RESUMO
The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
Assuntos
Genoma Bacteriano/genética , Genoma Humano/genética , Genômica/instrumentação , Genômica/métodos , Semicondutores , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/métodos , Escherichia coli/genética , Humanos , Luz , Masculino , Rodopseudomonas/genética , Vibrio/genéticaRESUMO
Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20-50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq DNA pol/TBD is thermostable, PCR competent and able to copy repetitive deoxynucleotide sequences six to seven times more faithfully than Taq DNA polymerase and makes 2-3-fold fewer AT-->GC transition mutations.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Taq Polimerase/metabolismo , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Bacteriófago T3/enzimologia , Sítios de Ligação/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Ativação Enzimática , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Homologia de Sequência de Aminoácidos , Estreptavidina/metabolismo , Taq Polimerase/genéticaRESUMO
Mutations in DNA accrue relentlessly, largely via stochastic processes. Random changes accumulate, eventually disabling genetic components which result in the formation of the cancer phenotype. Given the infrequency of measured nucleotide changes and the requirement for several mutations to occur in the same cell, it has been postulated that the rate of mutation must become elevated early in the course of evolution of the cancer. Recently, large scale sequencing of tumor DNA has sought to directly measure random mutations. We discuss the implications of these findings and the factors that must be considered in order for fruitful determination of whether a mutator phenotype is a necessary precursor for cancer.
