Conformation-sensitive gel electrophoresis as an ideal high-throughput strategy for accurate detection of sequence variations in DNA: screening hTomm and hTimm genes.
Conformation-sensitive gel electrophoresis is a heteroduplex-based method that is particularly well suited to high-throughput analyses. Its simplicity makes it amenable to various adaptations and modifications to enhance its applicability to genome-wide mutation scans. Technical aspects that markedly improve the conformation-sensitive gel electrophoresis performance by combining high throughput and high resolution of the bands facilitating the interpretation of the results are described here. The authors report some of the results they have obtained in the screening of the exon 1 of human Timm8A gene as an example of the suitability of the conformation-sensitive gel electrophoresis-based protocol that has been adapted to optimize its throughput, speed, and simplicity in the recognition of both heterozygous and homozygous DNA mutations. The higher throughput is achieved by using 12 batches per gel. The length of the gel is sufficient for an adequate well-to-read distance for each batch that allows a clear distinction and resolution of the conformation-sensitive gel electrophoresis bands. Standardization of the procedure using multichannel pipettes reduces the preparation time of the 96-well PCRs to 10 min and also accelerates the gel loading. The resulting bands give high-quality images, allowing easy detection of known as well as novel mutations.
Fundación Valenciana de Investigaciones Biomédicas, Instituto de Investigaciones Citológicas, Amadeo de Saboya, 4, 46010 Valencia, Spain. jrblesa@ochoa.fib.es
José Rafael
JR
Blesa
Jesús Angel
JA
Prieto-Ruiz
José
J
Hernández-Yago
D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D011956:Receptors, Cell Surface / Q000235:genetics*
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D011956:Receptors, Cell Surface; C118122:TIMM8A protein, human; C084008:TOMM20 protein, human; C489971:TOMM22 protein, human; C108485:TOMM34 protein, human; D004247:DNA