Plastic microchip electrophoresis for genetic screening: the analysis of polymerase chain reactions products of fragile X (CGG)n alleles.
Clinical screening of abnormal chromosomes associated with fragile X syndrome (FXS) demands a high-throughput method including DNA sizing and detection of the amplified products. This study is to explore the use of polymer microchip electrophoresis for the analysis of polymerase chain reaction (PCR) products of fragile X (CGG)n alleles to facilitate a fast exclusion test of FXS. The sequences flanking the CGG-repeat of FMR1 gene was amplified by betaine-PCR and the amplified products were desalted and then analyzed by microchips which were fabricated on poly(methyl methacrylate) (PMMA) substrate. The PCR bands with more than six CGG-repeats in difference could be clearly distinguished in less than 3 min by microchip electrophoresis with a separation length of 6 cm. It was found that the signal was greatly enhanced with the use of both covalent (Cy5) and intercalating dye (TORRO-3), which has never been demonstrated before. We tested the method by reanalysis of twelve samples from males and six samples from females. For female samples with less than six repeat differences, Southern blotting method was performed to confirm or exclude the findings from microchips. It was found that the test results from all male and female samples show a 100% correlation between the microchip electrophoresis and the existing methods.
Sung
W C
WC
Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
Lee
G B
GB
Tzeng
C C
CC
Chen
S H
SH
D019075:Electrophoresis, Capillary / Q000379:methods; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D016601:RNA-Binding Proteins; D018911:Trinucleotide Repeats
D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human