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Protocol
In situ genotyping of mitochondrial DNA using padlock probes and target-primed RCA
Keywords: In situ, mitochondrial DNA, SNP genotyping, padlock probes, RCA, FISH, hybridization, fluorescence, imaging
This method is used for detection and genotyping of mitochondrial DNA in fixed cultured cells or fresh frozen tissue sections.
This method is used for detecting and genotyping mitochondrial DNA (mtDNA) in fixed cell materials. The method was first described in 2004 (Larsson et al, 2004) and utilizes padlock probes amplified by rolling circle amplification (RCA) to detect and visualize localized mtDNA molecules in situ.
Padlock probes are linear oligonucleotides which upon recognition of a perfectly matched target sequence are converted into circular DNA molecules (Nilsson et al, 1994). The circular DNA molecules provide excellent templates for amplification by RCA. The produced RCA products consist of long single stranded DNA molecules consisting of a tandemly repeated sequence. These long molecules coil up randomly into discrete balls of DNA which can be visualized as bright fluorescent spots after hybridization of short fluorescence labeled detection probes. By utilizing differentially labeled detection probes towards different padlock probes, different sequences present in the same sample can be separated from each other. By preparing the target sequence with a combination of a restriction enzyme and an exonuclease, the RCA can be efficiently primed from the target sequence and heat denaturation of the sample is avoided, preserving sample morphology.
The output data consists of digital images. By performing image analysis relative amounts of different molecular species in the sample can be determined. A suitable software for detection and counting of RCA products in digital images is BlobFinder. This software can be freely downloaded from www.cb.uu.se/~amin/BlobFinder/.
Nilsson, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085-2088 (1994).
Larsson, C. et al. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat Methods 1, 227-232 (2004).
Main steps
- Cell and tissue fixation (5YCDT3)
- Cell permeabilisation (5YCST5)
- Restriction digestion (5YD9CQ)
- Exonucleolysis (5YDJQ6)
- Combined restriction digestion and exonucleolysis (5YDVVT)
- Padlock probe hybridization and ligation (5YERA0)
- Rolling circle amplification (5YFEHM)
- Detection probe hybridization (5YFQHB)
- Nuclear stain and mounting of slides (5YFVH6)
Instructions
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Cell and tissue fixation (5YCDT3)
Several fixation methods are compatible with this method, including ethanol fixation, formaldehyde fixation and zinc fixation. For simplicity, the most commonly used in our lab are described.
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Cultured cells
Wash cells briefly in serum free cell culture medium. Fix cells in 70% ethanol for 20 minutes in room temperature. To store cells, replace the 70% ethanol with fresh ethanol and place the cells in the fridge. Cells can be stored like this for several months as long as the ethanol is not evaporated.
Alternatively: Wash cells in serum free cell culture medium and fix in 3-4% formaldehyde for 20 minutes in room temperature. After fixation wash 2x with 1xPBS. To store cells place slides in 70% ethanol in the fridge or dehydrate slides and store them dry.
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Tissue sections
In our hands thin sections (14-16 µm) are preferred. A good tissue fixative is zinc fixative. Fix sections in zinc fixative over night in room temperature. Wash 2x in 1xTBS after fixation. Important! Do not wash in PBS when using zinc fixative since this can lead to precipitation of salts in your sample.
Alternatively, fix sections in 70% ethanol in room temperature for 30 minutes or in 3-4% formaldehyde for 30 minutes. Wash in 1xPBS after fixation.
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Cell permeabilisation (5YCST5)
This is an essential step which is very important for the end result! Thus, this step needs to be optimized to fit your cell type and the fixation method that you have chosen. Here we provide a protocol used for in situ genotyping of the cell line 143B fixed in 70% ethanol.
In situ genotyping of the cell line 143B fixed in 70% ethanol.
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Add 0.01 g pepsin to 100 ml 0.1 M HCl in a couplin jar. Make sure that the powder dissolves properly. Warm the solution to 37 °C in a water bath.
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Wash your slides briefly 2x in 1xPBS to remove fixative. Take the slides out of the PBS and put them immediately in the pepsin-HCl solution. After exactly 90 seconds remove the slides from the pepsin bath and place them immediately back into the PBS couplin jar. Discard the PBS and replace with fresh 1xPBS.
The timing of this step is very critical! If you have more than one slide, add your slides in sequence with 5-10 seconds in-between every slide rather than at the same time to be able to keep an exact timing.
Tissue sections usually require longer pepsination time than cultured cells. The interval we use for different tissues is between 2.5-5 minutes.
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Restriction digestion (5YD9CQ)
NOTE! This step can in some cases be modified and included together with step 4 in order to save time. For description of the modified protocol see step 5.
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Mix in an eppendorf tube: 0.5 U/µl restriction enzyme, 1x restriction enzyme buffer, 0.2 µg/µl BSA and H2O.
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Add the mixture to your samples and cover with a coverslip (if used). Incubate for 30-60 minutes at 37 °C (use the longer incubation time for tissue sections).
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After incubation wash briefly in wash buffer.
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Exonucleolysis (5YDJQ6)
NOTE! This step can in some cases be modified and included together with step 3 in order to save time. For description of the modified protocol see step 5.
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Mix in an eppendorf tube: 0.2 U/µl exonuclease (? exonuclease or T7 exonuclease), 1x corresponding exonuclease buffer, 0.2 µg/µl BSA, 5% glycerol and H2O.
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Add the mixture to your samples and cover with a coverslip (if used). Incubate for 30-45 minutes at 37 °C (use the longer incubation time for tissue sections).
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After incubation wash briefly in wash buffer.
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Combined restriction digestion and exonucleolysis (5YDVVT)
NOTE! This step is a modification to the standard protocol which combines the two previous steps into one. It can replace 3 and 4 in some cases in order to save time. This is possible to do when the exonuclease is functional in the restriction enzyme buffer, so that the two enzymes can be added in the same reaction.
For example, one such functional combination is a restriction enzyme working in NEB4 buffer together with either exonuclease described above. Usually we use T7 exonuclease in such a combination since the New England Biolabs version of this enzyme has NEB4 as recommended buffer. To know if your restriction enzyme is compatible to use together with the exonuclease, do a side by side comparison between the two different protocols.
To perform the modified protocol (instead of steps 3 and 4):
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Mix in an eppendorf tube: 0.5 U/µl restriction enzyme, 0.2 U/µl exonuclease, 1x restriction enzyme buffer, 0.2 µg/µl BSA and H2O.
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Add the mixture to your samples and cover with a coverslip (if used).
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Incubate for 30-60 minutes at 37 °C (use the longer incubation time for tissue sections).
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Wash in wash buffer after incubation.
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Padlock probe hybridization and ligation (5YERA0)
For ligation of the padlock probe on the target DNA there are two different ligases that can be used. T4 DNA ligase is quick and can be incubated at low temperature, but is slightly less stringent in ligation of mismatches than Ampligase. For genotyping purposes we therefore recommend Ampligase, but if the material is very delicate or if target differences are more than a single nucleotide T4 DNA ligase is a good choice.
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Ligation with T4 DNA ligase (5YF2FK)
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Mix in an eppendorf tube: 0.1 U/µl T4 DNA ligase, 100 nM of each padlock probe, 1x T4 ligase buffer, 1 mM ATP, 250 nM NaCl, 0.2 µg/µl BSA and H2O.
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Add the mixture to your samples and cover with a coverslip (if used). Incubate for 15-30 minutes at 37 °C (use the longer incubation time for tissue sections).
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Wash the slides in 2xSSC containing 0.05% Tween 20 at 37 °C for 5 minutes. Then rinse the slides in wash buffer and dehydrate by putting the slides in 70%, 85% and 99% ethanol for 3 minutes each.
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Ligation with Ampligase (5YF6DX)
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Mix in an eppendorf tube: 0.25 U/µl Ampligase, 100 nM of each padlock probe, 1x Ampligase buffer, 125 mM KCl, 0.2 µg/µl BSA, 5% glycerol and H2O.
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Add the mixture to your samples and cover with a coverslip (if used). Incubate for 30-90 minutes at 55 °C (use longer incubation time for tissue sections).
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Wash slides in 2xSSC containing 0.05% Tween 20 and 10 mM EDTA at 37 °C for 5 minutes. Then rinse the slides in wash buffer and dehydrate by putting the slides in 70%, 85% and 100% ethanol for 3 minutes each. Air dry by putting the slides in an upright position. It is important that all ethanol is completely evaporated before proceeding.
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Rolling circle amplification (5YFEHM)
The efficiency of the RCA reaction is dramatically improved by using the “bubble-method” to apply the reaction mix. We therefore do not recommend using a coverslip for this step. The reaction volume should also be increased with 50-100% compared to the previous steps to experience the best result.
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In an eppendorf tube mix: 1 U/µl Phi29 DNA polymerase, 1x Phi29 buffer, 0.25 mM dNTP, 0.2 µg/µl BSA, 5% glycerol and H2O.
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Add the mixture to your samples. Incubate for 60-120 minutes at 37 °C (use longer incubation time for tissue sections).
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Wash briefly in wash buffer after incubation.
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Detection probe hybridization (5YFQHB)
A stock of 4xSSC and 40% formamide can be prepared in advance and stored in the freezer for a prolonged time period.
NOTE 2: Since this step does not involve any enzymatic action, in order to save reagents the reaction volume can be kept slightly lower than before if using the “bubble-approach”.-
Mix in an eppendorf tube: 250 nM of each detection probe, 2xSSC, 20% formamide and H2O.
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Add the mixture to your samples. Incubate for 15-30 minutes at 37 °C (use the longer incubation time for tissue sections).
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Wash in wash buffer for >1 minute. When genotyping tissue, wash at least two times to remove nonspecific fluorophore binding. Dehydrate using a series of 70%, 85% and 100% ethanol, 3 minutes in each solution. Air dry the slides standing in an upright position.
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Nuclear stain and mounting of slides (5YFVH6)
After this step, the slides are ready for fluorescence microscopy and imaging of the results. Mounted slides can be stored for a long time in the dark in room temperature or in the fridge without fluorescence bleaching completely. Results should be recorded properly within a few days though.
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Cultured cells
Mount your slides in Vectashield mounting medium containing 100 ng/ml of DAPI to counter-stain the cell nuclei. Do not use too much solution since this disturbs imaging, 20 µl to a 22x55 mm cover slip is enough for one slide if using standard objective slides. After application of DAPI and Vectashield, leave the slides in darkness for a few hours before imaging to allow the nuclei to be uniformly stained.
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Tissue sections
The same solution as above can be used, but in some cases the DAPI also gives a diffuse stain of the cytoplasm which sometimes can be avoided by instead performing a separate nuclear staining step with Hoechst. Dilute the Hoechst to an appropriate concentration (e.g. 1 µl to 500 µl) in 1xTBS. Apply the solution to your sample and incubate for 60 minutes in room temperature in the dark to protect the slides from bleaching. The optimal Hoechst concentration to be used must be determined experimentally for your tissue. Wash 3x in 1xTBS after incubation and dehydrate as described in step 7. When slides are dry, apply Vectashield and cover with a cover slip.
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