DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry.
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, alpha-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.
Kwon
Y
Y
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea and Sequenom, Inc., 11555 Sorrento Valley Road, San Diego, CA 92121, USA.
Tang
K
K
Cantor
C
C
Köster
H
H
Kang
C
C
D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D006801:Humans; D017241:MELAS Syndrome / Q000235:genetics; D017354:Point Mutation; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013873:Thionucleotides / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004272:DNA, Mitochondrial; D013873:Thionucleotides; D012313:RNA