APEX disease gene resequencing: mutations in exon 7 of the p53 tumor suppressor gene.

Detection of mutations in disease genes will be a significant application of genomic research. Methods for detecting mutations at the single nucleotide level are required in highly mutated genes such as the tumor suppressor p53. Resequencing of an individual patient's DNA by conventional Sanger methods is impractical, calling for novel methods for sequence analysis. Toward this end, an arrayed primer extension (APEX) method for identifying sequence alterations in primary DNA structure was developed. A two-dimensional array of immobilized primers (DNA chip) was fabricated to scan p53 exon 7 by single bases. Primers were immobilized with 200 microm spacing on a glass support. Oligonucleotide templates of length 72 were used to study individual APEX resequencing reactions. A template-dependent DNA polymerase extension was performed on the chip using fluorescein-labeled dideoxynucleotides (ddNTPs). Labeled primers were evanescently excited and the induced fluorescence was imaged by CCD. The average signal-to-noise ratio (S/N) observed was 30:1. Software was developed to analyze high-density DNA chips for sequence alterations. Deletion, insertion, and substitution mutations were detected. APEX can be used to scan for any mutation (up to two-base insertions) in a known region of DNA by fabricating a DNA chip comprising complementary primers addressing each nucleotide in the wild-type sequence. Since APEX is a parallel method for determining DNA sequence, the time required to assay a region is independent of its length. APEX has a high level of accuracy, is sequence-based, and can be miniaturized to analyze a large DNA region with minimal reagents.

Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development.

Neural tissue and smooth muscle appear early in the developing fetal lung, but little is known of their origin and subsequent distribution. To investigate the spatial and temporal distribution of nerves, ganglia, and airway smooth muscle during the early pseudoglandular stage, fetal mouse lungs at embryonic days (E) 11 to 14 were immunostained as whole-mounts and imaged by confocal microscopy. At E11, the primordial lung consisted of the future trachea and two budding epithelial tubules that were covered in smooth muscle to the base of the growing buds. The vagus and processes entering the lung were positive for the neural markers PGP 9.5 (protein gene product 9.5) and synapsin but no neurons were stained at this stage. An antibody to p75NTR revealed neural crest cells on the future trachea as well as in the vagus and in processes extending from the vagus to the lung. This finding indicates that even though neuronal precursors are already present at this stage, they are still migrating into the lung. By E12, neural tissue was abundant in the proximal part of the lung and nerves followed the smooth muscle-covered tubules to the base of the growing buds. At E13 and E14, a neural network of interconnected ganglia, innervated by the vagus, covered the trachea. The postganglionic nerves mainly followed the smooth muscle-covered tubules, but some extended out into the mesenchyme beyond the epithelial buds. Furthermore, we show in a model of cultured lung explants that neural tissue and smooth muscle persist and continue to grow and differentiate in vitro. By using fluorescent markers and confocal microscopy, we present the developing lung as a dynamic structure with smooth muscle and neural tissue in a prime position to influence growth and development.