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Table 1. Strategies for PCR-Based Tests Used for Clinical Preimplantation Genetic Diagnosis

Method Disorder to be diagnosed Mutation type Single PCR, agarose gel (+/- Y band) X-linked disorders2 Various (gender determination to exclude hemizygotes) Nested PCR, agarose gel (+/- X/Y) X-linked disorders6 Various (gender determination to exclude hemizygotes) Nested PCR, heteroduplexing Cystic fibrosis22 38 44 75 Tay-Sachs disease111 3 bp deletion (F508) 4 bp insertion Nested PCR, allele-specific amplification RhD blood typing3 Myotonic dystrophy127 +/- RhD gene determines Rh status Expansion of (CTG)n trinucleotide repeat Nested PCR, restriction enzyme Cystic fibrosis,23 Beta thalassemia,83 Marfan syndrome,107 Epidermolysis Bullosa,100 Lesch-Nyhan syndrome,101 Sickle cell anemia,102 Fanconi’s anemia,103 Ornithine transcarbamylase deficiency,104 Spinal muscular atrophy108 109 110 Various point mutations Deletion. Distinguish between gene and pseudogene Nested PCR, restriction enzyme (2 mutations in 1 fragment) Skin fragility ectodermal dysplasia syndrome66 Allows detection of ADO Whole genome amplification and comparative genome hybridization Aneuploidy screening96 NA Whole genome amplification (PEP) Familial adenomatous polyposis coli60 Multiple analyses from each sample Nested PCR, linked markers Duchenne muscular dystrophy10 125 Ornithine transcarbamylase deficiency104 Exon deletions Point mutation (linked marker for ADO detection) Nested PCR, SSCP Familial Adenomatous Polyposis Coli60 Point mutation Nested PCR, direct cycle sequencing Skin fragility ectodermal dysplasia syndrome66 Point mutations (cycle sequencing to confirm restriction digest) Nested PCR, DGGE Beta thalassemia115 Point mutations Heminested PCR, site specific mutagenesis Retinitis pigmentosa99 Ornithine transcarbamylase deficiency104 Point mutation Point mutation Heminested PCR, allele dependent length polymorphism Retinitis pigmentosa99 Point mutation Nested multiplex PCR (including linked markers) Marfan syndrome119 Epidermolysis Bullosa100 Beta thalassemia83 Unknown mutation Monitor allele dropout Nested multiplex PCR (including linked and non-linked markers) Sickle cell anemia,41 hemophilia B,41 cystic fibrosis,41 Gaucher disease,61 Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency61 Monitor allele dropout and contamination Fluorescent PCR, allele size (fragment analysis) Huntington disease32 Cystic fibrosis106 Myotonic dystrophy55 Fragile X syndrome128 Expansion of (CAG)n trinucleotide repeat 3 bp deletion Expansion of (CTG)n trinucleotide repeat Expansion of (CGG)n trinucleotide repeat Fluorescent PCR, SSCP Medium chain acyl CoA dehydrogenase deficiency76 Point mutation Fluorescent PCR, ARMS Spinal muscular atrophy77 Exon deletion in gene but not pseudogene Fluorescent PCR, restriction analysis Congenital adrenal hyperplasia,78 osteogenesis imperfecta,105 medium chain acyl CoA dehydrogenase deficiency,33 Sickle cell anemia34 Point mutations Fluorescent PCR, restriction analysis (2 mutations in 1 fragment) Beta thalassemia34 Point mutations, small deletion Multiplex Fluorescent PCR Beta thalassemia34 Point mutations, small deletion Multiplex Fluorescent PCR (including unlinked marker) Myotonic dystrophy130 Expansion of (CTG)n trinucleotide repeat/contamination control Multiplex Fluorescent PCR (including linked marker) Medium chain acyl CoA dehydrogenase deficiency76 Maternal mutation unknown Fluorescent PCR, linked markers only Fragile X syndrome120 Marfan syndrome121 Charcot Marie Tooth disease45 Cystic fibrosis123 Expanded (CGG)n repeat (refractory to PCR) Unknown mutation Gene duplication Heterogeneous mutations

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