RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis.
Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.
Hartmer
Ralf
R
SEQUENOM GmbH, Mendelssohnstrasse 15D, D-22761 Hamburg, Germany.
Storm
Niels
N
Boecker
Sebastian
S
Rodi
Charles P
CP
Hillenkamp
Franz
F
Jurinke
Christian
C
van den Boom
Dirk
D
D001055:Apolipoproteins B / Q000235:genetics; D006147:Guanine / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D006163:Ribonuclease T1 / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001055:Apolipoproteins B; D009841:Oligonucleotides; D006147:Guanine; D006163:Ribonuclease T1