RESUMO
A method for RNA analysis based on primer extension by reverse transcriptase is described.
Assuntos
Primers do DNA/química , DNA/química , Técnicas de Sonda Molecular , DNA Polimerase Dirigida por RNA/química , RNA/química , Pareamento Incorreto de Bases , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
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.
Assuntos
Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise Mutacional de DNA/instrumentação , Análise Mutacional de DNA/normas , Primers do DNA , DNA de Neoplasias/análise , Éxons , Fluoresceína , Genes p53/genética , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SoftwareRESUMO
[structure: see text]. The most powerful DNA microarrays would be prepared by photolithography with free 3'-ends that could be processed enzymatically. A photoremovable group that could be removed in quantitative yield would ensure high purity of the synthesized probes. We have developed new pyrimidine building blocks for 5' --> 3' DNA synthesis with high cycle yields using the NPPOC (3'-nitrophenylpropyloxycarbonyl) protecting group. These phosphoramidites were proved in automated photochemical DNA synthesis on a modified synthesizer.
