1. Step 1: amplification and cloning procedures
Genomic DNA
miR site
DNA cleavage using blunt end-cutting restriction enzymes
Adaptor ligation
DNA database construction
Sequencing of ~200 recombinant plasmids
non-target DNA
miRNA gene
>seq1
acatctagatcaagaaaagcctttgagccgt
>seq2
tggagaagcagggcacgtgtacagtctagt
>Seq3
tggagaagcagggcacgtgccccagtgatt
PCR amplification using one miR primer (red arrow) and one adaptor primer (blue arrow)
BLAST searching for
miR* sites
Re-amplification across miR sites of selected molecules
Secondary structure prediction of miRNAs
Bacterial cloning
Inefficient amplification of non-target DNA due to PCR suppression effect
Efficient amplification of mIRNA genes
Vector ligation +
Step 1: amplification and cloning procedures
Step 2: mainly in silico procedures