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Protocol
Padlock probe ligation and preparation for dual tag microarray readout
Keywords: Padlock probe, nucleic acid detection, mRNA analysis, RCA, MlyI
The method is used to convert nucleic acid target sequences into reporter molecules that can be analyzed by the dual tag microarray analysis platform.
This method encodes nucleic acid targets into single stranded reporter molecules comprising tag motifs in the 3’ and 5’ end. These reporter molecules can then be decoded with very high precision and sensitivity by the dual tag microarray readout platform.
The method requires design of a padlock probeset comprising two separate tag motifs in each probe along with the appropriate restriction enzyme sites for MlyI. For example, see the figure below. ? [figure 1]
Sequence design. The blue and red sequence elements are the tags, green the general detection cassette and grey the MlyI restriction digestion cassette. a) Padlock probe design; the general detection sequence is flanked by the two tag sequences in turn flanked by the restriction digestion cassette and target complementary sequences in the 3’ and 5’ ends. b) One segment of the concatemeric RCA product complementary to the padlock. Oligonucleotides are hybridized to the RCA product directing the MlyI restriction enzyme digestion to the junction of the tag and the restriction digestion cassette.
Instructions
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Padlock probe ligation (5YGL4X)
Padlock probe ligation is performed in 10 µl reactions comprising 0.1 nM of each probe, 1 µl sample and 5 U of Ampligase in Ampligase buffer (Epicentre Biotechnologies, WI, USA) for 4 cycles of 4 hrs at 50°C, separated by 2 min at 95°C.
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Rolling circle amplification (5YGNAH)
1 µl of 100 nM RCA primer oligonucleotide is added and RCA is performed at 37°C for 1 hr by addition of 5 µl S8 buffer (20 mM TRIS-Ac pH 8, 50 mM KAc, 10 mM (NH4)2SO4, 10 mM MgAc2, 1 mM DTT), with 0.1 µg/µl bovine serum albumin (BSA) (New England Biolabs, USA), 0.6 mM of each dNTP, and 3 U phi29 DNA polymerase (Fermentas, Lithuania).
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MlyI digestion of RCA products (5YGTJF)
RCA products are cleaved with MlyI by addition of 5 µl S8 buffer with 0.1 µg/µl BSA, 10 pmol of each of the two restriction oligonucleotides, along with 5 U of MlyI for 1 h at 37°C. The cleaved products are now ready for analysis using the dual-tag microarray analysis platform (see separate protocol).
