High capacity molecule detection
A target molecule and quantitative technology, applied in the field of high-capacity molecular detection, can solve the problem that the probe labeling scheme cannot expand in situ molecular detection and spatial imaging
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Embodiment 1
[0188] Embodiment 1. Intensity variation of single-molecule RNA FISH and simulation of 2-color HC-smFISH.
[0189] Single-molecule RNA FISH: Rapid RNA FISH was performed in HeLa cells using the TFRC mRNA probe from LGC Biosearch Technologies. The TFRC probe consists of 48 oligonucleotide probes. Each probe is 20 nt long and labeled with a Quasar 570 dye at the 5' end. An 8-well glass bottom culture chamber (Ibidi) was used for sample imaging. Cells were fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences) for 5–10 min at room temperature and then permeabilized in pure methanol (Sigma) for 5–10 min. Samples were then processed by: (1) thorough heat denaturation at 75°C for 10 min in a drying bath of 80%-formamide (Sigma) and 2 × SSC buffer; (2) in 10% formamide, 20 Add short DNA oligonucleotide probes to the hybridization buffer of % dextran sulfate (Sigma, MW>500,000) and 2×SSC, and the hybridization time is 10 minutes; (3) wash with 2×SSC buffer for 2-3 tim...
Embodiment 2
[0207] Embodiment 2. Generation of multiple dye-labeled oligonucleotide probes
[0208] Multiple dyes of the same color or different colors can be conjugated to each oligonucleotide probe for multicolor intensity encoding. For two dye labels per oligo we can use short oligos (typically 20-50nt) with end-labeling at both ends of each oligo. For three- and four-dye labeling, we will use longer oligonucleotide probes, but still ≤100 nt to accommodate multiple dye conjugations per oligonucleotide. Each long probe consists of a hybridization sequence and a readout sequence. Hybridizing sequences are 20 to 40 nt in length to native nucleotides. Reads are typically at least 15 nt in length. Dye labeling density will be controlled to be greater than 10 nt per dye (typically around 20 nt per dye) to minimize energy transfer and quenching between adjacent dyes.
[0209] Oligonucleotide-Dye Conjugates : There are various approaches to conjugating multiple different dyes to the s...
Embodiment 3
[0210] Embodiment 3. Screening for intensity encoding with labeling protocol-3 and DNA FISH
[0211] This example uses repetitive DNA sequences in mouse cells to screen for good dye pairs for labeling protocol-3. Each dye pair was tested individually in the experiment.
[0212] Probe sequences for telomeric and centromeric repeat regions in the mouse genome were ordered from IDT:
[0213] Telomere probe, Tel-Cy5-Cy3: CCCTAACCCTAACCCTAA, 5' labeled with Cy5; 3' labeled with Cy3;
[0214] Centromere probe-1, Cen-Cy5.5-Cy3: ATTCGTTGGAAACGGGA, 5' labeled with Cy5.5; 3' labeled with Cy3;
[0215] Centromere probe-2, Cen-Cy5-A532: ATTCGTTGGAAACGGGA, 5' labeled with Cy5; 3' labeled with Alexa532;
[0216] Centromere probe-3, Cen-Cy5-A546: ATTCGTTGGAAACGGGA, 5' labeled with Cy5; 3' labeled with Alexa546;
[0217] Centromeric probe-4, Cen-Cy5-Atto590: ATTCGTTGGAAACGGGA, 5' labeled with Cy5; 3' labeled with Atto590.
[0218] DNA hybridization on mouse embryonic fibroblast (MEF) ...
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