The MassARRAY Comparative Sequence Analysis application sets a new precedent in molecular typing. Recently, a new and optimized protocol has been developed based on the MassCLEAVE™ chemistry to enable accurate comparative sequence analysis. The application uses reference sequences as a comparative measure to identify microbes, viruses, or other haploid organisms with sensitivity down to a single nucleotide.
The new MassCLEAVE assay is based on an in vitro transcription ribonuclease enzymatic cleavage of 500–800 bp PCR products from the target region of interest. A protocol overview is provided in Figure 1.
Figure 1. (Click to enlarge)
The MassCLEAVE process is homogeneous and does not require purification of the PCR product or subsequent analytes. The process is automatable and thus amenable to high throughput.
Base-specific cleavage generates a defined experimental mass signal pattern of the sample. For analysis, the experimental pattern is subsequently compared to an in silico reference mass signal pattern derived from an individual or set of reference sequences. Differences between the expected and the observed mass signal pattern are interpreted and enable identification of sequence variations.
The three step protocol is PCR based and allows for the amplification of a target region of interest.
Following SAP treatment, the samples are subject to in vitro transcription and enzymatic base-specific cleavage. After sample conditioning, the cleavage mixture is dispensed onto a 384-well SpectroChip® The SpectroChip is introduced into the MassARRAY Compact™ Analyzer and the cleavage products are automatically analyzed by MALDI-TOF mass spectrometry.
Data analysis is performed on the Comparative Sequence Analysis software. A graphical software interface of the ID results and mass spectra is provided in Figure 2.
Figure 2. (Click to enlarge)
The Comparative Sequence Analysis software automatically generates a report containing ID results listing the best matching reference sequences for each target region of interest, as well as sequence variations and new sequences.
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