Letters to the Editor
Am. J. Hum. Genet. 47:1023-1024, 1990
Rapid Detection of the Hemoglobin C Mutation by Allele-specific Polymerase Chain Reaction To the Editor: One of every 625 black couples in the United States is at risk for having a child with hemoglobin SC disease. Prenatal diagnosis for the sickle cell mutation is possible within a few hours (Chehab et al. 1987). The region around the sickle cell mutation is amplified by polymerase chain reaction (PCR) and is digested by a restriction endonuclease whose recognition site is abolished by the A-to-T mutation at codon 6 in the sickle gene, and the resulting abnormal fragment is detected
with ethidium bromide staining after electrophoresis. Detection of the G-to-A mutation at codon 6 in the hemoglobin C gene however is more difficult- since no known restriction-endonuclease site is abolished or created by the mutation-and usually requires allelespecific oligonucleotide hybridization (Studencki et al. 1985). Allele-specific amplification by PCR with primers, the 3' ends of which are placed at the mutation site, has been shown to be possible in some instances (Okayama et al. 1989; Wu et al. 1989), while in other instances the amplification efficiency is not impaired by terminal 3' nucleotide mismatches (Kwok et al. 1990). We describe here a rapid allele-specific PCR amplification technique that allows detection of the hemoglobin C mutation in an even shorter time span than the one required for detection of the hemoglobin S mutation. o 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4706-0022$02.00
For each DNA sample two PCR reactions were performed. In one of them the primers used were beta 5'-a (5'-GTACGGCTGTCATCACTTAGACCTCA-3') and beta A-b (5'-TAACGGCAGACTTCTCCTC-3'), corresponding to the wild-type allele, and in the other the primers used were beta 5'-a and beta C-b (5'-TAACGGCAGACTTCTCCTT-3'), corresponding to the hemoglobin C allele. In each case a 216-bp product is expected if amplification occurs. The wild-type betaglobin primer beta 5'-b (5'-AGGGGAAAGAAAACATCAA-3') was included in each reaction (660-bp product). PCR was performed (Saiki et al. 1988) with 100 ng of purified genomic DNA, 10 pM of each primer, 11.5 mM Mg Cl2, 200 gM dNTPs, and 5.0 units of Taq polymerase in each 100-gl reaction. After the initial denaturing step at 950C for 5 min, 35 cycles were performed, including a 30-s denaturing step at 941C, a 30-s annealing step at 600C, and a 120-s extension step at 650C. Aliquots (10 gl) of each reaction were electrophoresed for 30 min at 50 mA on an ethidium bromide-stained 2.0% standard agarose gel and were photographed on a UV transilluminator. Figure 1 shows that in four individuals who had previously been characterized by standard hemoglobin electrophoresis it is possible to detect specific alleles. The normal allele is only detected with beta 5'-a and beta A-b, while the hemoglobin C allele is only detected with beta 5'-a and beta C-b. The hemoglobin S allele, which has a one-nucleotide mismatch with both beta A-b and beta C-b, is not detected with either of those primers. The unusually high MgCl2 concentration used in our experiment was found to give the minimum amount 1023
Letters to the Editor
1024
660bp
SC
AC
AA
1
2
1
2
1
SM
Ss 2
1
2
*0i
216bp
NATHAN FISCHEL-GHODSIAN, PHYLLIS C. HIRSCH, AND MARLENE C. BOHLMAN Divisions of Pediatric Hematology/Oncology and Medical Genetics Cedars-Sinai Medical Center Los Angeles
References Ballabio A, Gibbs RA, Caskey CT (1990) PCR test for cystic fibrosis deletion. Nature 343:220 Chehab FF, Doherty M, Cai S, Kan YW, Cooper S, Rubin EM (1987) Detection of sickle cell anaemia and thalassaemias. Nature 329:293
Identification of hemoglobin C mutation by alleleFigure I specific PCR, for individuals with hemoglobin AA, AC, SC, and SS. Lanes 1, Primers specific for hemoglobin A (beta 5'-a and beta A-b). Lanes 2, Primers specific for hemoglobin C (beta 5'-a and beta C-b). In lanes 1 and lanes 2, control primer beta 5'-b is added. Lane SM, Size marker (Phi X cleaved with the restriction enzyme HaeIII).
of background bands. It is, however, not necessary for discrimination of the point mutations. The use of a coamplification control within the beta-globin gene can further be used to diagnose the hemoglobin S mutation in fetuses at risk for sickle cell disease. Thus the same amplification reaction product used to detect the presence of the hemoglobin C mutation can be digested with the restriction enzyme DdeI and can be analyzed for the specific 398-bp sickle cell mutation band. This process is used in addition to the fact that the 216-bp band is expected to be absent when primers beta S'-a, beta 5'-b, and beta A-b are used. The use of allele-specific PCR has recently been described for the 3-bp deletion at amino acid position 508 in the CF gene (Ballabio et al. 1990), for the sickle cell mutation (Wu et al. 1989; Chehab et al. 1990), and for several mutations causing alpha-1-antitrypsin deficiency (Newton et al. 1989; Okayama et al. 1989). However, not all point mutations will be detectable by this method (Kwok et al. 1990). Despite this limitation, allele-specific PCR might find general application in the diagnosis of many small mutations that so far have required allele-specific oligonucleotide hybridization or sequencing. In particular, coamplification with several different primers might become a rapid method for pinpointing mutant alleles in diseases in which limited numbers of point mutations are responsible for the majority of cases.
Chehab FF, Kan YW (1990) Detection of sickle cell anaemia mutation by colour DNA amplification. Lancet 335:15 Kwok S, Kellog DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 18:999 Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, et al (1989) Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids Res 17:2503 Okayama H, Curiel DT, Brantly ML, Holmes MD, Crystal RG (1989) Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification. J Lab Clin Med 114:105 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis HA, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487 Studencki AB, Conner BJ, Impraim CC, Teplitz RL, Wallace RB (1985) Descrimination among the human DA Ps, and D3C-globin genes using allele-specific oligonucleotide hybridization probes. Am J Hum Genet 37:42-51 Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989) Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA 86:2757
Am. J. Hum. Genet. 47:1024-1028, 1990
The Slash Sheet: A Simple Procedure for Risk Analysis in Cystic Fibrosis To the Editor:
When you have eliminated the impossible whatever remains, however improbable, must be the truth. [CONAN DOYLE, "The Sign of the Four"]
Am. J. Hum. Genet. 47:1023-1024, 1990
Rapid Detection of the Hemoglobin C Mutation by Allele-specific Polymerase Chain Reaction To the Editor: One of every 625 black couples in the United States is at risk for having a child with hemoglobin SC disease. Prenatal diagnosis for the sickle cell mutation is possible within a few hours (Chehab et al. 1987). The region around the sickle cell mutation is amplified by polymerase chain reaction (PCR) and is digested by a restriction endonuclease whose recognition site is abolished by the A-to-T mutation at codon 6 in the sickle gene, and the resulting abnormal fragment is detected
with ethidium bromide staining after electrophoresis. Detection of the G-to-A mutation at codon 6 in the hemoglobin C gene however is more difficult- since no known restriction-endonuclease site is abolished or created by the mutation-and usually requires allelespecific oligonucleotide hybridization (Studencki et al. 1985). Allele-specific amplification by PCR with primers, the 3' ends of which are placed at the mutation site, has been shown to be possible in some instances (Okayama et al. 1989; Wu et al. 1989), while in other instances the amplification efficiency is not impaired by terminal 3' nucleotide mismatches (Kwok et al. 1990). We describe here a rapid allele-specific PCR amplification technique that allows detection of the hemoglobin C mutation in an even shorter time span than the one required for detection of the hemoglobin S mutation. o 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4706-0022$02.00
For each DNA sample two PCR reactions were performed. In one of them the primers used were beta 5'-a (5'-GTACGGCTGTCATCACTTAGACCTCA-3') and beta A-b (5'-TAACGGCAGACTTCTCCTC-3'), corresponding to the wild-type allele, and in the other the primers used were beta 5'-a and beta C-b (5'-TAACGGCAGACTTCTCCTT-3'), corresponding to the hemoglobin C allele. In each case a 216-bp product is expected if amplification occurs. The wild-type betaglobin primer beta 5'-b (5'-AGGGGAAAGAAAACATCAA-3') was included in each reaction (660-bp product). PCR was performed (Saiki et al. 1988) with 100 ng of purified genomic DNA, 10 pM of each primer, 11.5 mM Mg Cl2, 200 gM dNTPs, and 5.0 units of Taq polymerase in each 100-gl reaction. After the initial denaturing step at 950C for 5 min, 35 cycles were performed, including a 30-s denaturing step at 941C, a 30-s annealing step at 600C, and a 120-s extension step at 650C. Aliquots (10 gl) of each reaction were electrophoresed for 30 min at 50 mA on an ethidium bromide-stained 2.0% standard agarose gel and were photographed on a UV transilluminator. Figure 1 shows that in four individuals who had previously been characterized by standard hemoglobin electrophoresis it is possible to detect specific alleles. The normal allele is only detected with beta 5'-a and beta A-b, while the hemoglobin C allele is only detected with beta 5'-a and beta C-b. The hemoglobin S allele, which has a one-nucleotide mismatch with both beta A-b and beta C-b, is not detected with either of those primers. The unusually high MgCl2 concentration used in our experiment was found to give the minimum amount 1023
Letters to the Editor
1024
660bp
SC
AC
AA
1
2
1
2
1
SM
Ss 2
1
2
*0i
216bp
NATHAN FISCHEL-GHODSIAN, PHYLLIS C. HIRSCH, AND MARLENE C. BOHLMAN Divisions of Pediatric Hematology/Oncology and Medical Genetics Cedars-Sinai Medical Center Los Angeles
References Ballabio A, Gibbs RA, Caskey CT (1990) PCR test for cystic fibrosis deletion. Nature 343:220 Chehab FF, Doherty M, Cai S, Kan YW, Cooper S, Rubin EM (1987) Detection of sickle cell anaemia and thalassaemias. Nature 329:293
Identification of hemoglobin C mutation by alleleFigure I specific PCR, for individuals with hemoglobin AA, AC, SC, and SS. Lanes 1, Primers specific for hemoglobin A (beta 5'-a and beta A-b). Lanes 2, Primers specific for hemoglobin C (beta 5'-a and beta C-b). In lanes 1 and lanes 2, control primer beta 5'-b is added. Lane SM, Size marker (Phi X cleaved with the restriction enzyme HaeIII).
of background bands. It is, however, not necessary for discrimination of the point mutations. The use of a coamplification control within the beta-globin gene can further be used to diagnose the hemoglobin S mutation in fetuses at risk for sickle cell disease. Thus the same amplification reaction product used to detect the presence of the hemoglobin C mutation can be digested with the restriction enzyme DdeI and can be analyzed for the specific 398-bp sickle cell mutation band. This process is used in addition to the fact that the 216-bp band is expected to be absent when primers beta S'-a, beta 5'-b, and beta A-b are used. The use of allele-specific PCR has recently been described for the 3-bp deletion at amino acid position 508 in the CF gene (Ballabio et al. 1990), for the sickle cell mutation (Wu et al. 1989; Chehab et al. 1990), and for several mutations causing alpha-1-antitrypsin deficiency (Newton et al. 1989; Okayama et al. 1989). However, not all point mutations will be detectable by this method (Kwok et al. 1990). Despite this limitation, allele-specific PCR might find general application in the diagnosis of many small mutations that so far have required allele-specific oligonucleotide hybridization or sequencing. In particular, coamplification with several different primers might become a rapid method for pinpointing mutant alleles in diseases in which limited numbers of point mutations are responsible for the majority of cases.
Chehab FF, Kan YW (1990) Detection of sickle cell anaemia mutation by colour DNA amplification. Lancet 335:15 Kwok S, Kellog DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 18:999 Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, et al (1989) Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids Res 17:2503 Okayama H, Curiel DT, Brantly ML, Holmes MD, Crystal RG (1989) Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification. J Lab Clin Med 114:105 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis HA, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487 Studencki AB, Conner BJ, Impraim CC, Teplitz RL, Wallace RB (1985) Descrimination among the human DA Ps, and D3C-globin genes using allele-specific oligonucleotide hybridization probes. Am J Hum Genet 37:42-51 Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989) Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA 86:2757
Am. J. Hum. Genet. 47:1024-1028, 1990
The Slash Sheet: A Simple Procedure for Risk Analysis in Cystic Fibrosis To the Editor:
When you have eliminated the impossible whatever remains, however improbable, must be the truth. [CONAN DOYLE, "The Sign of the Four"]
