Letters to the Editor
~M
Am. J. Hum. Genet. 47:169, 1990
Am. J. Hum. Genet. 47:169-170, 1990
Polymerase Chain Reaction-generated Heteroduplexes from Ashkenazi Tay-Sachs Carriers with an Insertion Mutation Can Be Detected on Agarose Gels To the Editor: The Ashkenazi Jewish population is an ethnic isolate which carries the gene for a severe form of Tay-Sachs disease at a reasonably high frequency (one in 30). We found that 70% of the Ashkenazi Jewish carriers bear a 4-bp insertion in the a-chain gene encoding a subunit of the lysosomal enzyme 0-hexosaminidase A (Myerowitz and Costigan 1988). In a recent letter, "Diagnostic Heteroduplexes: Simple Detection of Carriers of a 4-bp Insertion Mutation in Tay-Sachs Disease;' Triggs-Raine and Gravel (1990) report that, as a consequence of polymerase chain reaction amplification of a short segment of the a-chain gene surrounding the
Heteroduplex Formation in Polymerase Chain Reaction To the Editor:
Recently it has been pointed out that the identification of heteroduplexes in polymerase chain reaction (PCR) can provide a convenient test for identifying heterozygous individuals (Rommens et al. 1990; Triggs-Raine and Gravel 1990). The generation of heteroduplex will theoretically occur in any PCR reaction in which two homologous DNA segments that differ for a point mutation or insertion/deletion are amplified. Heteroduplexes form during the later cycles of PCR probably because the DNA concentration increases so much that the complementary strands reanneal and outcompete
4-bp insertion, heteroduplexes that form between the normal and mutant alleles are diagnostic for the carrier state. The authors caution, however, that these heteroduplex molecules are visible only on polyacrylamide gels and not on agarose gels. Contrary to the TriggsRaine and Gravel report, we have found that 4% NuSieve agarose minigels effectively separate the heteroduplex products from the normal and mutant homoduplex products (Shore and Myerowitz, in press), thereby facilitating the assay considerably. SHIRAH SHORE AND RACHEL MYEROWITZ Laboratory of Biochemistry and Metabolism National Institutes of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, MD References Myerowitz R, Costigan FC (1988) The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the a-chain of 0-hexosaminidase. J Biol Chem 263:18587-18589 Shore S, Myerowitz R. A gel electrophoretic assay for detecting the insertion defect in Ashkenazi Jewish carriers of TaySachs disease. Anal Biochem (in press) Triggs-Raine BL, Gravel RA (1990) Diagnostic heteroduplexes: simple detection of carriers of a 4-bp insertion mutation in Tay-Sachs disease. Am J Hum Genet 46:183-184
169
C-
bp
5O 47 4123 1 Figure I
2
3
4
Amplification and analysis of CF deletion. The PCR protocol has been described elsewhere (Mattew et al. 1989). The amplified DNA of the three members of a cystic fibrosis family was electrophoresed through vertical 12% polyacrylamide gel for 2 h at 90 mA and stained in 5% ethidium bromide. Lane 1-3, Family with the affected child compound heterozygote for AF508 mutation. Lane 4, pBR322 DNA-HaeIII.
Letters to the Editor
170
bp
/| C 50 47
M C
/I|\ 50
l
47 50
A,B-_
2
3
4
5
6
7
8
Figure 2
Gel electrophoresis on denaturing polyacrylamide gel and silver staining of isolated DNA segments from the PCR products. The separated DNA segments of each family member were isolated from the gel and denatured for 10 min at 900C in denaturing buffer (90% formamide, 1 x TBE, 0.02% bromophenol blue), quick chilled on ice, and loaded on denaturing gel (12% polyacrylamide, 8 M urea, 1 x TBE). Vertical electrophoresis was carried out for 2 h at 130 mA through a 0.75-mm-thick minigel, followed by silver staining (Merril et al. 1981). Lanes 2, 6, and 8, 50-bp homoduplex N/N. Lanes 1 and 5, Heteroduplex N/AE Lanes 3 and 7, 47-bp homoduplex AF/AF.
the hybridization of the oligonucleotides with their template strand. The phenomenon of heteroduplex generation from amplified products of homologous loci was identified by Nagamine et al. (1989) during the studies of the candidate sequence for the mouse testis-determining Y gene (mYfin). Furthermore, denaturing gradient-gel-electrophoresis studies have long exploited heteroduplexes to identify single-base mutation (Sheffield et al. 1989). We report the formation of heteroduplex during the PCR amplification of the coding region surrounding the AF508 mutation in cystic fibrosis (Kerem et al. 1989). We have amplified 50 bp of the DNA, in the region of 3-bp deletion, of affected and carrier individuals, and we have analyzed the products on polyacrylamide gel (Mathew et al. 1989). As shown in figure
1, the vertical PAGE has resolved three bands in the heterozygotes for the AF508 mutation: a 47-bp band, a 50-bp band, and an additional band (C) that migrates in the gel a little more slowly than do the other bands. The three fragments have been isolated from the gel and analyzed by denaturing gel electrophoresis followed by silver staining to improve detection of the small singlestranded DNA fragments (Merril et al. 1981). The result obtained (fig. 2) confirmed that the extra band was a hybrid DNA molecule formed by a normal strand (A) and a 3-bp deleted strand (B). The spurious migration of the N/AF heteroduplex in polyacrylamide gel may be due to the secondary DNA structure that is generated by the annealing of the two strands. FRANCA ANGLANI, LUIGI PICCI, CRISTINA CAMPORESE, AND FRANCO ZACCHELLO Department of Pediatrics, University of Padua, Padua References Kerem B-S, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, et al. (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:1073-1080 Mathew CG, Roberts RG, Harris A, Bentley DR, Bobrow M (1989) Rapid screening for AF508 deletion in cystic fibrosis. Lancet 2:1346 Merril CR, Goldman D, Sedman SA, Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437-1438 Nagamine CM, Chan K, Lau Y-FC (1989) A PCR artifact: generation of heteroduplexes. Am J Hum Genet 45: 337-339 Rommens J, Kerem B-S, Greer W, Chang P, Tsui L-C, Ray P (1990) Rapid nonradioactive detection of the major cystic fibrosis mutation. Am J Hum Genet 46:395-396 Sheffield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 86:232-236 Triggs-Raine BL, Gravel RA (1990) Diagnostic heteroduplexes: simple detection of carriers of a 4-bp insertion mutation in Tay-Sachs disease. Am J Hum Genet 46:183-184
~M
Am. J. Hum. Genet. 47:169, 1990
Am. J. Hum. Genet. 47:169-170, 1990
Polymerase Chain Reaction-generated Heteroduplexes from Ashkenazi Tay-Sachs Carriers with an Insertion Mutation Can Be Detected on Agarose Gels To the Editor: The Ashkenazi Jewish population is an ethnic isolate which carries the gene for a severe form of Tay-Sachs disease at a reasonably high frequency (one in 30). We found that 70% of the Ashkenazi Jewish carriers bear a 4-bp insertion in the a-chain gene encoding a subunit of the lysosomal enzyme 0-hexosaminidase A (Myerowitz and Costigan 1988). In a recent letter, "Diagnostic Heteroduplexes: Simple Detection of Carriers of a 4-bp Insertion Mutation in Tay-Sachs Disease;' Triggs-Raine and Gravel (1990) report that, as a consequence of polymerase chain reaction amplification of a short segment of the a-chain gene surrounding the
Heteroduplex Formation in Polymerase Chain Reaction To the Editor:
Recently it has been pointed out that the identification of heteroduplexes in polymerase chain reaction (PCR) can provide a convenient test for identifying heterozygous individuals (Rommens et al. 1990; Triggs-Raine and Gravel 1990). The generation of heteroduplex will theoretically occur in any PCR reaction in which two homologous DNA segments that differ for a point mutation or insertion/deletion are amplified. Heteroduplexes form during the later cycles of PCR probably because the DNA concentration increases so much that the complementary strands reanneal and outcompete
4-bp insertion, heteroduplexes that form between the normal and mutant alleles are diagnostic for the carrier state. The authors caution, however, that these heteroduplex molecules are visible only on polyacrylamide gels and not on agarose gels. Contrary to the TriggsRaine and Gravel report, we have found that 4% NuSieve agarose minigels effectively separate the heteroduplex products from the normal and mutant homoduplex products (Shore and Myerowitz, in press), thereby facilitating the assay considerably. SHIRAH SHORE AND RACHEL MYEROWITZ Laboratory of Biochemistry and Metabolism National Institutes of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, MD References Myerowitz R, Costigan FC (1988) The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the a-chain of 0-hexosaminidase. J Biol Chem 263:18587-18589 Shore S, Myerowitz R. A gel electrophoretic assay for detecting the insertion defect in Ashkenazi Jewish carriers of TaySachs disease. Anal Biochem (in press) Triggs-Raine BL, Gravel RA (1990) Diagnostic heteroduplexes: simple detection of carriers of a 4-bp insertion mutation in Tay-Sachs disease. Am J Hum Genet 46:183-184
169
C-
bp
5O 47 4123 1 Figure I
2
3
4
Amplification and analysis of CF deletion. The PCR protocol has been described elsewhere (Mattew et al. 1989). The amplified DNA of the three members of a cystic fibrosis family was electrophoresed through vertical 12% polyacrylamide gel for 2 h at 90 mA and stained in 5% ethidium bromide. Lane 1-3, Family with the affected child compound heterozygote for AF508 mutation. Lane 4, pBR322 DNA-HaeIII.
Letters to the Editor
170
bp
/| C 50 47
M C
/I|\ 50
l
47 50
A,B-_
2
3
4
5
6
7
8
Figure 2
Gel electrophoresis on denaturing polyacrylamide gel and silver staining of isolated DNA segments from the PCR products. The separated DNA segments of each family member were isolated from the gel and denatured for 10 min at 900C in denaturing buffer (90% formamide, 1 x TBE, 0.02% bromophenol blue), quick chilled on ice, and loaded on denaturing gel (12% polyacrylamide, 8 M urea, 1 x TBE). Vertical electrophoresis was carried out for 2 h at 130 mA through a 0.75-mm-thick minigel, followed by silver staining (Merril et al. 1981). Lanes 2, 6, and 8, 50-bp homoduplex N/N. Lanes 1 and 5, Heteroduplex N/AE Lanes 3 and 7, 47-bp homoduplex AF/AF.
the hybridization of the oligonucleotides with their template strand. The phenomenon of heteroduplex generation from amplified products of homologous loci was identified by Nagamine et al. (1989) during the studies of the candidate sequence for the mouse testis-determining Y gene (mYfin). Furthermore, denaturing gradient-gel-electrophoresis studies have long exploited heteroduplexes to identify single-base mutation (Sheffield et al. 1989). We report the formation of heteroduplex during the PCR amplification of the coding region surrounding the AF508 mutation in cystic fibrosis (Kerem et al. 1989). We have amplified 50 bp of the DNA, in the region of 3-bp deletion, of affected and carrier individuals, and we have analyzed the products on polyacrylamide gel (Mathew et al. 1989). As shown in figure
1, the vertical PAGE has resolved three bands in the heterozygotes for the AF508 mutation: a 47-bp band, a 50-bp band, and an additional band (C) that migrates in the gel a little more slowly than do the other bands. The three fragments have been isolated from the gel and analyzed by denaturing gel electrophoresis followed by silver staining to improve detection of the small singlestranded DNA fragments (Merril et al. 1981). The result obtained (fig. 2) confirmed that the extra band was a hybrid DNA molecule formed by a normal strand (A) and a 3-bp deleted strand (B). The spurious migration of the N/AF heteroduplex in polyacrylamide gel may be due to the secondary DNA structure that is generated by the annealing of the two strands. FRANCA ANGLANI, LUIGI PICCI, CRISTINA CAMPORESE, AND FRANCO ZACCHELLO Department of Pediatrics, University of Padua, Padua References Kerem B-S, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, et al. (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:1073-1080 Mathew CG, Roberts RG, Harris A, Bentley DR, Bobrow M (1989) Rapid screening for AF508 deletion in cystic fibrosis. Lancet 2:1346 Merril CR, Goldman D, Sedman SA, Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437-1438 Nagamine CM, Chan K, Lau Y-FC (1989) A PCR artifact: generation of heteroduplexes. Am J Hum Genet 45: 337-339 Rommens J, Kerem B-S, Greer W, Chang P, Tsui L-C, Ray P (1990) Rapid nonradioactive detection of the major cystic fibrosis mutation. Am J Hum Genet 46:395-396 Sheffield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 86:232-236 Triggs-Raine BL, Gravel RA (1990) Diagnostic heteroduplexes: simple detection of carriers of a 4-bp insertion mutation in Tay-Sachs disease. Am J Hum Genet 46:183-184
