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Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: Application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia

Cantàfora, Alfredo ; Blotta, Ida ; Bruzzese, Nicodemo ; Calandra, Sebastiano ; Bertolini, Stefano

Electrophoresis, 2001-10, Vol.22 (18), p.4012-4015 [Periódico revisado por pares]

Hoboken: Wiley Subscription Services, Inc., A Wiley Company

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Título:
Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: Application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia
Autor: Cantàfora, Alfredo ; Blotta, Ida ; Bruzzese, Nicodemo ; Calandra, Sebastiano ; Bertolini, Stefano
Assuntos: Alleles ; Cosegregation analysis ; DNA - blood ; DNA - genetics ; Electrophoresis, Capillary - instrumentation ; Electrophoresis, Capillary - methods ; Exons - genetics ; Familial hypercholesterolemia ; Genetic Predisposition to Disease ; Heterozygote ; Humans ; Hyperlipoproteinemia Type II - epidemiology ; Hyperlipoproteinemia Type II - genetics ; Italy - epidemiology ; Lab-on-a-chip ; Leukocytes - chemistry ; Low-density lipoprotein ; Microchemistry - instrumentation ; Microchemistry - methods ; Microchip electrophoresis ; Microsatellite Repeats ; Microsatellite sizing ; Mutagenesis, Insertional ; Mutation, Missense ; Point Mutation ; Polymerase Chain Reaction ; Receptors, LDL - genetics ; Reproducibility of Results ; Short tandem repeats
É parte de: Electrophoresis, 2001-10, Vol.22 (18), p.4012-4015
Notas: ArticleID:ELPS4012
istex:F988D1524F2A2D115A64028E7E9D8159773AF77E
ark:/67375/WNG-NBB99D9X-2
Descrição: This study evaluated the applicability of microchip electrophoresis to the sizing of microsatellites suitable to genetic, clinical and forensic applications. The evaluation was performed with the D19S394 tetranucleotide (AAAG) repeat characterized by a wide variation in the repeat number (1–17) and a short recombination distance from the low‐density lipoprotein (LDL)‐receptor gene that makes it suitable to cosegregation analysis of familial hypercholesterolemia (FH). The study was performed with 70 carriers of two LDL‐receptor mutations common in northern Italy (i.e., the 4 bp insertion in exon 10 known as FH‐Savona and the D200G missense mutation in the exon 4, known as FH‐Padova 1) and 100 healthy controls. The polymerase chain reaction (PCR) amplification products prepared with a cosolvent PCR protocol and an antibody‐protected polymerase were directly analyzed with an apparatus for high‐voltage capillary electrophoresis on microchips and laser‐induced fluorescence detection equipped with chips for the analysis of 25–500 bp dsDNA fragments. The test could not be extended to dinucleotide repeats due to the resolution characteristics of the available microchip. This novel approach was able to distinguish 17 microsatellite alleles varying from 0 to 17 repeats. Many of these alleles were quite rare, but the seven more abundant accounted for over the 70% of allele distribution in control population. The standard deviation in the sizing of the most abundant alleles ranged from ±0.60 to ±0.75 bp. This indicated that the size attribution to a conventional allele using the ±1 bp range around it allowed a confidence limit above the 80 %. The sizing of D19S394 obtained this way allowed the cosegregation analysis with both the FH mutations tested. Therefore, this innovative approach to microsatellite sizing was much simpler, but equally effective as traditional capillary electrophoresis, at least with tetranucleotide repeats.
Editor: Hoboken: Wiley Subscription Services, Inc., A Wiley Company
Idioma: Inglês

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Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: Application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia

Cantàfora, Alfredo ; Blotta, Ida ; Bruzzese, Nicodemo ; Calandra, Sebastiano ; Bertolini, Stefano

Hoboken: Wiley Subscription Services, Inc., A Wiley Company

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