Results on single cell PCR for Huntington's gene and WAVE product analysis for preimplantation genetic diagnosis.
Triple repeat base pair amplification is the basis for a number of prevalent genetic diseases such as Huntington's, Fragile X, Myotonic Dystrophy and others. We have chosen to investigate the use of PCR to amplify a portion of the Huntington's gene in single cells in order to develop a clinical test system for preimplantation genetic diagnosis (PGD). Amplification of CAG triple repeat sequences poses difficulties due to resistance of GC melting for amplification. Special PCR modifications are necessary to carry out the amplification of GC rich areas found in most triple base pair expansions. We have used a modified polymerase chain reaction (PCR) protocol to amplify the expanded repeat sequence of the Huntington's gene with satisfactory efficiency. Detection of the amplified expanded CAG repeats is shown to be possible using both agarose gel electrophoresis and high definition denaturing high pressure liquid (DHPLC) chromatography. The incidence of allele dropout (ADO) is documented.
Drury
K C
KC
Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL, USA. druryk@obgyn.ufl.edu
Liu
M C
MC
Lilleberg
S
S
Kipersztok
S
S
Williams
R S
RS
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D020732:Cytogenetic Analysis; D017931:DNA Primers; D004622:Embryo, Mammalian / Q000473:pathology; D005260:Female; D005347:Fibroblasts / Q000473:pathology; D005819:Genetic Markers; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000175:diagnosis* / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods; D019680:Trinucleotide Repeat Expansion
D017931:DNA Primers; D005819:Genetic Markers; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins