DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry
AUTOR(ES)
Kwon, Young-Soo
FONTE
Oxford University Press
RESUMO
Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3′-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt. It is also demonstrated that A→G and C→T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis. As a step towards single-tube sequencing reactions, α-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=30412Documentos Relacionados
- Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification
- Classification and Identification of Petroleum Microorganisms by MALDI-TOF Mass Spectrometry
- Hierarchical high-throughput SNP genotyping of the human Y chromosome using MALDI-TOF mass spectrometry
- Characterization of a multi-functional metal-mediated nuclease by MALDI-TOF mass spectrometry
- A new MALDI-TOF based mini-sequencing assay for genotyping of SNPS