Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.
Nat Biotechnol
; 30(4): 349-53, 2012 Mar 25.
Article
em En
| MEDLINE
| ID: mdl-22446694
ABSTRACT
Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of â¼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sequenciamento de Nucleotídeos em Larga Escala
/
Nanoporos
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article