and reliable DNA-based procedure to screen samples for HLA-B27, we have modified the specific exon 3 PCR method initially described5 in the following ways: (a) using a rapid method of obtaining DNA from blood samples; (b) using an annealing PCR temperature of 50°C to increase the specificity of the reaction; (c) using an internal control to test amplification reactivity; (d) analysing PCR products by agarose electrophoresis only, because the 135 bp product obtained (even without a further specific hybridization step) is a reliable tool for typing HLA-B27; and (e) using minigel for rapid detection of PCR products in numerous samples. This modified method, which relies on a single-step PCR targeting a sequence of the HLA-B gene unique to the B27 allele, proved to be a simple and reliable method for routine analysis. Another method of typing HLA-B27 by PCR was recently published.7 The sensitivity of our PCR assay was 100%; all 10 B27-positive samples tested were identified correctly. The specificity was tested by characterizing as B27negative the 90 other samples of our panel with
B39,B62
HLA-B classification of 100 samples
B27,B62
Table 1.
B8,B62
314
135
Fig. 1. HLA-B27 typing by allele-specific amplification on minigel with primers E91s and E136a. The expected 135 bp band was detectable only in the B27-positive samples. M=DNA marker.
DNA typing of HLA-B27
different non-B27 alleles, confirming the high specificity of the reaction. This sort of PCR assay offers some advantage over the flow cytometric technique: (a) it is possible to test by PCR about one hundred samples/day (depending on the capacity of the thermocycler used); (b) concerning the time for analysis, 20 samples can be processed conveniently within a maximal time of 4 h; (c) once obtained, genomic DNA can be conserved, in contrast with flow cytometry where samples have to be prepared as soon as they arrive; (d) the PCR procedure requires approximately US$2.5 per sample for material costs, which is comparable to the cost of flow cytometry, but PCR requires no expensive investment, except for the PCR thermocycler; (e) overall, unequivocal results were obtained with our PCR assay; in contrast, 2–5% of the flow cytometry results were, in a first step, uninterpretable because of crossreactivity of the monoclonal antibodies used. Nevertheless the risk of PCR-induced contamination,8 especially sample-to-sample contamination, requires drastic precautions.
315
REFERENCES 1. Schlosstein, L., Terasaki, P. J., Bluestone, R. & Pearson, C. (1973) High association of HLA antigen W27 with ankylosing spondylitis. New England Journal of Medicine 288, 704–6. 2. Saiki, R. K., Scharf, S, Faloona, F. et al. (1985). Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–56. 3. Yoshida, M., Kimura, A., Numano, F. & Susazuki,, T. (1992). Polymerase chain reaction based analysis of polymorphism in the HLA-B gene. Human Immunology 34, 257–66. 4. Wu, D. Y., Ugozzoli, L., Pal, B. K. & Wallace, R. B. (1989) Allele specific enzymatic amplification of bglobin genomic DNA diagnosis of sickle cell anemia. Proceedings of the National Academy of Science, USA 86, 2757–64. 5. Dominguez, O., Coto, E., Martinez-Naves, E., Choo, S. Y. & Lo´pez-Larrea, C. (1992). Molecular typing of HLA-B27 alleles. Immunogenetics 36, 277–82. 6. Albrecht, A. & Mu¨ller, H. A. G. (1987). HLA-B27 typing by use of flow cytofluorometry. Clinical Chemistry 33, 1619–23. 7. Olerup, O. (1994). HLA-B27 typing by a group specific PCR amplification. Tissue Antigens 43, 253–6. 8. Kwok, S. & Higuchi, R. (1989). Avoiding false positives with PCR. Nature 339, 237–8.