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SAME-DAY, FIRST-TRIMESTER ANTENATAL DIAGNOSIS FOR CYSTIC FIBROSIS BY GENE AMPLIFICATION
102 PRENATAL DIAGNOSIS OF CYSTIC FIBROSIS BY DNA AMPLIFICATION FOR DETECTION OF KM-19 POLYMORPHISM
SIR,-Prenatal diagnosis for cystic fibrosis (CF) is now done routinely by use of tightly linked restriction fragment length polymorphisms (RFLP).1.2 RFLPs in strong linkage disequilibrium with the CF mutation have lately been identified.3’ The KM-19 probe detects an RFLP with PstI, and this polymorphism is among the most useful for prenatal diagnosis of CF. Amplification of DNA by the polymerase chain reaction (PCR) allows rapid analysis without the use of radioactivity. 5,6 We report the characterisation of DNA sequences surrounding the KM-19 polymorphism and describe the use of PCR to analyse this RFLP for prenatal diagnosis of CF. Cosmid CNX.43 was subcloned, and restriction fragments were identified which flank the polymorphic Pstl site. DNA sequencing was done to prepare oligonucleotides which flank the polymorphic site. By use of two oligonucleotides, it was possible to do a diagnostic analysis with a single amplification process followed by restriction enzyme digestion and agarose gel eiectrophoresis. The figure demonstrates the polymorphism in two heterozygous parents with an affected CF child who is homozygous for the presence of PstI restriction site, while the fetus is homozygous for absence of the restriction site. The product of the amplification reaction is approximately 0-95 kb, and digestion with Pstl results in fragments of 0-65 and 0-30 kb. Homozygotes for the absence of the restriction site demonstrate the 0-95 kb fragment, homozygotes for the presence of the site demonstrate the 0-65 and 0-30 kb fragments, and heterozygotes demonstrate all three fragments. Both manual and automated procedures were used for amplification. Several fetuses were studied by chorionic villus sampling and amniocentesis, and
the results with PCR were unambiguous and in agreement with Southern blotting results in all cases. Because of the linkage disequilibrium with the KM-19 polymorphism,4.7 this RFLP is diagnostic in a large proportion of families at risk for CF. Of 85 couples with a 1-in-4 risk for CF studied in Houston, 48% were fully informative with this polymorphism, 41% were informative for one parent, and 11% were uninformative. Adequate prenatal diagnosis will be provided by PCR in 48 % of families which are fully informative and in about half of the fetuses where the informative parent is predicted to have transmitted the normal gene to the fetus. This means that the use of PCR with the KM-19 polymorphism would give an adequate prenatal diagnosis in about 70% of families at risk for CF. PCR is also available for the neighbouring linked polymorphic marker CS.7. Although very rare, crossovers between the KM-19 probe and the CF locus are known,’ and development of PCR for additional polymorphisms flanking the CF locus is desirable until such time as the gene is cloned. Institute for Molecular Genetics, Baylor College of Medicine, Houston
Regional DNA Laboratory, Department of Molecular Genetics, St Mary’s Hospital Medical School,
GERALD L. FELDMAN
North West Thames
London W2
Hughes Medical Institute Institute for Molecular Genetics,
ROBERT WILLIAMSON
Howard
Baylor College of Medicine, Houston, Texas 77030, USA 1 Beaudet
ARTHUR L. BEAUDET WILLIAM E. O’BRIEN
AL, Buffone GJ Prenatal diagnosis of cystic fibrosis. J Pediatr 1987, 111:
630-33.
M, Roedek C, Stanier P, et al First-trimester prenatal diagnosis of cystic fibrosis with linked DNA probes. Lancet 1986, ii: 1402-04 Estivill X, Farrall M, Scambler PJ, et al A candidate for the cystic fibrosis locus isolated by selection for methylation-free islands. Nature 1987, 326: 840-45 Estivill X, Scambler PJ, Wainwright BJ, et al. Patterns of polymorphism and linkage disequilibrium for cystic fibrosis Genomics 1987; 1: 257-63 Mullis KB, Faloona FA Specific synthesis of DNA in vitro visa a polymerase-catalyzed chain reaction Methods Enzymol 1987, 155: 335-50. Saiki RK, Gelfand DH, Stoffels, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 1988, 239: 487-91 Beaudet AL, Spence JE, Montes M, et al Experience with new DNA markers for the diagnosis of cystic fibrosis N Engl J Med 1988 318: 50-51 Farrall M, Stanier P, Beaudet A, et al. Recombination between IRP and cystic fibrosis Am J Hum Genet (in press) Kogan SC, Doherty M, Gitschier J An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences N Engl J Med 1987, 318: 985-90
2 Farrall
3 4 5
6 7
8 9
SAME-DAY, FIRST-TRIMESTER ANTENATAL DIAGNOSIS FOR CYSTIC FIBROSIS BY GENE AMPLIFICATION
Use of PCR for analysis of KM-19 diagnosis of CF.
Genomic DNA fluid cells. PCR
was was
polymorphism
and
prenatal
isolated from peripheral blood or cultured amnioticdone as described, with minor modifications.9
Oligonucleotide sequences were 5’-GCTGCATCATATAAGTTGCC-3’ 75 5’-AAGGCTACACTGTTAATTTT-3’. pmol of each oligonucleotide was used with 1 ).1g genomic DNA and 2 5 units of Taq DNA polymerase in total volume of 50 1. Second addition of 2 5 units of Taq polymerase was made after 15 cycles. Annealing conditions were 2 min at 55 °C, extension was 5 mm at 72’C, and denaturation was 1 min at 94°C for a total of 30 cycles. Samples of products of the reaction were digested with PstI and run on 1 5% agarose gel. Fragments were visualised with ethidium bromide and ultraviolet light. Approximate fragment sizes are indicated in kb. Extraneous fragment (c) of varying intensity sometimes occurs during amplification process, but this fragment does not interfere with the analysis. and
SiR,—DNA diagnosis is now available for about 170 inherited diseases,’ including the haemoglobinopathies, Duchenne muscular dystrophy, cystic fibrosis (CF), phenylketonuria, and the haemophilias. It is usual nowadays to attempt antenatal diagnoses on DNA from first-trimester trophob1ast,2 which has the advantage that, if a termination is requested, it can be done before fetal quickening. However, Southern blot analysis of trophoblast DNA can take up to 10 days. The polymerase chain reaction (PCR) technique permits simplified and rapid carrier testing by probes closely linked to CFand Dr Feldman and colleagues (see accompanying letter) have shown that it is applicable to about 70% of at-risk couples. Using PCR for gene amplification 3we have done a same-day antenatal diagnosis for CF. A couple who had a child with CF sought antenatal diagnosis, and trophoblast was obtained at 9 weeks’ amenorrhoea. Half of a single villus was removed from the specimen, the rest being used for Southern blot analysis. The small amount of villus was amplified 1Il parallel with DNA from the parents and the affected sibling, and haplotyped for CF with oligonucleotide primers specific for a
identifying the CS.7 locus.6 amplified products were analysed by digestion with HhaI, recognises a DNA polymorphism where one allele containing the cleavage site is common to most CF chromosomes.
sequence
PCR which
Each parent has one chromosome with the restriction site and one without, while the CF child has two chromosomes both of which
103
Fig 1-Hhal restricted and non-restricted gene amplification products for analysis of the CS.7 polymorphism and antenatal diagnosis of CF. Upper: genomic DNA for parents and child with CF was isolated from blood and buccal epithelial cells obtained by mouthwash. CVS and the buccal cell material was extracted," cells were suspended in 0-5 ml 0-0 mol/1 NaOH, and boiled for 5-10 min. Alkali suspension was neutralised with 3 mol/I sodium acetate and centrifuged to pellet denatured protein and cell debris. 5-10 pl supernatant used for each amplification. Primer oligonucleotide sequences were: 5’-CCCAGCTTCAGGGAGAGAAGCGAAGCAATG-3’ and 5’-AAACGCGGGGTTTTAGACACGGGTGCATGA-3’. Reaction was done in 1 x Taq DNA polymerase buffer (50 mmol/1 KC1, 10 mmol/1 "Tris"-HCI pH 8 3, 1-5 mmot/1 MgCl2, 0-01% gelatin), 20 pmol dNTPs, 50 pmol of each
oligonucleotide primer, and 0-4 ug
or less of genomic DNA, made up to 100 ul with water. Before first addition of enzyme the reaction mix was boiled for 6 min. 1-5-20 units of TaqI DNA polymerase (Cetus) was added per reaction. Amplification was for thirty cycles, each consisting of a denaturation step at 93-94’C for 90 s, followed by annealing and elongation combined at 68’C for 2 min without further addition of enzyme. Amplification product is 330 bp. Restriction with either BssHII or HhaI gives fragments of 164 and 166 bp which superimpose and are seen as a single band. DNA from persons with the Al/A2 haplotype shows the 330,164, and 166 bp fragments. DNA from homozygotes for Al have only the 330 bp doublet band, while homozygotes for A2 have doublet bands at 166 and 164 bp due to restriction of both alleles. All analyses of amplified products were done using 2% agarose gels stained with ethidium bromide and visualised with ultraviolet light. Lanes 2, 4, 6, 8, and 10 are unrestricted amplified products from father, mother, affected child, chorionic villus sample (CVS), and affected child buccal cell DNA, respectively. Lanes 3, 5, 7, and I are the HhaI restriction digest products from the same samples, respectively. Lane 1 contains a 1 kb ladder of DNA size markers.
have the site (fig 1). The fetus is heterozygous for the polymorphic site, and therefore is highly likely to be unaffected, although a carrier. The result was confirmed by Southern blotting (fig 2). The pregnancy is continuing. Buccal epithelial cells from the parents and affected child were also collected, and amplified with the same primers.’ Restriction enzyme digestion of amplified buccal cell preparations gave identical results to those obtained with whole blood (fig 1, lanes 10 and 11). Blood or mouthwash samples can be collected either before or simultaneously with the chorionic villus biopsy. While only one antenatal diagnosis has been done by same-day technology so far, many control chorion samples have been amplified in this way. The use of these primers and those reported by Feldman et al for the adjacent DNA probe KM-19, together with buccal cell analysis, permits combined simple and rapid carrier testing and antenatal diagnosis. The reduction of time for analysis to a single day would permit counselling on the evening of the day the biopsy is done. Termination could then be arranged for the following morning, though couples will often prefer to take their time,’ and return several days later when they have decided what do do. Not all families at high risk are informative; only about 50% of CF high-risk families are fully informative with CS.7 alone, but probes such as KM-19 increase informativeness. The technology has been automated (by Perkin Elmer/Cetus and Cambio) but the temperature cycling can also be done manually with two water baths (as for this diagnosis) or with a computer controlled robot arm. As informative probes are developed for many severe inherited diseases same-day diagnosis with PCR may become the aim of antenatal diagnostic services. We thank Nick Lench for advice on PCR methodology, and Kyproula Christodoulou for practical help. This research was supported by the Medical Research Council, the Wellcome Trust, and the Cystic Fibrosis Research Trust. North West Thames Regional DNA Department of Molecular Genetics, St Mary’s Hospital Medical School, London W2 1PG
Laboratory,
CAROLYN WILLIAMS ROBERT WILLIAMSON
Department of Human Molecular Genetics, Central Institute of Molecular Biology, Academy of Sciences, Berlin, East Germany
CHARLES COUTELLE
Department of Obstetrics and Gynaecology, St Mary’s Hospital, London W2
FRANK LOEFFLER JOHN SMITH
DNA Laboratory, Department of Medical Genetics, St Mary’s Hospital, Manchester
ADRIAN IVINSON
1
Cooper DN, Schmitke J. Diagnosis of genetic disease using recombinant DNA. Hum
Genet 1987; 77: 66-75 RE, Eskdale J, Coleman DV, Naizi M, Loeffler FE, Modell BM. Direct gene analysis of chorionic villus a possible technique for first trimester antenatal diagnosis of haemoglobinopathies. Lancet 1981; ii: 1125-27. 3. Lench N, Stanier P, Williamson R. A simple non-invasive method to obtain DNA for gene analysis. Lancet 1988; i: 1356-58. 4. Saiki R, Scharf S, Faloona F, Mullis B, Horn G, Erlich H, Arnheim, N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-54. 5. Kogan S, Doherty M, Gitschier J. An improved method for pre-natal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 1987; 316: 985-90. 6. Estivill X, Farrall M, Scambler P, et al. A candidate for the cystic fibrosis locus isolated by selection of methylation free islands. Nature 1987; 326: 840-15. 7. Sjogren B, Uddenberg N Decision making during the prenatal diagnostic procedure: a questionnaire and interview study of 211 women participating in prenatal diagnosis. Prenatal Diagnosis 1988; 8: 263-73 2. Williamson
Fig 2-Dot-bIot analysis. DNA from each amplified product was blotted under suction and probed separately with two 32P-dA TP-labelled oligonucleotides. Each probe spanned the polymorphic site. Upper row=Al, specific for "’1"’ in this region. Lower row = A2, for "G". Probes were hybridised at 55° and 57°C, respectively. Lanes 1 and 2 are father and mother, respectively; both A1and A2 hybridised, indicating heterozygosity for this polymorphic site. Lane 3 is the affected child (homozygous A2 A2) and lane 4 is CVS (heterozygous Al A2). Lanes 5 and 6 are control samples (A A2 and A2 A2, respectively).
NOVEL VIRUSES IN HUMAN FAECES
SiR,—Polyacrylamide gel electrophoresis (PAGE) to detect rotaviruses1 has revealed, in a small proportion of human faeces, two electrophoretic bands (figure) thought to represent the genome of a new group of viruses. The two bands are formed by double stranded (ds) RNA molecules, co-sedimenting in caesium chloride gradients with uniform particles which have an average diameter of
SIR,-Prenatal diagnosis for cystic fibrosis (CF) is now done routinely by use of tightly linked restriction fragment length polymorphisms (RFLP).1.2 RFLPs in strong linkage disequilibrium with the CF mutation have lately been identified.3’ The KM-19 probe detects an RFLP with PstI, and this polymorphism is among the most useful for prenatal diagnosis of CF. Amplification of DNA by the polymerase chain reaction (PCR) allows rapid analysis without the use of radioactivity. 5,6 We report the characterisation of DNA sequences surrounding the KM-19 polymorphism and describe the use of PCR to analyse this RFLP for prenatal diagnosis of CF. Cosmid CNX.43 was subcloned, and restriction fragments were identified which flank the polymorphic Pstl site. DNA sequencing was done to prepare oligonucleotides which flank the polymorphic site. By use of two oligonucleotides, it was possible to do a diagnostic analysis with a single amplification process followed by restriction enzyme digestion and agarose gel eiectrophoresis. The figure demonstrates the polymorphism in two heterozygous parents with an affected CF child who is homozygous for the presence of PstI restriction site, while the fetus is homozygous for absence of the restriction site. The product of the amplification reaction is approximately 0-95 kb, and digestion with Pstl results in fragments of 0-65 and 0-30 kb. Homozygotes for the absence of the restriction site demonstrate the 0-95 kb fragment, homozygotes for the presence of the site demonstrate the 0-65 and 0-30 kb fragments, and heterozygotes demonstrate all three fragments. Both manual and automated procedures were used for amplification. Several fetuses were studied by chorionic villus sampling and amniocentesis, and
the results with PCR were unambiguous and in agreement with Southern blotting results in all cases. Because of the linkage disequilibrium with the KM-19 polymorphism,4.7 this RFLP is diagnostic in a large proportion of families at risk for CF. Of 85 couples with a 1-in-4 risk for CF studied in Houston, 48% were fully informative with this polymorphism, 41% were informative for one parent, and 11% were uninformative. Adequate prenatal diagnosis will be provided by PCR in 48 % of families which are fully informative and in about half of the fetuses where the informative parent is predicted to have transmitted the normal gene to the fetus. This means that the use of PCR with the KM-19 polymorphism would give an adequate prenatal diagnosis in about 70% of families at risk for CF. PCR is also available for the neighbouring linked polymorphic marker CS.7. Although very rare, crossovers between the KM-19 probe and the CF locus are known,’ and development of PCR for additional polymorphisms flanking the CF locus is desirable until such time as the gene is cloned. Institute for Molecular Genetics, Baylor College of Medicine, Houston
Regional DNA Laboratory, Department of Molecular Genetics, St Mary’s Hospital Medical School,
GERALD L. FELDMAN
North West Thames
London W2
Hughes Medical Institute Institute for Molecular Genetics,
ROBERT WILLIAMSON
Howard
Baylor College of Medicine, Houston, Texas 77030, USA 1 Beaudet
ARTHUR L. BEAUDET WILLIAM E. O’BRIEN
AL, Buffone GJ Prenatal diagnosis of cystic fibrosis. J Pediatr 1987, 111:
630-33.
M, Roedek C, Stanier P, et al First-trimester prenatal diagnosis of cystic fibrosis with linked DNA probes. Lancet 1986, ii: 1402-04 Estivill X, Farrall M, Scambler PJ, et al A candidate for the cystic fibrosis locus isolated by selection for methylation-free islands. Nature 1987, 326: 840-45 Estivill X, Scambler PJ, Wainwright BJ, et al. Patterns of polymorphism and linkage disequilibrium for cystic fibrosis Genomics 1987; 1: 257-63 Mullis KB, Faloona FA Specific synthesis of DNA in vitro visa a polymerase-catalyzed chain reaction Methods Enzymol 1987, 155: 335-50. Saiki RK, Gelfand DH, Stoffels, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 1988, 239: 487-91 Beaudet AL, Spence JE, Montes M, et al Experience with new DNA markers for the diagnosis of cystic fibrosis N Engl J Med 1988 318: 50-51 Farrall M, Stanier P, Beaudet A, et al. Recombination between IRP and cystic fibrosis Am J Hum Genet (in press) Kogan SC, Doherty M, Gitschier J An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences N Engl J Med 1987, 318: 985-90
2 Farrall
3 4 5
6 7
8 9
SAME-DAY, FIRST-TRIMESTER ANTENATAL DIAGNOSIS FOR CYSTIC FIBROSIS BY GENE AMPLIFICATION
Use of PCR for analysis of KM-19 diagnosis of CF.
Genomic DNA fluid cells. PCR
was was
polymorphism
and
prenatal
isolated from peripheral blood or cultured amnioticdone as described, with minor modifications.9
Oligonucleotide sequences were 5’-GCTGCATCATATAAGTTGCC-3’ 75 5’-AAGGCTACACTGTTAATTTT-3’. pmol of each oligonucleotide was used with 1 ).1g genomic DNA and 2 5 units of Taq DNA polymerase in total volume of 50 1. Second addition of 2 5 units of Taq polymerase was made after 15 cycles. Annealing conditions were 2 min at 55 °C, extension was 5 mm at 72’C, and denaturation was 1 min at 94°C for a total of 30 cycles. Samples of products of the reaction were digested with PstI and run on 1 5% agarose gel. Fragments were visualised with ethidium bromide and ultraviolet light. Approximate fragment sizes are indicated in kb. Extraneous fragment (c) of varying intensity sometimes occurs during amplification process, but this fragment does not interfere with the analysis. and
SiR,—DNA diagnosis is now available for about 170 inherited diseases,’ including the haemoglobinopathies, Duchenne muscular dystrophy, cystic fibrosis (CF), phenylketonuria, and the haemophilias. It is usual nowadays to attempt antenatal diagnoses on DNA from first-trimester trophob1ast,2 which has the advantage that, if a termination is requested, it can be done before fetal quickening. However, Southern blot analysis of trophoblast DNA can take up to 10 days. The polymerase chain reaction (PCR) technique permits simplified and rapid carrier testing by probes closely linked to CFand Dr Feldman and colleagues (see accompanying letter) have shown that it is applicable to about 70% of at-risk couples. Using PCR for gene amplification 3we have done a same-day antenatal diagnosis for CF. A couple who had a child with CF sought antenatal diagnosis, and trophoblast was obtained at 9 weeks’ amenorrhoea. Half of a single villus was removed from the specimen, the rest being used for Southern blot analysis. The small amount of villus was amplified 1Il parallel with DNA from the parents and the affected sibling, and haplotyped for CF with oligonucleotide primers specific for a
identifying the CS.7 locus.6 amplified products were analysed by digestion with HhaI, recognises a DNA polymorphism where one allele containing the cleavage site is common to most CF chromosomes.
sequence
PCR which
Each parent has one chromosome with the restriction site and one without, while the CF child has two chromosomes both of which
103
Fig 1-Hhal restricted and non-restricted gene amplification products for analysis of the CS.7 polymorphism and antenatal diagnosis of CF. Upper: genomic DNA for parents and child with CF was isolated from blood and buccal epithelial cells obtained by mouthwash. CVS and the buccal cell material was extracted," cells were suspended in 0-5 ml 0-0 mol/1 NaOH, and boiled for 5-10 min. Alkali suspension was neutralised with 3 mol/I sodium acetate and centrifuged to pellet denatured protein and cell debris. 5-10 pl supernatant used for each amplification. Primer oligonucleotide sequences were: 5’-CCCAGCTTCAGGGAGAGAAGCGAAGCAATG-3’ and 5’-AAACGCGGGGTTTTAGACACGGGTGCATGA-3’. Reaction was done in 1 x Taq DNA polymerase buffer (50 mmol/1 KC1, 10 mmol/1 "Tris"-HCI pH 8 3, 1-5 mmot/1 MgCl2, 0-01% gelatin), 20 pmol dNTPs, 50 pmol of each
oligonucleotide primer, and 0-4 ug
or less of genomic DNA, made up to 100 ul with water. Before first addition of enzyme the reaction mix was boiled for 6 min. 1-5-20 units of TaqI DNA polymerase (Cetus) was added per reaction. Amplification was for thirty cycles, each consisting of a denaturation step at 93-94’C for 90 s, followed by annealing and elongation combined at 68’C for 2 min without further addition of enzyme. Amplification product is 330 bp. Restriction with either BssHII or HhaI gives fragments of 164 and 166 bp which superimpose and are seen as a single band. DNA from persons with the Al/A2 haplotype shows the 330,164, and 166 bp fragments. DNA from homozygotes for Al have only the 330 bp doublet band, while homozygotes for A2 have doublet bands at 166 and 164 bp due to restriction of both alleles. All analyses of amplified products were done using 2% agarose gels stained with ethidium bromide and visualised with ultraviolet light. Lanes 2, 4, 6, 8, and 10 are unrestricted amplified products from father, mother, affected child, chorionic villus sample (CVS), and affected child buccal cell DNA, respectively. Lanes 3, 5, 7, and I are the HhaI restriction digest products from the same samples, respectively. Lane 1 contains a 1 kb ladder of DNA size markers.
have the site (fig 1). The fetus is heterozygous for the polymorphic site, and therefore is highly likely to be unaffected, although a carrier. The result was confirmed by Southern blotting (fig 2). The pregnancy is continuing. Buccal epithelial cells from the parents and affected child were also collected, and amplified with the same primers.’ Restriction enzyme digestion of amplified buccal cell preparations gave identical results to those obtained with whole blood (fig 1, lanes 10 and 11). Blood or mouthwash samples can be collected either before or simultaneously with the chorionic villus biopsy. While only one antenatal diagnosis has been done by same-day technology so far, many control chorion samples have been amplified in this way. The use of these primers and those reported by Feldman et al for the adjacent DNA probe KM-19, together with buccal cell analysis, permits combined simple and rapid carrier testing and antenatal diagnosis. The reduction of time for analysis to a single day would permit counselling on the evening of the day the biopsy is done. Termination could then be arranged for the following morning, though couples will often prefer to take their time,’ and return several days later when they have decided what do do. Not all families at high risk are informative; only about 50% of CF high-risk families are fully informative with CS.7 alone, but probes such as KM-19 increase informativeness. The technology has been automated (by Perkin Elmer/Cetus and Cambio) but the temperature cycling can also be done manually with two water baths (as for this diagnosis) or with a computer controlled robot arm. As informative probes are developed for many severe inherited diseases same-day diagnosis with PCR may become the aim of antenatal diagnostic services. We thank Nick Lench for advice on PCR methodology, and Kyproula Christodoulou for practical help. This research was supported by the Medical Research Council, the Wellcome Trust, and the Cystic Fibrosis Research Trust. North West Thames Regional DNA Department of Molecular Genetics, St Mary’s Hospital Medical School, London W2 1PG
Laboratory,
CAROLYN WILLIAMS ROBERT WILLIAMSON
Department of Human Molecular Genetics, Central Institute of Molecular Biology, Academy of Sciences, Berlin, East Germany
CHARLES COUTELLE
Department of Obstetrics and Gynaecology, St Mary’s Hospital, London W2
FRANK LOEFFLER JOHN SMITH
DNA Laboratory, Department of Medical Genetics, St Mary’s Hospital, Manchester
ADRIAN IVINSON
1
Cooper DN, Schmitke J. Diagnosis of genetic disease using recombinant DNA. Hum
Genet 1987; 77: 66-75 RE, Eskdale J, Coleman DV, Naizi M, Loeffler FE, Modell BM. Direct gene analysis of chorionic villus a possible technique for first trimester antenatal diagnosis of haemoglobinopathies. Lancet 1981; ii: 1125-27. 3. Lench N, Stanier P, Williamson R. A simple non-invasive method to obtain DNA for gene analysis. Lancet 1988; i: 1356-58. 4. Saiki R, Scharf S, Faloona F, Mullis B, Horn G, Erlich H, Arnheim, N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-54. 5. Kogan S, Doherty M, Gitschier J. An improved method for pre-natal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 1987; 316: 985-90. 6. Estivill X, Farrall M, Scambler P, et al. A candidate for the cystic fibrosis locus isolated by selection of methylation free islands. Nature 1987; 326: 840-15. 7. Sjogren B, Uddenberg N Decision making during the prenatal diagnostic procedure: a questionnaire and interview study of 211 women participating in prenatal diagnosis. Prenatal Diagnosis 1988; 8: 263-73 2. Williamson
Fig 2-Dot-bIot analysis. DNA from each amplified product was blotted under suction and probed separately with two 32P-dA TP-labelled oligonucleotides. Each probe spanned the polymorphic site. Upper row=Al, specific for "’1"’ in this region. Lower row = A2, for "G". Probes were hybridised at 55° and 57°C, respectively. Lanes 1 and 2 are father and mother, respectively; both A1and A2 hybridised, indicating heterozygosity for this polymorphic site. Lane 3 is the affected child (homozygous A2 A2) and lane 4 is CVS (heterozygous Al A2). Lanes 5 and 6 are control samples (A A2 and A2 A2, respectively).
NOVEL VIRUSES IN HUMAN FAECES
SiR,—Polyacrylamide gel electrophoresis (PAGE) to detect rotaviruses1 has revealed, in a small proportion of human faeces, two electrophoretic bands (figure) thought to represent the genome of a new group of viruses. The two bands are formed by double stranded (ds) RNA molecules, co-sedimenting in caesium chloride gradients with uniform particles which have an average diameter of