Bisulfite padlock probes (BSPP) are a method for the targeted quantification of DNA methylation in mammalian genomes. They can simultaneously characterize the level of methylcytosine modification in a large number of targeted regions at single-base resolution. 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.
In the design phase, the researcher decides on the genomic targets to capture and design padlock probes to capture those targets. Previous works have established the design parameters that work best for bisulfite padlock probes to enable robust and consistent capture results. Ex ppDesigner can be downloaded from http://genome-tech.ucsd.edu/public/Gen2_BSPP/ppDesigner/ppDesigner.php.
Oligonucleotides to used for Padlock probes can then be ordered in bulk from LC Sciences.
Bisulfite padlock probe preparation(1) Schematic view of a synthesized oligonucleotide. (2) Amplification of oligonucleotides is carried out using a forward primer containing multiple phosphorothioate bonds (marked with a star) to prohibit 5′ digestion by lambda exonuclease, and a uracil base at the 3′ end and a reverse primer that is phosphorylated at the 5′ end to promote lambda exonuclease digestion and a DpnII recognition site at the 3′ end. (3) Digestion of the reverse complement strand is carried out by lambda exonuclease to generate single stranded probe. (4) The 5′ adaptor is cleaved by the USER cleavage system, which contains Uracil DNA glycosylase (UDG, not pictured) that catalyzes the removal of the uracil base and Endonuclease VIII (pictured as a square) that breaks the phosphodiester backbone. (5) The 3′ adaptor is cleaved by DpnII (pictured as a hexagon) with the help of a guide oligonucleotide to make the cleavage region double-stranded
Bisulfite padlock probe capture(1) 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
Once padlock probes are ready, it is possible to move to the experiment phase, which involves the processing of samples. Generally, up to 96 or 384 samples may be processed in parallel in 96-well or 384-well plates to minimize batch-to-batch variability. Each sample is prepared for capture in separate tubes.
Finally, the samples are prepared for sequencing with a two-step PCR protocol that enables multiplexing of individual samples using barcoded primers and pooled size exclusion to enrich for targeted fragment based on the expected fragment sizes. The resulting libray is sequenced via next-gen technology.