A major advantage of BSPP is that it allows the flexible capture of an arbitrary subset of genomic regions (hundreds to hundreds of thousands of genomic loci) in single-tube reactions. Large number of samples can be processed efficiently and converted into multiplexed sequencing libraries with only three enzymatic steps, without the conventional library preparation procedures. BSPP are applicable to clinical studies, screening cell lines, and for quantifying low abundance regions using deep sequencing.
The two annealing arms of padlock probes “capture” bisulfite converted DNA during annealing incubation. (2) A mixture of dNTPs, polymerase, ligase, and buffer is added to generate a circular product in extension and ligation. (3) The genomic DNA and leftover padlock probes are removed by exonucleases. (4) Next, hybrid primers are used to label individual samples by appending barcodes (barcode regions are highlighted by dashed circles) and to additionally append common adaptors for sequencing. (5) PCR amplification is performed on capture products to generate multiple copies of the captured DNA
Read more about the process in our application note: Large-Scale Targeted DNA Methylation Analysis Using Bisulfite Padlock Probes