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 disorders 2	Various (gender determination to exclude hemizygotes)
Nested PCR, agarose gel (+/− X/Y)	X-linked disorders 6	Various (gender determination to exclude hemizygotes)
Nested PCR, heteroduplexing	Cystic fibrosis 22, 38, 44, 75 Tay-Sachs disease 111	3 bp deletion (ΔF508) 4 bp insertion
Nested PCR, allele-specific amplification	RhD blood typing 3 Myotonic dystrophy 127	+/− 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 atrophy 108, 109, 110	Various point mutations Deletion. Distinguish between gene and pseudogene
Nested PCR, restriction enzyme (2 mutations in 1 fragment)	Skin fragility ectodermal dysplasia syndrome 66	Allows detection of ADO
Whole genome amplification and comparative genome hybridization	Aneuploidy screening 96	NA
Whole genome amplification (PEP)	Familial adenomatous polyposis coli 60	Multiple analyses from each sample
Nested PCR, linked markers	Duchenne muscular dystrophy 10, 125 Ornithine transcarbamylase deficiency 104	Exon deletions Point mutation (linked marker for ADO detection)
Nested PCR, SSCP	Familial Adenomatous Polyposis Coli 60	Point mutation
Nested PCR, direct cycle sequencing	Skin fragility ectodermal dysplasia syndrome 66	Point mutations (cycle sequencing to confirm restriction digest)
Nested PCR, DGGE	Beta thalassemia 115	Point mutations
Heminested PCR, site specific mutagenesis	Retinitis pigmentosa 99 Ornithine transcarbamylase deficiency 104	Point mutation Point mutation
Heminested PCR, allele dependent length polymorphism	Retinitis pigmentosa 99	Point mutation
Nested multiplex PCR (including linked markers)	Marfan syndrome 119 Epidermolysis Bullosa 100 Beta thalassemia 83	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 deficiency 61	Monitor allele dropout and contamination
Fluorescent PCR, allele size (fragment analysis)	Huntington disease 32 Cystic fibrosis 106 Myotonic dystrophy 55 Fragile X syndrome 128	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 deficiency 76	Point mutation
Fluorescent PCR, ARMS	Spinal muscular atrophy 77	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 anemia 34	Point mutations
Fluorescent PCR, restriction analysis (2 mutations in 1 fragment)	Beta thalassemia 34	Point mutations, small deletion
Multiplex Fluorescent PCR	Beta thalassemia 34	Point mutations, small deletion
Multiplex Fluorescent PCR (including unlinked marker)	Myotonic dystrophy 130	Expansion of (CTG)n trinucleotide repeat/contamination control
Multiplex Fluorescent PCR (including linked marker)	Medium chain acyl CoA dehydrogenase deficiency 76	Maternal mutation unknown
Fluorescent PCR, linked markers only	Fragile X syndrome 120 Marfan syndrome 121 Charcot Marie Tooth disease 45 Cystic fibrosis 123	Expanded (CGG)n repeat (refractory to PCR) Unknown mutation Gene duplication Heterogeneous mutations