title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome.
abstract: Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10,000 and 5/100,000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 x 10(-4). Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10(-6) using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (approximately 100 bp) juxtaposed with a 'clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent approximately 9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 x 10(-5) in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 x 10(4) turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10(5) persons.
authors.affiliation: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. akim@fhcrc.org
authors.forename: Andrea S
authors.initials: AS
authors.lastname: Kim
authors.forename: William G
authors.initials: WG
authors.lastname: Thilly
mesh_terms: D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D015894:Genome, Human; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase