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Sep 2020

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Identification and Monitoring of Nucleotide Repeat Expansions Using Southern Blotting in Drosophila Models of C9orf72 Motor Neuron Disease and Frontotemporal Dementia
应用Southern Blotting鉴定和监测C9orf72运动神经元疾病和额颞叶痴呆模型的核苷酸重复扩增    

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Abstract

Repeat expansion diseases, including fragile X syndrome, Huntington’s disease, and C9orf72-related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide sequences. These expansions are highly repetitive and often hundreds to thousands of repeats in length, making accurate identification and determination of repeat length via PCR or sequencing challenging. Here we describe a protocol for monitoring repeat length in Drosophila models carrying 1,000 repeat C9orf72-related dipeptide repeat transgenes using Southern blotting. This protocol has been used regularly to check the length of these lines for over 100 generations with robust and repeatable results and can be implemented for monitoring any repeat expansion in Drosophila.

Keywords: Southern blotting (DNA印迹术), Drosophila (果蝇), C9orf72 (C9orf72), Dipeptide repeats (二肽重复), Repeat expansions (重复扩展)

Background

Repeat expansion diseases are a class of genetic disorders associated with the expansion of tandem repeat DNA sequences. The polymorphic nature and inherent instability of tandem repeats renders them prone to mutation, and as such they are one of the most abundant causes of variation in the human genome (Gymrek, 2017). The length and location of repeat sequences can vary, ranging from a single nucleotide to several nucleotides per repeat, situated within both coding and non-coding regions. There have been over 50 repeat expansion disorders identified, most of which primarily affect the central nervous system (Depienne and Mandel, 2021).


Repeat expansions in coding regions tend to be trinucleotide repeats, resulting in abnormally long repetitive amino acid sequences within proteins. In contrast, expansions in the UTRs or introns of genes are more varied in terms of sequence and how they confer toxicity. The repeat sequence itself can vary in length, the longest reported being a dodecamer in the 5’UTR of the CSTB (cystatin B) gene, which results in progressive myoclonic epilepsy type 1 (EPM1) (Lalioti et al., 1997). Moreover, whilst GC-rich repeat expansions in the 5’UTR tend to cause loss of function toxicity via epigenetic mechanisms such as persistent DNA hypermethylation, expansions situated within 3’UTRs and introns more frequently lead to a gain of function toxicity through RNA toxicity or polypeptide synthesis via repeat associated non-AUG (RAN) translation. An example of a repeat expansion that both reduces gene expression and elicits RNA and polypeptide repeat toxicity is the intronic GGGGCC expansion in the C9orf72 gene, the leading genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND) (Renton et al., 2011; DeJesus-Hernandez et al., 2011). Research using genetic models, including fruit flies, has determined that, whilst the C9orf72 expansion does reduce gene expression and cause haploinsufficiency, the main driver of toxicity in this case is the production of dipeptide repeat proteins (DPRs) from non-canonical RAN translation of the repeat itself. As translation occurs in all frames and from both sense and antisense RNA, five different DPRs are produced: glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), alanine-proline (AP), and proline-arginine (PR) (Mori et al., 2013). These DPRs have been shown to aggregate in patient brains and spinal cord and are toxic in multiple model systems (May et al., 2014; Mizielinska et al., 2014; Zhang et al., 2014; Moens et al., 2019; West et al., 2020).


To further our understanding of expansion disorders and the mechanisms underpinning them, a range of transgenic model systems have been developed. For each expansion disorder, there is usually a consensus as to what constitutes a pathogenic repeat length. For example, the intronic hexanucleotide GGGGCC repeat in the C9orf72 gene is normally under 30 repeats in healthy individuals, whereas in those who develop disease it comprises over 500 and often thousands of repeats (Renton et al., 2011; DeJesus-Hernandez et al., 2011). In West et al. (2020), we developed the first Drosophila models expressing each DPR, individually and without repeat RNA, at over 1,000 repeats. To do this, alternative coding sequences were designed to produce the DPRs independently of the GGGGCC repeat (Callister et al., 2016). However, the transgene is still highly repetitive, GC-rich, and over 6000 bp in length, making accurate genotyping of repeat length via PCR or sequencing challenging. Furthermore, tandem repeats are increasingly unstable in a length dependent manner (Depienne and Mandel, 2021), and repetitive sequences introduced into bacteria and animal models are known to retract or be excised completely through generations (Bichara et al., 2006; Ryan et al., 2019). Therefore, it is important to be able to determine the length of repeat sequences in transgenic models and monitor them to check for retractions. The most common method, and widely considered gold standard, is Southern blotting.


Southern blotting is a highly sensitive technique used to detect specific DNA sequences in a blood or tissue sample. It involves the digestion of DNA with site-specific restriction endonucleases to isolate the genomic region of interest, followed by the separation of DNA fragments by size using gel electrophoresis and transfer onto a porous, positively charged nylon or nitrocellulose membrane via capillary action. The DNA of interest is detected using molecular hybridisation with specific nucleic acid probes, which are tagged with either radioisotopes or non-isotopic reagents (e.g., to facilitate chemiluminescent detection) (Southern, 2006). Whilst Southern blotting has been largely replaced by modern sequencing techniques and is no longer widely used, it remains an essential technique for researchers working with long and repetitive DNA sequences. Repeat sequences have always proved technically challenging for sequence alignment and assembly, and this is exacerbated when the repeat exceeds the sequencing fragment length (typically 350–500 bp). Moreover, traditional PCR amplification libraries have an inherent GC-bias, which can result in under-representation of reads from repeat expansions, a large proportion of which have 100% GC content (Treangen and Salzberg, 2012). These inaccuracies can lead to under-estimation of repeat sizes and mischaracterisation of repeat expansions (Rajan-Babu et al., 2021). It has been reported that PCR-based techniques used to detect the C9orf72 expansion in FTD/MND patients are unreliable, with both a high false-positive and false-negative rate (Akimoto et al., 2014). Therefore, whilst there are promising developments in long-read sequencing analysis, at present Southern blotting is considered the gold standard for genotyping GC-rich repeat expansions. Here we have developed and refined a protocol for the Southern blotting of repeat sequences in Drosophila. We optimized this protocol using Drosophila C9orf72 DPR models published in West et al. (2020), but it can be applied to other repeat expansions with alterations to the procedure required only for the oligonucleotide probes and restriction endonucleases.

Materials and Reagents

  1. Whatman® 3MM filter paper (Whatman®, GE Healthcare, catalog number: WHA30306185)

  2. Extra thick blotting paper (2.5 mm) (ThermoFisher Scientific, Thermo ScientificTM, catalog number 88605)

  3. AmershamTM HybondTM-N 0.45 µm pore, neutral nylon membrane (GE Amersham, catalog number: RPN203N)

  4. 15 mL Falcon tubes (e.g., Corning® 15 mL centrifuge tubes, Sigma-Aldrich, Merck, catalog number: CLS430790)

  5. 50 mL Falcon tubes (e.g., Corning® 50 mL centrifuge tubes, Sigma-Aldrich, Merck, catalog number: CLS430829)

  6. 1.5 mL microfuge tubes (e.g., Eppendorf® Safe-Lock microcentrifuge tubes, Sigma-Aldrich, Merck, catalog number: T9661)

  7. Plastic film (e.g., clingfilm) (Scientific Laboratory Supplies, catalog number: FIL1003)

  8. Acetate sheets (Rapid Electronics Ltd, Diacel, catalog number: 34-0303)

  9. Petri dish or weighing boat (for picking heads as shown in Figure 1, e.g., small square weighing boat, Sigma-Aldrich, Merck, catalog number: Z708542-500EA)

  10. Phenol-chloroform-isoamylalcohol (25:24:1) saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma-Aldrich, Merck, catalog number: P3803)

  11. Chloroform (analytical reagent grade) (Sigma-Aldrich, Merck, catalog number: 366927)

  12. Ethanol (for molecular biology) (Sigma-Aldrich, Merck, catalog number: 51976)

  13. Ethidium Bromide, Molecular Biology-grade Aqueous Solution (500 mg/mL) (Sigma-Aldrich, Merck, catalog number: E1385)

  14. Concentrated (36.5–38.0%) hydrochloric acid (HCl) (Sigma-Aldrich, Merck, catalog number: H1758)

  15. Sodium chloride (NaCl) (analytical reagent grade) (Sigma-Aldrich, Merck, catalog number: S9888)

  16. Sodium hydroxide (NaOH) pellets (anhydrous) (Sigma-Aldrich, Merck, catalog number: S5881)

  17. Sodium citrate (Sodium citrate tribasic dihydrate) (Sigma-Aldrich, Merck, catalog number: C8532)

  18. TRIS hydrochloride (TRIS HCl) pH 8.0 (Sigma-Aldrich, Merck, catalog number: 10812846001)

  19. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, Merck, catalog number: L3771)

  20. Ethylenediaminetetraacetic acid (EDTA) (Merck, Sigma-Aldrich, catalog number: E9884)

  21. Drosophila stocks to be tested (50–60 flies per genotype (see Notes for applying this to other tissues/protocols), including a negative wild-type control such as Canton S or Oregon R). Wild-type flies can be acquired from Bloomington Drosophila Stock Centre (BDSC, Indiana University) (Canton S: BDSC 9515, Oregon R BDSC 2376) or other stock centre. Transgenic stocks used in our example were generated by microinjection of pUAST-DPR-EGFP into VK00005 embryos by Cambridge Microinjection Facility [see West et al. (2020); available upon request].

  22. Plasmids used to generate the transgenic fly lines, for use as positive controls [e.g., pUAST-DPR(1000)], see West et al., 2020, available upon request

  23. Dry ice

  24. Nuclease free water (molecular biology grade) (Sigma-Aldrich, Merck, catalog number: W4502)

  25. Proteinase K (ThermoFisher Scientific, Thermo ScientificTM, catalog number: EO0491), divide into 1 mL aliquots and store at -20°C

  26. Restriction enzymes to excise DNA of interest from the genome with appropriate buffer; for our example, we used Dde1 (New England Biolabs, catalog number: R0175) and NlaIII (New England Biolabs, catalog number: R0125), along with CutSmart® buffer (New England Biolabs, catalog number: B7204).

  27. TBE Buffer (Tris-borate-EDTA) (10×) (ThermoFisher Scientific, Thermo ScientificTM, catalog number: B52)

  28. Agarose for molecular biology (Sigma-Aldrich, Merck, catalog number: A9539)

  29. 6× loading dye containing bromophenol blue (New England Biolabs, catalog number: B7021S)

  30. DNA Molecular Weight Marker II, DIG-labeled (Roche, Merck, catalog number: 11218590910)

  31. 1 kb Plus DNA Ladder (NEB, catalog number: N3200)

  32. Probes specific to target sequence [The probes used in West et al., (2020), synthesised by Eurofins UK, are listed in the notes section of this protocol].

  33. DIG easy hyb (Roche, Merck, catalog number: 11603558001)

  34. Salmon sperm DNA (Agilent Technologies, catalog number: 201190), divide into aliquots of 300 µL and 150 µL and store at -20°C.

  35. DIG Wash and Block Buffer Set (Roche, Merck, catalog number: 15857 62001), aliquot DIG block (e.g., 50 mL aliquots) and store at -20°C for long term (one aliquot can be stored for up to 3 months at 4°C).

  36. Anti-Digoxigenin-AP, Fab fragments (Roche, Merck, catalog number: 11093274910, RRID:AB_2734716), store at 4°C

  37. Ultrapure water (for example from Milli-Q® Purification System)

  38. CDP-Star® chemiluminescent substrate (Roche, Merck, catalog number: CAS160081-62-9), store at 4°C

  39. 70% Ethanol (see Recipes)

  40. Depurination solution (see Recipes)

  41. Gel denaturing solution (see Recipes)

  42. Gel neutralizing solution (see Recipes)

  43. 20× SSC stock (see Recipes)

  44. 10% SDS Solution (see Recipes)

  45. 2× SSC, 0.1% SDS (see Recipes)

  46. 0.5× SSC, 0.1% SDS (see Recipes)

  47. 0.1× SSC, 0.1% SDS (see Recipes)

  48. Positive controls (see Recipes)

  49. Genomic extraction buffer (see Recipes)

  50. 1× TBE (see Recipes)

  51. Ladder mix (see Recipes)

  52. 1× DIG block (see Recipes)

  53. 1× maleic acid buffer (see Recipes)

  54. 1× maleic acid wash buffer (see Recipes)

  55. 1× detection buffer (see Recipes)

Equipment

  1. Pipettes and tips (P1000, P200, P20, P10, and P2)

  2. Fine paintbrush (e.g., RS PRO Thin 6.4 mm paintbrush, RS Components Ltd, catalog number: 2379190)

  3. Vortex mixer (e.g., Vortex-Genie® 2 mixer, Sigma-Aldrich, Merck, catalog number: Z258415)

  4. Fume hood

  5. pH meter

  6. Standard 700W–1000W microwave

  7. Standard laboratory microfuge (e.g., Sigma 1-14 Microfuge, Sciquip, catalog number: 90616)

  8. Tabletop mini microfuge (e.g., SciSpin MINI Microfuge, Sciquip, catalog number: SS-6050)

  9. Wheel tube rotator (e.g., Cole-ParmerTM Stuart TM Rotator Disk, Fisher Scientific, catalog number: 11496548)

  10. PowerPacTM Basic Power Supply (Bio-Rad, catalog number: 1645050)

  11. Sub-Cell GT Electrophoresis Cell with 20-well combs and gel casting tray (15 × 15 cm) (Bio-Rad, catalog number: 1704402)

  12. Clean plastic trays and sandwich boxes for incubating the membrane and gel, and assembling the Southern blot (Figure 2)

  13. Large sandwich container (e.g., 250 × 150 mm) to use as the basin for Southern blotting apparatus (Figure 2)

  14. Chemiluminescence and fluorescence imaging system (e.g., G:box imaging unit, Syngene)

  15. HB pencil

  16. Scissors/guillotine for cutting filter paper and membrane

  17. UV Transilluminator (e.g., dual wavelength (302/365) 8W transilluminator LM-20, VWR, catalog number: 732-4388)

  18. Standard boiling water bath (e.g., VWR, SBB Aqua 5 plus, catalog number: 462-0171) or hot block capable of reaching >100°C

  19. Standard hybridisation oven (e.g., HB-1000 Hybridiser, VWR, catalog number: 732-4300)

  20. Glass hybridisation bottles to fit in hybridisation oven (e.g., 35 × 150 mm hybridisation bottles, VWR, catalog number: 732-4350)

  21. Flat edged forceps (e.g., S MurrayTM Stainless Steel Forceps L325/01, Fisher Scientific, catalog number: 12342158)

  22. Rocker (e.g., Cole-ParmerTM StuartTM See-Saw Rocker, catalog number: 10470655)

Software

  1. Imaging software for imaging the blot, for example: GeneSys and GeneTools (Syngene, https://www.syngene.com/support/software-downloads/)

Procedure

  1. Prepare Reagents

    Before starting the protocol ensure that you have prepared the positive controls, ladder mixture, and all buffers and solutions described in the recipes section. All recipes, excluding 1× DIG block and 1× detection buffer, which must be made up from 10× on the day of use, can be made in advance and stored for at least 1 year. Proteinase K must be added fresh to the genomic extraction buffer immediately before use.


  2. DNA extraction from Drosophila heads (whole flies or other tissues could be used, but we find that using heads gives a better signal due to the lack of contaminants from, for example, gut contents).

    DAY 1

    1. Collect 50–60 flies (yielding ~25–30 µg genomic DNA), per genotype, in 15 mL Falcon tubes and place on dry ice. In addition to test samples, collect wild-type flies, for example Canton S or Oregon R, to use as a negative control.

    2. To separate the fly heads, place 3–4 pieces of dry ice into a 50 mL Falcon tube and place the 15 mL Falcon tube containing the flies inside the 50 mL Falcon tube (see Figure 1A). Do not put the lid back on the 50 mL Falcon tube. Vortex at top speed until heads detach (~30 s) (see Video 1). Keep on dry ice at all times when not vortexing.


      Video 1. Detaching Drosophila heads by vortexing.


    3. Pick heads and transfer them to a 1.5 mL microcentrifuge tube on dry ice. Heads can be picked manually using whichever method is easiest. Our recommended method is to tip the decapitated flies onto a petri dish lined with filter paper sitting in a large tray or weighing boat containing dry ice (see Figure 1B). Use a fine paintbrush to pick up the heads (see Figure 1C) and transfer them to a 1.5 mL tube on dry ice. This procedure is easier when everything is kept as cold as possible.

    4. Defrost proteinase K and add to genomic extraction buffer (see Recipes).

    5. Homogenize the heads by “squishing” with a 1–200 µL pipette tip filled with genomic extraction buffer (1 µL per head) without expelling the liquid (sufficient buffer will be expelled during the process to efficiently homogenize the heads without them floating around) (~10 s). Then expel the remaining extraction buffer and put in the hybridisation oven (or another incubator) and set to 57°C. Incubate overnight.



      Figure 1. Example apparatus for collecting Drosophila heads.

      A. Apparatus for detaching Drosophila heads using a vortex mixer (see also Video 1). B. Apparatus for picking heads using a paintbrush. C. Heads detached from the rest of the fly.


    DAY 2

    1. The following day, in a fume hood, add one volume (equivalent to the volume used in step 5) phenol-chloroform-isoamylalcohol (25:24:1) and incubate at room temperature for 15 min with rotation (using a wheel tube rotator, for example).

    2. Centrifuge for 5 min at ~13,000 × g.

    3. Transfer the aqueous (top) layer to a new tube.

    4. Add one volume of 100% chloroform to the recovered aqueous phase from step 8, incubate at room temperature for 15 min with rotation (using a wheel tube rotator, for example), and repeat steps 7–8.

    5. Recover DNA with standard ethanol precipitation:

      1. Add 1 mL of 100% ethanol, invert for one minute, and place at -20°C overnight.


      DAY 3
      1. The following day, spin at 13,000 × g for 10 min and discard supernatant.

      2. Wash the pellet with 800 µL of 70% ethanol.

      3. Centrifuge at 13,000 × g for 5 min and discard supernatant.

      4. Leave the pellet to air dry for 15–30 min at room temperature and then resuspend in 100 µL of nuclease free water.


    6. Allow to dissolve overnight at 4°C or 1 h at room temperature. At this point DNA can be frozen and stored at -20°C until ready to move on to section C.


  3. DNA digests

    DAY 4

    Note: Restriction enzymes were chosen to give efficient digestion of genomic DNA without cutting the target sequence. For our example, DdeI and NlaII were chosen as they efficiently cut the vector used to insert our construct into the fly genome, without cutting the DPR sequence [see Figure 2 and West et al., (2020)]. Whilst the approximate cutting frequency of each enzyme within the genome should be considered (for example, NlaIII cuts a 4 bp site so has a cutting frequency of ~44), the most critical factor is that the enzyme does not cut your target sequence. The enzymes used in our example are listed in Materials and Reagents and detailed in Table 1. Digests are carried out in a large volume of 500 µL to allow complete digestion of genomic DNA. All the DNA from a 50–60 head extraction is used (~25–30 µg DNA).



    Figure 2. Plasmid map indicating cut sites of restriction enzymes NIaIII and Dde1.

    pUASt-DPR1000-GFP plasmid map showing mini-white element, DPR sequence, and EGFP tag. Restriction sites for NlalII and Dde1 are indicated (made using Snapgene Viewer).


    1. Set hybridisation oven (or other incubator) to required temperature for your restriction enzymes.

    2. Prepare reaction mixtures (example shown in Table 1).

    3. Mix by pipetting and settle contents by centrifugation in a tabletop microfuge (10 s at 2,680 × g).

    4. Incubate in optimal conditions for your chosen enzymes (in our example, overnight at 37°C).


    DAY 5

    1. Recover DNA with standard ethanol precipitation:

      1. Add 1 mL of 100% ethanol, invert for one minute, and place at -20°C overnight.

    DAY 6

    1. The following day, spin at 13,000 × g for 10 min and discard supernatant.

    2. Wash the pellet with 800 µL of 70% ethanol.

    3. Centrifuge at 13,000 × g for 5 min and discard supernatant.

    4. Leave the pellet to air dry for 15-30 min at room temperature.

  1. Resuspend the pellet in 17 µL of nuclease free water, mix well, and centrifuge briefly in a tabletop microfuge (10 s at 2,680 × g) to ensure all the DNA is at the bottom of the tube.

  2. Allow the DNA to dissolve for 2–3 days at 4°C before running the Southern blot.


    Table 1. DNA digestion mixture example

    Volume (µL)
    DNA 100
    Cutsmart buffer (10×) 50
    NIaIII 2
    Dde1 2
    Nuclease free water 346
    Total Volume 500


  1. Blotting samples

    DAY 7 (2–3 days after Day 6)

    1. Make an agarose gel:

      1. Mix 130 mL 1× TBE with an appropriate mass of agarose to make a gel of the percentage required to resolve your construct (see Table 2 for a guide to gel percentages and resolving capability based on product size; in our example, we use 1% for a 6 kb product).

      2. Boil the TBE agarose mix in a microwave.

      3. Add 0.5 µg/mL ethidium bromide and swirl to mix.

      4. Pour into a 15 × 15 cm casting tray using a 20 well comb and leave to set.

    2. Prepare samples for electrophoresis:

      1. Defrost the ladder mix and 10 ng/µL positive control stock.

      2. Add 0.5 µL of positive control to 9.5 µL of nuclease free water and 2 µL of 6× loading dye to give a total volume of 12 µL.

      3. For test samples and negative controls prepared in sections B and C, add 3 µL of 6× loading dye to give a total volume of 20 µL, which is the maximum that can be loaded into the wells.

    3. Prepare to run the gel:

      1. Place the gel into the tank, fill the tank with 1× TBE, and remove the comb.

      2. Load 7 µL of ladder mix, 12 µL of positive control, and 20 µL of samples to individual wells. It can be beneficial to leave blank wells either side of the positive control to avoid false positives resulting from spill over between wells.

    4. Electrophorese DNA samples at 100 V for long enough to give good separation between ladder bands around target DNA (for a 6 kb product this is approximately 2.5 h).


      Table 2. Guide for % agarose in TBE gel based on expected product size

      Agarose % (w/v) Resolution
      0.50% 1,000–30,000 bp
      0.70% 800–12,000 bp
      1.00% 500–10,000 bp
      1.20% 400–7,000 bp
      1.50% 200–3,000 bp
      2.00% 50–2,000 bp


    5. Image the gel using a gel documentation system such as a G-box. This is useful to ascertain whether the DNA has digested fully (digests should appear as smears down the lanes) and to refer to later if the signal is weak in certain lanes when imaging the final blot. It is also helpful to use the UV transilluminator to help trim the blot.

    6. The gel can be trimmed smaller, using the ladder as a guide to ensure DNA of interest is not removed, if desired. Cut one corner of the gel to help identifying the gel orientation and transfer the gel into a clean plastic container.

    7. Depurinate the gel: add enough depurination solution to cover the gel and shake slowly at room temperature on a see-saw rocker or orbital shaker, until the bromophenol blue in the loading dye turns yellow or for no longer than 10 min.

    8. Pour off the depurination solution and briefly rinse the gel in distilled water before the next step.

    9. Denature the DNA in the gel: add enough gel denaturing solution to cover the gel and incubate at room temperature for 30 min with shaking.

    10. Neutralize the gel: pour off denaturing solution and replace with the same volume of neutralising solution. Incubate at room temperature with shaking for 30 min.

    11. Pour off the neutralising solution and replace with the same volume of 20× SSC. Incubate for 20 min at room temperature with shaking before blotting. This helps to equilibrate the gel and remove background.

    12. Meanwhile, prepare 3 sheets of Whatman 3 MM paper, a sheet of Hybond-N 0.45 µm pore nylon membrane, and 15 sheets of extra thick blotting paper, all cut to the same size as the gel. Also cut 3 strips of 3MM paper slightly wider than the gel, and longer than the length of the support to use as wicks (see Figure 2).

    13. At this point, you can cut a corner off the membrane to allow the blot to be orientated after hybridisation, and put a pencil mark on the side that will face the gel to identify the DNA side.

    14. Clean flat edged forceps using 70% ethanol to remove alkaline phosphatase before using them to handle the nylon membrane (alkaline phosphatase reacts with chemiluminescent substrates used in the detection process).

    15. Assemble the transfer as shown in Figure 3. For this step, 20× SSC can be reused.

      1. Place a small plastic container (the bridge/support) upside down inside a larger container to form the basis of your transfer apparatus.

      2. Place the longer strips of filter paper over the bridge/support to act wicks, wicking the 20× SSC in the buffer reservoir up into the gel. Pour a little 20× SSC in to wet the wicks.

      3. Place the gel onto the wick covered bridge/support (we typically place our gels well side up; however, this is not important as transfer can occur in either orientation), followed by the nylon membrane (pencil mark down facing the gel and with the cut corner of the membrane matching the cut corner of the gel, to aid with orientation). Be careful to remove all bubbles (gently with gloved fingers) between the gel and membrane.

      4. Follow with the sheets of 3MM paper and extra thick blotting paper. To avoid short circuiting the transfer, make sure the buffer cannot bypass the gel and membrane as it is drawn up to the thick blotting paper, by ensuring the wicks do not touch the blotting paper above the gel and membrane.

      5. Place a weight (~100–300 g, for example a small plastic container filled with enough water to cover the base but not spill) on top of the paper and leave the gel overnight at room temperature to transfer. Ensure the wicks are immersed and the weight is level. If the room temperature is warm and you have problems with evaporation of the 20× SSC, cover the apparatus with plastic film to prevent this.



    Figure 3. Southern blotting assembly.

    A. Schematic. B. Photo. Place a small plastic container upside down in a larger plastic container to form a bridge. Place the longer strips of filter paper over the container to allow the 20× SSC in the buffer reservoir to wick up into the gel. Pour a little 20× SSC in to wet the wicks. Place gel on the bridge, followed by nylon membrane, being careful to remove all bubbles between the gel and membrane. Carefully place the sheets of 3MM paper and extra thick blotting paper on top. Place a weight (for example, a plastic container filled with water) on top of the paper and leave the gel overnight to transfer. Ensure that the wicks are covered and the weight level. If you are worried about evaporation you can cover in plastic film.


  2. Hybridisation

    DAY 8

    1. The following day, set the boiling water bath/hot block to 100°C and the hybridisation oven to 42°C.

    2. Place 30 mL DIG easy hyb at 42°C to prewarm. Defrost 300 µL salmon sperm DNA.

    3. Disassemble the blot (keep 20× SSC for reuse) and gently wash the membrane in ~20 mL of 2× SSC.

    4. Immobilise the DNA on the membrane using UV (302 nm, “Hi” intensity setting): place plastic film over the surface of the UV transilluminator, place the membrane DNA side down, turn on the illuminator, and leave to fix for 180 s (Figure 4). If using a UV crosslinker such as a Stratalinker, use a standard autocrosslinking setting at 1,200 µJ.


      Figure 4.UV Fixing the membrane - schematic.

      Ensure you are wearing correct personal protective equipment for using UV. Place plastic film over the surface of the UV transilluminator and the membrane DNA side down on top of the plastic film. Turn on the UV and leave to fix for 180 s.

    5. Transfer to a hybridisation bottle (DNA side facing inwards; ensure the DNA side of the membrane is fully exposed and not folded over on itself) and add ~50 mL of 2× SSC to stop the membrane drying out*.

      Note: * The membrane can be left in 2× SSC overnight, but if it is to be left longer before hybridisation, rinse the membrane twice for 10 min in ultrapure water, then gently air dry the membrane and store between sheets of 3MM paper for later hybridisation. This washing is crucial as it removes any traces of salt that may dry on the membrane and result in background signal.

    6. Prepare pre-hybridisation solution: boil 300 µL of salmon sperm DNA stock to denature (this is 3,000 µg DNA) for 10 min and then place on ice to prevent re-annealing; add to 30 mL of prewarmed DIG easy hyb.

    7. Pour off 2× SSC if using/place dry membrane in hybridisation bottle DNA side inwards and add the pre-hybridisation solution. Pre-hybridise with rotation for 4 h at 42°C.

    8. Prepare hybridisation solution: defrost 150 µL (1,500 µg) salmon sperm DNA, prewarm 15 mL DIG easy hyb to 42°C; denature salmon sperm DNA as in step 6 and add this and 7.5 µL 10 ng/μL oligo probe stock to DIG easy hyb, giving a final salmon sperm DNA concentration of 100 µg/µL and probe concentration of 0.005 ng/µL.

    9. Pour off pre-hybridisation solution and replace with hybridisation solution. Hybridise overnight at 42°C with rotation.


  3. Detection

    DAY 9

    1. The following day, remove the hybridisation bottles from the oven and set to 65°C.

    2. Put 2 × 50 mL aliquots of 2× SSC; 0.1% SDS and 1 × 50 mL 0.5× SSC; 0.1% SDS (and 50 mL 0.1× SSC; 0.1% SDS if using for optional extra final wash (step 7)) at 65 °C to prewarm. Defrost 10× DIG block and prepare 1× block (see Recipes).

    3. Pour off the probe, and rinse the membrane in the hybridisation bottle with approximately 50 mL 2× SSC; 0.1% SDS.

    4. Add 50 mL of prewarmed 2× SSC; 0.1% SDS to the membrane in the bottle and wash for 15 min at 65°C with rotation.

    5. Replace with 50 mL of fresh prewarmed 2× SSC; 0.1% SDS and wash for 15 min.

    6. Replace with 50 mL of prewarmed 0.5× SSC; 0.1% SDS and wash for 15 min.

    7. Optional extra wash (if background is high and your signal is weak): replace with 50 mL of prewarmed 0.1× SSC; 0.1% SDS and wash for 15 min.

    8. Pour off the last of the hybridisation wash solutions and rinse the bottle out with ~50 mL 1× maleic acid buffer (shake vigorously before use).

    9. Transfer the membrane to a clean dish and incubate the membrane at room temperature with shaking for 2 min in maleic acid buffer.

    10. Prepare the antibody solution by centrifuging the anti-digoxigenin-AP antibody for 15 min at 13,000 × g, to remove antibody complexes. Pipette antibody from the top to make 20 mL 1:20,000 anti-DIG in 1× DIG block.

    11. Pour off the maleic acid buffer and incubate the membrane for 30 min at room temperature in 50 mL 1× DIG block with shaking.

    12. Pour off the blocking solution and incubate the membrane at room temperature for 30 min in 20 mL antibody solution with shaking.

    13. Wash the membrane twice (2 × 15 min, at room temperature, and with shaking) in approximately 50 mL 1× maleic acid wash buffer.

    14. Prepare 1× detection buffer (see Recipes). Equilibrate the membrane for 5 min in 20 mL 1× detection buffer. Leave in detection buffer at room temperature until ready to image.

    15. Place the membrane DNA side up on a sheet of clean acetate. Add 1–2 mL (to cover the membrane) CDP-Star® chemiluminescent substrate, distributed evenly over membrane. Place another sheet of clean acetate over the top, sandwiching the membrane in between. Be careful to eliminate bubbles as you place the second sheet of acetate over the membrane. Proceed to imaging immediately.

    16. Image the membrane using the G-Box or equivalent system initially for 1 min to check the blot has worked (should see the ladder) and then again at the highest quality.

    17. For enhanced detection of weak bands, highlight the area of interest and set the auto exposure time based on these bands. Image again. This may take up to 30 min using the G-Box system listed here. See Figure 5 for an example final Southern blot image.



    Figure 5. An example Southern blot used to length check UAS-GA1000-EGFP fly lines.

    A 6 kb band corresponds to 1000 dipeptide repeats. Lanes in order: positive control (+, DNA from wild-type flies spiked with 1000 repeat linearized plasmid DNA), ladder, negative control (-, DNA from wild-type flies), and two independent GA1000 lines (1 and 2). This blot was imaged for 30 min.

Notes

We found that extraction from 50-60 heads was optimal (25–30 µg genomic DNA), but if using other genomic DNA extraction protocols, or are extracting DNA from other tissues, this may require optimizing. We recommend a minimum of 5 µg genomic DNA should be used.

Different DIG ladders can be used if the DNA of interest is of a shorter/longer repeat length.

If the target DNA is different to the examples given, optimisation of the following will be required:

  1. Gel percentage and running time

  2. Amounts of positive control loaded, based on the intensity relative to samples


    For further optimisation of background reduction, we recommend adding an additional stringent wash after hybridisation, as noted in procedures section F step 7. A third 15 min wash in maleic acid wash buffer after antibody incubation (section F step 12) may also help reduce high background levels.

    For Southern blotting of Drosophila generated in West et al. (2020) the following probes were used:

    GA probe: DIG-GGCAGGAGCTGGAGCTGGCGCAGGAGCTGGTGCTGGG-DIG

    GR probe: DIG-AGGCAGAGGTCGTGGGAGAGGCAGGGGTCGCGGACGTGGA-DIG

    AP probe: DIG-AGCACCAGCACCGGCGCCAGCTCCAGCACCAGCACCC-DIG

    PR probe: DIG-AGACCCCGTCCTCGTCCTCGTCCAAGACCAAGGCCGAGGC-DIG


    The probe design will vary depending on your target sequence. Generally, oligonucleotide probes should be between 18 and 50 bases to balance yield and specificity. An optimal GC content is between 40–50%, and computational analysis is recommended to optimize specificity.

Recipes

Unless otherwise stated, buffers and solutions should be kept at room temperature for up to one year.

  1. Genomic extraction buffer

    25 mM NaCl, 10 mM Tris-HCl pH 8.2, 1 mM EDTA, and Proteinase K 200 μg/mL

    Store buffer without Proteinase K at room temperature; proteinase K should be stored at –20°C in 1 mL aliquots and added to the appropriate volume of buffer immediately before use.

  2. Positive controls

    Wild-type Drosophila DNA spiked with 157 ng of linearized plasmid containing target repeat sequence per 1 µg genomic DNA, to produce the equivalent of haploid DNA. Keep a stock of 10 ng/µL at -20°C.

  3. Ladder mix

    1:1 DIG-labelled DNA marker II: 1 kb plus DNA ladder, plus 1× loading dye (the visible ladder is used as a reference for the size of the DNA fragments when the gel is trimmed after electrophoresis). Keep a stock of this mixture at -20°C.

  4. 70% Ethanol (make up in nuclease free water)

    Make up in nuclease free water

  5. 1× TBE

    Make up in ultrapure water

  6. Depurination solution

    0.25 M HCl

  7. Gel denaturing solution

    0.6 M NaCl, 0.2 N NaOH

    17.53 g of NaCl, 4 g NaOH in 500 mL water

  8. Gel neutralizing solution

    1.5 M NaCl, 0.5 M Tris-HCl pH 8.0

    43.8 g NaCl, 30.30 g Tris, in 500 mL water plus HCl to pH (approx. 30 mL)

  9. 20× SSC stock

    3 M NaCl, 300 mM Sodium citrate pH 7.4

    175.3 g NaCl, 88.2 g of sodium citrate per L distilled water

  10. 10% SDS Solution

    50 g sodium dodecyl sulfate in a total volume of 500 mL water

  11. 2× SSC, 0.1% SDS

    50 mL of 20× SSC, 5 mL of 10% SDS in a total volume of 500 mL of distilled water

  12. 0.5× SSC, 0.1% SDS

    12.5 mL 20× SSC, 5 mL of 10% SDS in a total volume of 500 mL of distilled water

  13. 0.1× SSC, 0.1% SDS

    2.5 mL 20× SSC, 5 mL of 10% SDS in a total volume of 500 mL of distilled water

    This can be used for a final 15 min stringent wash if your blot has a lot of background, and the signal is weak.

  14. 1× DIG block (prepared on the day)

    Dilute 10× DIG block (DIG Wash and Block Buffer Set) in 1× maleic acid buffer

  15. 1× maleic acid buffer

    Dilute 10× malic acid buffer (DIG Wash and Block Buffer Set) in distilled water

  16. 1× maleic acid wash buffer

    Dilute 10× malic acid wash buffer (DIG Wash and Block Buffer Set) in distilled water

  17. 1× detection buffer (prepared on the day)

    Dilute 10× detection buffer (DIG Wash and Block Buffer Set) in distilled water

Acknowledgments

We thank Dr Sara Rollinson for her help and expertise with optimising this protocol.

This work was supported by an Alzheimer’s Society fellowship awarded to RJHW (AS-JF-16b-004 (510)), The Royal Society (RGS\R2\212216), a Medical Research Council studentship awarded to JLS and a Jean Corsan Foundation studentship awarded to NSH. This protocol is derived from our previous work (West et al., 2020; doi: 10.1186/s40478-020-01028-y).

Competing interests

The authors declare no competing interests

References

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  6. Gymrek, M. (2017). A genomic view of short tandem repeats. Curr Opini Genet Dev 44: 9-16.
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  9. Mizielinska, S., Gronke, S., Niccoli, T., Ridler, C. E., Clayton, E. L., Devoy, A., Moens, T., Norona, F. E., Woollacott, I. O. C., Pietrzyk, J., et al. (2014). C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345(6201): 1192-1194.
  10. Moens, T. G., Niccoli, T., Wilson, K. M., Atilano, M. L., Birsa, N., Gittings, L. M., Holbling, B. V., Dyson, M. C., Thoeng, A., Neeves, J., et al. (2019). C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A. Acta Neuropathol 137(3): 487-500.
  11. Mori, K., Arzberger, T., Grasser, F. A., Gijselinck, I., May, S., Rentzsch, K., Weng, S. M., Schludi, M. H., van der Zee, J., Cruts, M., et al. (2013). Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol 126(6): 881-893.
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简介



【摘要】重复扩增疾病,包括脆性X综合征、亨廷顿舞蹈病、 C9orf72相关运动神经元病和额颞叶痴呆,是一组与串联重复核苷酸序列多态性扩增相关的疾病。这些扩展是高度重复的,并且通常有数百到数千个重复长度,这使得通过 PCR 或测序准确识别和确定重复长度具有挑战性。在这里,我们描述了一种使用 Southern 印迹监测携带 1,000 个重复C9orf72相关二肽重复转基因的果蝇模型中重复长度的协议。该协议已被定期用于检查这些行的长度超过 100 代,结果可靠且可重复,并可用于监测果蝇中的任何重复扩展。
关键词: Southern印迹,果蝇, C9orf72 ,二肽重复,重复扩增

[背景] 重复扩增疾病是一类与串联重复 DNA 序列扩增相关的遗传疾病。串联重复的多态性和固有的不稳定性使它们容易发生突变,因此它们是人类基因组变异最丰富的原因之一(Gymrek,2017) 。重复序列的长度和位置可以变化,范围从单个核苷酸到每个重复的几个核苷酸,位于编码区和非编码区。已经确定了 50 多种重复扩张障碍,其中大部分主要影响中枢神经系统(Depienne 和 Mandel,2021 年) 。
编码区的重复扩增往往是三核苷酸重复,导致蛋白质内异常长的重复氨基酸序列。相比之下,基因的 UTR 或内含子的扩展在序列和它们如何赋予毒性方面更加多样化。重复序列本身的长度可以变化,最长的报道是CSTB (胱抑素 B) 基因的 5'UTR 中的十二聚体,这导致进行性肌阵挛性癫痫 1 型 (EPM1) (Lalioti et al. , 1997) 。此外,虽然 5'UTR 中富含 GC 的重复扩增倾向于通过表观遗传机制(如持续的 DNA 高甲基化)导致功能毒性丧失,但位于 3'UTR 和内含子内的扩增更频繁地通过 RNA 毒性或通过重复相关的非 AUG (RAN) 翻译合成多肽。一个既能降低基因表达又能引起 RNA 和多肽重复毒性的重复扩增的例子是C9orf72基因中的内含子 GGGGCC 扩增,这是额颞叶痴呆 (FTD) 和运动神经元疾病 (MND) 的主要遗传原因(Renton et al. ,2011 年;DeJesus-Hernandez等人,2011 年) 。使用包括果蝇在内的遗传模型进行的研究已经确定,虽然C9orf72扩增确实会降低基因表达并导致单倍体不足,但在这种情况下,毒性的主要驱动因素是从非规范 RAN 翻译中产生二肽重复蛋白 (DPR)。重复本身。由于翻译发生在所有框架中以及从正义和反义 RNA 中,产生了五种不同的 DPR:甘氨酸-丙氨酸 (GA)、甘氨酸-脯氨酸 (GP)、甘氨酸-精氨酸 (GR)、丙氨酸-脯氨酸 (AP) 和脯氨酸-精氨酸 (PR) (Mori et al. , 2013) 。这些 DPR 已显示在患者大脑和脊髓中聚集,并且在多个模型系统中具有毒性(May等人,2014;Mizielinska等人,2014;Zhang等人,2014;Moens等人,2019;West等人,2020 年) 。
为了进一步了解扩张障碍及其背后的机制,已经开发了一系列转基因模型系统。对于每种扩张障碍,通常对什么构成致病性重复长度达成共识。例如, C9orf72基因中的内含子六核苷酸 GGGGCC 重复在健康个体中通常低于 30 个重复,而在患有疾病的个体中,它包含超过 500 个甚至数千个重复(Renton等人,2011;DeJesus-Hernandez等人。 , 2011) 。在韦斯特等人。 (2020),我们开发了第一个果蝇模型,以超过 1,000 次重复表达每个 DPR,单独且没有重复 RNA。为此,设计了替代编码序列以产生独立于 GGGGCC 重复的 DPR (Callister等人,2016) 。然而,转基因仍然高度重复、富含 GC 且长度超过 6000 bp,这使得通过 PCR 或测序对重复长度进行准确的基因分型具有挑战性。此外,串联重复序列在长度依赖性方面越来越不稳定(Depienne 和 Mandel,2021),并且已知引入细菌和动物模型的重复序列会在几代人之间完全收缩或切除(Bichara等人,2006 年;Ryan等人) . , 2019) .因此,重要的是能够确定转基因模型中重复序列的长度并监测它们以检查撤回。最常见的方法,被广泛认为是金标准,是Southern印迹。
Southern 印迹是一种高度敏感的技术,用于检测血液或组织样本中的特定 DNA 序列。它涉及用位点特异性限制性内切酶消化 DNA 以分离感兴趣的基因组区域,然后使用凝胶电泳按大小分离 DNA 片段,并通过毛细作用转移到多孔、带正电的尼龙或硝酸纤维素膜上。使用与特定核酸探针的分子杂交来检测感兴趣的DNA,这些探针用放射性同位素或非同位素试剂标记(例如,以促进化学发光检测) (Southern,2006) 。虽然 Southern 印迹已在很大程度上被现代测序技术取代并且不再广泛使用,但它仍然是研究人员处理长且重复的 DNA 序列的一项基本技术。重复序列在序列比对和组装方面一直被证明在技术上具有挑战性,当重复超过测序片段长度(通常为 350 – 500 bp)时,这种情况会更加严重。此外,传统的 PCR 扩增文库具有固有的 GC 偏差,这可能导致重复扩增的读数代表性不足,其中很大一部分具有 100% 的 GC 含量(Treangen 和 Salzberg,2012) 。这些不准确可能导致重复大小的低估和重复扩展的错误表征(Rajan-Babu等人,2021) 。据报道,用于检测FTD/MND 患者中C9orf72扩增的基于 PCR 的技术不可靠,假阳性率和假阴性率都很高(Akimoto等人,2014 年) 。因此,虽然在长读长测序分析方面取得了有希望的发展,但目前 Southern 印迹被认为是对富含 GC 的重复扩增进行基因分型的金标准。在这里,我们开发并完善了果蝇重复序列的南方印迹协议。我们使用 West等人发表的果蝇 C9orf72 DPR 模型优化了该协议。 (2020),但它可以应用于其他重复扩增,只需改变寡核苷酸探针和限制性内切酶所需的程序。

关键字:DNA印迹术, 果蝇, C9orf72, 二肽重复, 重复扩展



材料和试剂


1. Whatman ® 3MM 滤纸(Whatman ® ,GE Healthcare,目录号:WHA30306185)
2. 超厚吸墨纸 (2.5 mm) ( ThermoFisher Scientific, Thermo Scientific TM ,目录号88605 )
3. Amersham TM Hybond TM -N 0.45 µm 孔,中性尼龙膜(GE Amersham,目录号:RPN203N)
4. 15 mL Falcon 管(例如,Corning ® 15 mL 离心管,Sigma-Aldrich,Merck,目录号:CLS430790)
5. 50 mL Falcon 管(例如,Corning ® 50 mL 离心管, Sigma-Aldrich,Merck,目录号:CLS430829)
6. 1.5 mL 微量离心管(例如,Eppendorf ® Safe-Lock 微量离心管,Sigma-Aldrich,Merck,目录号:T9661)
7. 塑料薄膜(例如保鲜膜)(Scientific Laboratory Supplies,目录号:FIL1003)
8. 醋酸片(Rapid Electronics Ltd,Diacel,目录号:34-0303)
9. 培养皿或称重船(用于如图1所示的采摘头,例如,小方形称重船,Sigma-Aldrich,Merck,目录号:Z708542-500EA)
10. 用10 mM Tris,pH 8.0,1 mM EDTA(Sigma-Aldrich, Merck ,目录号:P3803)饱和的苯酚-氯仿-异戊醇(25:24:1)
11. 氯仿(分析试剂级)(Sigma-Aldrich,Merck,目录号:366927)
12. 乙醇(用于分子生物学)(Sigma-Aldrich,Merck,目录号:51976)
13. 溴化乙锭,分子生物学级水溶液(500 mg/mL)(Sigma-Aldrich, Merck,目录号:E1385)
14. 浓(36.5-38.0%)盐酸(HCl)(Sigma-Aldrich,Merck,目录号:H1758)
15. 氯化钠(NaCl)(分析试剂级)(Sigma-Aldrich,Merck,目录号:S9888)
16. 氢氧化钠(NaOH)丸(无水)(Sigma-Aldrich,Merck,目录号:S5881)
17. 柠檬酸钠(柠檬酸钠三水合物)(Sigma-Aldrich,Merck,目录号:C8532)
18. TRIS盐酸盐(TRIS HCl)pH 8.0(Sigma-Aldrich,Merck,目录号:10812846001)
19. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,Merck,目录号:L3771)
20. 乙二胺四乙酸(EDTA) (Merck,Sigma-Aldrich,目录号:E9884)
21. 要测试的果蝇种群(每种基因型 50 – 60 只苍蝇(参见将其应用于其他组织/协议的说明),包括阴性野生型对照,例如 Canton S 或 Oregon R)。野生型苍蝇可以从布卢明顿果蝇种群中心(BDSC,印第安纳大学)(Canton S:BDSC 9515,俄勒冈州 R BDSC 2376)或其他种群中心获得。我们示例中使用的转基因原种是通过剑桥显微注射设施将pUAST -DPR-EGFP 显微注射到 VK00005 胚胎中产生的 [参见 West等人。 (2020);可应要求提供]。
22. 用于生成转基因蝇系的质粒,用作阳性对照 [例如, pUAST - DPR( 1000)],参见 West等人,2020,可根据要求提供
23. 干冰
24. 无核酸酶水(分子生物学级)(Sigma-Aldrich,Merck,目录号:W4502)
25. 蛋白酶 K( ThermoFisher Scientific, Thermo Scientific TM ,目录号:EO0491),分成 1 mL 等分试样并储存在 -20°C
26. 限制性内切酶,用适当的缓冲液从基因组中切除感兴趣的 DNA;对于我们的示例,我们使用了 Dde1(New England Biolabs,目录号:R0175)和 NlaIII ( New England Biolabs,目录号:R0125),以及 CutSmart ®缓冲液(New England Biolabs,目录号:B7204)。
27. TBE 缓冲液 (Tris-borate-EDTA) (10 × ) ( ThermoFisher Scientific, Thermo Scientific TM ,目录号:B52)
28. 用于分子生物学的琼脂糖(Sigma-Aldrich, Merck,目录号:A9539)
29. 6 ×上样染料(New England Biolabs,目录号:B7021S)
30. DNA 分子量标记 II,DIG标记 (罗氏,默克,目录号:11218590910)
31. 1 kb Plus DNA Ladder(NEB,目录号:N3200)
32. 特定于目标序列的探针 [West et al ., (2020 ) 中使用的探针,由 Eurofins UK 合成,列在本协议的注释部分]。
33. DIG easy hyb(Roche,Merck,目录号:11603558001)
34. 鲑鱼精子 DNA(Agilent Technologies,目录号:201190),分成 300 µL 和 150 µL 的等分试样,并在 -20°C 下储存。
35. DIG Wash and Block Buffer Set(Roche,Merck,目录号:15857 62001) ,一个等分 DIG 块(例如,50 mL 等分试样)并在 -20°C 下长期储存(一个等分试样可储存长达 3 个月在 4°C)。
36. 抗地高辛-AP,Fab 片段(Roche,Merck,目录号:11093274910, RRID:AB _2734716),储存于 4°C
37. 超纯水(例如来自 Milli-Q ® Purification System)
38. CDP - Star ®化学发光底物(Roche,Merck,目录号:CAS160081-62-9),储存于 4°C
39. 70% 乙醇(见食谱)
40. 脱嘌呤溶液(见配方)
41. 凝胶变性溶液(见配方)
42. 凝胶中和溶液(见配方)
43. 20 × SSC 库存(见食谱)
44. 10% SDS 溶液(见配方)
45. 2 × SSC,0.1% SDS(见配方)
46. 0.5 × SSC,0.1% SDS(见配方)
47. 0.1 × SSC,0.1% SDS(见配方)
48. 阳性对照(见食谱)
49. 基因组提取缓冲液(见配方)
50. 1 × TBE(见配方)
51. 梯子混合(见食谱)
52. 1 × DIG 块(见配方)
53. 1 ×马来酸缓冲液(见配方)
54. 1 ×马来酸洗涤缓冲液(参见配方)
55. 1 ×检测缓冲液(参见配方)


设备


1. 移液器和吸头(P1000、P200、P20、P10 和 P2)
2. 精细画笔(例如,RS PRO Thin 6.4 mm 画笔,RS Components Ltd,目录号:2379190)
3. 涡流混合器(例如,Vortex-Genie ® 2 混合器,Sigma-Aldrich,Merck,目录号:Z258415)
4. 通风柜
5. 酸碱度计
6. 标准 700W – 1000W 微波
7. 标准实验室微量离心机(例如,Sigma 1-14 Microfuge, Sciquip ,目录号:90616)
8. (例如, SciSpin MINI Microfuge, Sciquip ,目录号:SS-6050)
9. 轮管旋转器(例如,Cole- Parmer TM Stuart TM Rotator Disk,Fisher Scientific,目录号:11496548)
10. PowerPac TM基本电源(Bio-Rad,目录号:1645050)
11. × 15 cm)的Sub-Cell GT 电泳池(Bio-Rad,目录号:1704402)
12. 清洁塑料托盘和三明治盒,用于孵育膜和凝胶以及组装 Southern 印迹(图 2)
13. 大三明治容器(例如250 × 150 mm)用作 Southern 印迹装置的盆(图 2)
14. 化学发光和荧光成像系统(例如, G:box成像单元, Syngene )
15. HB铅笔
16. 用于切割滤纸和膜的剪刀/断头台
17. UV Transilluminator(例如,双波长(302/365)8W transilluminator LM-20,VWR,目录号:732-4388)
18. 标准沸水浴(例如,VWR,SBB Aqua 5 plus,目录号:462-0171)或能够达到> 100°C的热块
19. 标准杂交烘箱(例如,HB-1000 Hybridiser,VWR,目录号:732-4300)
20. 适合杂交炉的玻璃杂交瓶(例如,35 × 150 mm 杂交瓶,VWR,目录号:732-4350)
21. 平刃钳(例如, S Murray TM不锈钢钳 L325/01,Fisher Scientific,目录号:12342158)
22. 摇杆(例如, Cole- Parmer TM Stuart TM See-Saw Rocker,目录号:10470655)


软件 


1. 用于对印迹进行成像的成像软件,例如: GeneSys和GeneTools ( Syngene , https ://www.syngene.com/support/software-downloads/ )


程序


A. 准备试剂
在开始方案之前,请确保您已准备好阳性对照、梯形混合物以及配方部分中描述的所有缓冲液和溶液。除 1 × DIG 块和 1 ×检测缓冲液外,所有配方必须在使用当天由 10 ×补足,可提前制作并保存至少 1 年。蛋白酶 K 必须在使用前立即新鲜添加到基因组提取缓冲液中。


B. 果蝇头部提取 DNA (可以使用整只果蝇或其他组织,但我们发现使用头部可以提供更好的信号,因为缺乏来自肠道内容物等污染物)。


第一天
1. 收集 50 – 60 只苍蝇(产生约 25 – 30 µg 基因组 DNA),每个基因型,在 15 mL Falcon 管中,放在干冰上。除了测试样品,收集野生型苍蝇,例如Canton S或Oregon R ,用作阴性对照。
2. 要分离飞头,将3-4块干冰放入 50 mL Falcon 管中,并将含有苍蝇的 15 mL Falcon 管放入 50 mL Falcon 管内(参见图 1A)。不要将盖子放回 50 mL Falcon 管上。以最高速度涡旋直到头部分离(~30 s)(参见视频 1)。不涡旋时始终保持在干冰上。




视频 1.通过涡旋分离果蝇头。


3. 挑选头并将它们转移到干冰上的 1.5 mL 微离心管中。可以使用最简单的方法手动拾取头部。我们推荐的方法是将被斩首的苍蝇倒在衬有滤纸的培养皿上,该滤纸位于大托盘或装有干冰的称重船上(参见图 1B)。使用细画笔拾取头部(参见图 1C)并将它们转移到干冰上的 1.5 mL 管中。当一切都尽可能保持低温时,此过程会更容易。
4. 解冻蛋白酶 K 并添加到基因组提取缓冲液中(参见食谱)。
5. 通过用 1 – 200 µL 移液器吸头“挤压”来均质化磁头,其中填充有基因组提取缓冲液(每头 1 µL)而不排出液体(在此过程中将排出足够的缓冲液以有效地均质化磁头而不会使磁头四处飘浮)(约 10 秒)。然后排出剩余的提取缓冲液并放入杂交烘箱(或另一个培养箱)并设置为 57°C。孵育过夜。




图1 。用于收集果蝇头部的示例装置。
A.使用涡流混合器分离果蝇头的装置(另请参见视频 1)。 B.使用画笔拾取头部的设备。 C.头部与苍蝇的其余部分分离。


第 2 天
6. 第二天,在通风橱中,加入一体积(相当于步骤 5 中使用的体积)苯酚-氯仿-异戊醇(25:24:1)并在室温下旋转孵育 15 分钟(使用轮管旋转器) , 例如)。
7. × g下离心 5 分钟。
8. 将水(顶部)层转移到新管中。
9. 将 1 体积的 100% 氯仿添加到步骤 8 中回收的水相中,在室温下旋转孵育 15 分钟(例如,使用轮管旋转器),然后重复步骤7-8 。
10. 用标准乙醇沉淀法回收 DNA:
a. 加入 1 mL 100% 乙醇,颠倒 1 分钟,然后在 -20°C 下放置过夜。


第 3 天
b. 第二天,以 13,000 × g旋转10 分钟并弃去上清液。
c. 用 800 μL 的 70% 乙醇清洗颗粒。
d. 以 13,000 × g离心5 分钟并弃去上清液。
e. 在室温下风干15-30 分钟,然后重新悬浮在 100 μL 的无核酸酶水中。
11. 允许在 4°C 下溶解过夜或在室温下 1 小时。此时可以将 DNA 冷冻并储存在 -20°C 下,直到准备好进入 C 部分。


C. DNA 消化
第 4 天
注意:选择限制性内切酶可有效消化基因组 DNA,而不会切割靶序列。对于我们的示例,选择DdeI和NlaII是因为它们有效地切割了用于将我们的构建体插入果蝇基因组的载体,而不切割 DPR 序列 [参见图 2 和 West 等人,(2020)]。虽然应考虑基因组中每种酶的大致切割频率(例如, NlaIII切割 4 bp 位点,因此切割频率约为 4 4 ),但最关键的因素是酶不会切割您的目标序列。我们示例中使用的酶在材料和试剂中列出,并在表 1中进行了详细说明。消化在 500 μL 的大体积中进行,以便完全消化基因组 DNA。使用 50 – 60 头提取的所有 DNA(~ 25–30 µg DNA) 。




图 2. 显示限制性酶NIaIII和 Dde1 切割位点的质粒图谱。
pUASt-DPR1000-GFP 质粒图谱显示了 mini-white 元素、DPR 序列和 EGFP 标签。指示了NlalII和 Dde1 的限制位点(使用Snapgene Viewer 制作)。


1. 将杂交炉(或其他培养箱)设置为限制酶所需的温度。
2. 准备反应混合物(如表 1 所示的示例)。
3. 通过移液混合并通过在台式微量离心机中离心来沉淀内容物(10 秒,2,680 × g )。
4. 在您选择的酶的最佳条件下孵育(在我们的示例中,在 37°C 下过夜)。


第 5 天
5. 用标准乙醇沉淀法回收 DNA:
a. 加入 1 mL 100% 乙醇,颠倒 1 分钟,然后在 -20°C 下放置过夜。
第 6 天
b. 第二天,以 13,000 × g旋转10 分钟并弃去上清液。
c. 用 800 μL 的 70% 乙醇清洗颗粒。
d. 以 13,000 × g离心5 分钟并弃去上清液。
e. 让颗粒在室温下风干 15-30 分钟。
6. 将颗粒重新悬浮在 17 μL 的无核酸酶水中,充分混合,然后在台式微量离心机中短暂离心(10 秒,2,680 × g ),以确保所有 DNA 都位于管底。
7. 在进行 Southern 印迹之前,让 DNA 在 4°C 下溶解 2 – 3 天。


表1 。 DNA 消化混合物示例
体积 (µL)
脱氧核糖核酸 100
Cutsmart缓冲区 (10 × ) 50
NIaIII 2
Dde1 2
无核酸酶水 346
总容积 500


D. 印迹样品
第 7 天(第 6 天后 2 – 3 天)
1. 制作琼脂糖凝胶:
a. 将 130 mL 1 × TBE 与适当质量的琼脂糖混合,制成分离构建体所需百分比的凝胶(有关凝胶百分比和基于产品大小的分离能力的指南,请参见表 2;在我们的示例中,我们使用 1%对于 6 kb 的产品)。
b. 在微波炉中煮沸 TBE 琼脂糖混合物。
c. 加入 0.5 µg/mL 溴化乙锭并旋转混合。
d. 倒入 15 × 15 厘米的浇铸托盘中并静置。
2. 准备电泳样品:
a. 解冻梯形混合物和 10 ng/µL 阳性对照库存。
b. ×上样染料中加入 0.5 μL 的阳性对照,总体积为 12 μL。
c. 对于 B 和 C 部分中制备的测试样品和阴性对照,添加 3 μL 的 6 ×上样染料,使总体积为 20 μL,这是可以装入孔中的最大值。
3. 准备运行凝胶:
a. 将凝胶放入槽中,用 1 × TBE 填充槽,然后取下梳子。
b. 将 7 μL 的梯形组合、12 μL 的阳性对照和 20 μL 的样品加载到单个井中。将空白孔留在阳性对照的任一侧可能是有益的,以避免因孔之间溢出而导致误报。
4. 在 100 V 下对 DNA 样品进行足够长的电泳,以在目标 DNA 周围的梯带之间提供良好的分离(对于 6 kb 的产品,这大约是 2.5 小时)。


表2 。基于预期产品大小的 TBE 凝胶中琼脂糖百分比指南
琼脂糖 % (w/v) 解析度
0.50% 1,000–30,000 碱基对
0.70% 800–12,000 碱基对
1.00% 500–10,000 碱基对
1.20% 400–7,000 碱基对
1.50% 200–3,000 碱基对
2.00% 50–2,000 碱基对


5. 使用凝胶文档系统(如 G-box)对凝胶进行成像。这有助于确定 DNA 是否已完全消化(消化应显示为泳道上的涂片),并在对最终印迹成像时参考某些泳道中的信号是否较弱。使用紫外线透射仪帮助修剪印迹也很有帮助。
6. 如果需要,可以使用梯子将凝胶修剪得更小,以确保不会去除感兴趣的 DNA。切开凝胶的一个角以帮助识别凝胶方向,然后将凝胶转移到干净的塑料容器中。
7. 凝胶脱嘌呤:加入足够的脱嘌呤溶液覆盖凝胶,室温下在跷跷板或定轨摇床上缓慢摇动,直至上样染料中的溴酚蓝变黄或不超过 10 分钟。
8. 在下一步之前,倒掉脱嘌呤溶液并在蒸馏水中短暂冲洗凝胶。
9. 使凝胶中的 DNA 变性:加入足够的凝胶变性溶液以覆盖凝胶,并在室温下摇动孵育 30 分钟。
10. 中和凝胶:倒出变性溶液,换上等体积的中和溶液。在室温下振荡孵育 30 分钟。
11. 倒掉中和溶液,换上等体积的 20 × SSC。在室温下孵育 20 分钟,在吸墨前摇动。这有助于平衡凝胶并去除背景。
12. 同时,准备 3 张 Whatman 3 MM 纸、一张Hybond -N 0.45 µm 孔尼龙膜和 15 张超厚吸水纸,均切成与凝胶相同的尺寸。还剪下 3 条 3MM 纸,略宽于凝胶,长于支撑的长度,用作灯芯(见图 2)。
13. 此时,您可以从膜上剪下一个角,让杂交后的印迹能够定向,并在将面对凝胶的一侧画一个铅笔标记,以识别 DNA 侧。
14. 使用 70% 乙醇清洁平刃镊子以去除碱性磷酸酶,然后再使用它们处理尼龙膜(碱性磷酸酶与检测过程中使用的化学发光底物发生反应) 。
15. 如图 3 所示组装传输。对于这一步,可以重复使用20 × SSC。
a. 将一个小的塑料容器(桥/支架)倒置在一个较大的容器中,以形成您的转移装置的基础。
b. 将较长的滤纸条放在桥/支架上以起到吸水作用,将缓冲液储液罐中的 20 × SSC 吸到凝胶中。倒入少许 20 × SSC 以润湿灯芯。
c. 将凝胶放在灯芯覆盖的桥/支架上(我们通常将凝胶正面朝上放置;然而,这并不重要,因为转移可以在任何方向发生),然后是尼龙膜(铅笔标记朝下,面向凝胶,膜的切角与凝胶的切角相匹配,以帮助定向)。小心去除凝胶和膜之间的所有气泡(用戴手套的手指轻轻地)。
d. 紧随其后的是 3MM 纸和超厚吸墨纸。为避免转移短路,确保缓冲液在被吸至厚吸墨纸时不会绕过凝胶和膜,方法是确保吸液芯不接触凝胶和膜上方的吸墨纸。
e. 在纸上放置一个重物(约 100–300 克,例如一个装满水以覆盖底部但不会溢出的小塑料容器),并将凝胶在室温下放置过夜以进行转移。确保灯芯浸入水中并且重量是水平的。如果室温是温暖的并且您对 20 × SSC 的蒸发有疑问,请用塑料薄膜盖住设备以防止这种情况发生。




图 3.Southern 印迹组装。
A.示意图。 B.照片。将一个小的塑料容器倒置在一个较大的塑料容器中,形成一个桥梁。 将较长的滤纸条放在容器上,让缓冲液槽中的 20 × SSC 芯吸到凝胶中。倒入少许 20 × SSC 以润湿灯芯。将凝胶放在桥上,然后是尼龙膜,小心去除凝胶和膜之间的所有气泡。小心地将 3MM 纸和超厚吸墨纸放在上面。将重物(例如,装满水的塑料容器)放在纸上,让凝胶过夜以进行转移。确保灯芯被覆盖和重量水平。如果你担心蒸发,你可以用塑料薄膜覆盖。


E. 杂交
第 8 天
1. 第二天,将沸水浴/热块设置为 100°C,杂交烘箱设置为 42°C。
2. 将 30 mL DIG easy hyb置于 42°C 预热。解冻 300 µL 鲑鱼精子 DNA。
3. 拆卸印迹(保留 20 × SSC 以供重复使用)并在 ~ 20 mL 的 2 × SSC 中轻轻清洗膜。
4. 使用 UV(302 nm,“Hi”强度设置)将 DNA 固定在膜上:将塑料薄膜放在 UV 透照器表面,将膜 DNA 面朝下,打开照明器,固定 180 秒(图 4)。如果使用 UV 交联剂,例如 Stratalinker,请使用 1200 µJ 的标准自动交联设置。




图 4. 紫外固定膜 - 示意图。
确保您佩戴正确的个人防护设备以使用紫外线。将塑料薄膜放在紫外线透照仪的表面上,膜 DNA 面朝下放在塑料薄膜的顶部。打开 UV 并离开修复 180 秒。


5. 转移到杂交瓶中(DNA 面朝内;确保膜的 DNA 面完全暴露且不折叠)并加入约 50 mL 的 2 × SSC 以防止膜变干* 。
注意:* 膜可以在 2 × SSC 中放置过夜,但如果在杂交前放置更长时间,请在超纯水中冲洗膜两次 10 分钟,然后轻轻风干膜并在 3MM 纸之间存放后期杂交。这种洗涤是至关重要的,因为它可以去除任何可能在膜上干燥并产生背景信号的微量盐分。
6. 准备预杂交溶液:将 300 µL 鲑鱼精子 DNA 原液煮沸 10 分钟使其变性(这是 3,000 µg DNA),然后置于冰上以防止重新退火;加入 30 mL 预热的 DIG easy hyb。
7. 倒掉 2 × SSC,然后加入预杂交溶液。在 42°C 下旋转预杂交 4 小时。
8. 制备杂交溶液:解冻 150 µL (1,500 µg) 鲑鱼精子 DNA,将 15 mL DIG easy hyb预热至 42°C;如步骤 6 中所述使鲑鱼精子 DNA 变性,并将其和 7.5 µL 10 ng/ µL寡核苷酸探针原液添加到 DIG easy hyb 中,最终鲑鱼精子 DNA 浓度为 100 µg/µL,探针浓度为 0.005 ng/µL。
9. 倒掉预杂交溶液并更换为杂交溶液。在 42°C 下旋转杂交过夜。


F. 检测
第 9 天
1. 第二天,从烤箱中取出杂交瓶并设置为 65°C。
2. 放入 2 × SSC的 2 × 50 mL 等分试样; 0.1% SDS 和 1 × 50 mL 0.5 × SSC; 0.1% SDS(和 50 mL 0.1 × SSC;如果用于可选的额外最终洗涤(步骤 7),则为 0.1% SDS)在 65 °C 下预热。解冻 10 × DIG 块并准备 1 ×块(参见食谱)。
3. 倒掉探针,用大约 50 mL 2 ×冲洗杂交瓶中的膜 SSC; 0.1% SDS。
4. 加入 50 mL 预热的 2 × SSC;将 0.1% SDS 加到瓶中的膜上,并在 65°C 下旋转洗涤 15 分钟。
5. 更换为 50 mL 新鲜预热的 2 × SSC; 0.1% SDS 洗涤 15 分钟。
6. 更换为 50 mL 预热的 0.5 × SSC; 0.1% SDS 洗涤 15 分钟。
7. 可选的额外清洗(如果背景高且信号较弱):替换为 50 mL 预热的 0.1 × SSC; 0.1% SDS 洗涤 15 分钟。
8. ×马来酸缓冲液冲洗瓶子(使用前剧烈摇晃)。
9. 将膜转移到干净的盘子中,在室温下将膜在马来酸缓冲液中振荡 2 分钟。
10. × g下将抗地高辛-AP 抗体离心 15 分钟来制备抗体溶液,以去除抗体复合物。从顶部移取抗体以在 1 × DIG 块中制备 20 mL 1:20,000 抗DIG。
11. 倒出马来酸缓冲液并在室温下将膜在 50 mL 1 × DIG 块中摇动孵育 30 分钟。
12. 倒出封闭溶液,在室温下将膜在 20 mL 抗体溶液中振荡孵育 30 分钟。
13. ×马来酸洗涤缓冲液中洗涤膜两次(2 × 15 分钟,在室温下摇动) 。
14. 准备 1 ×检测缓冲液(参见配方)。在 20 mL 1 ×检测缓冲液中平衡膜 5 分钟。在室温下留在检测缓冲液中,直到准备好成像。
15. 将膜 DNA 面朝上放在一张干净的醋酸盐上。加入 1–2 mL(覆盖膜)CDP- Star ®化学发光底物,均匀分布在膜上。在顶部放置另一张干净的醋酸盐,将膜夹在中间。当您将第二张醋酸盐片放在膜上时,请小心消除气泡。立即进行成像。
16. 最初使用 G-Box 或等效系统对膜进行 1 分钟成像,以检查印迹是否有效(应该看到梯子),然后再次以最高质量成像。
17. 为了增强对弱波段的检测,突出显示感兴趣的区域并根据这些波段设置自动曝光时间。再上图。使用此处列出的 G-Box 系统最多可能需要 30 分钟。请参见图 5,了解最终的 Southern 印迹图像示例。




图 5. 用于长度检查 UAS-GA1000-EGFP 飞线的 Southern 印迹示例。
一个 6 kb 的条带对应于 1000 个二肽重复。泳道按顺序排列:阳性对照(+,来自野生型果蝇的 DNA,掺有 1000 个重复线性化质粒 DNA)、阶梯、阴性对照(-,来自野生型果蝇的 DNA)和两个独立的 GA1000 系(1 和 2)。该印迹成像 30 分钟。


笔记


我们发现从 50-60 个头部中提取是最佳的(25-30 µg 基因组 DNA),但如果使用其他基因组 DNA 提取方案,或者从其他组织中提取 DNA,这可能需要优化。我们建议至少应使用 5 µg 基因组 DNA。


如果感兴趣的 DNA 具有更短/更长的重复长度,则可以使用不同的 DIG 梯。
如果目标 DNA 与给出的示例不同,则需要优化以下内容:
凝胶百分比和运行时间
加载的阳性对照量,基于相对于样品的强度
为了进一步优化背景降低,我们建议在杂交后添加额外的严格洗涤,如程序部分 F 步骤 7 中所述。抗体孵育后在马来酸洗涤缓冲液中第三次洗涤 15 分钟(F 部分步骤 12)也可能有助于降低高背景水平。


在 West等人中产生的果蝇的 Southern 印迹。 (2020) 使用了以下探针:
GA探针:DIG-GGCAGGAGCTGGAGCTGGCGCAGGAGCTGGTGCTGGG-DIG
GR 探头:DIG-AGGCAGAGGTCGTGGGAGAGGCAGGGGTCGCGGACGTGGA-DIG
AP探头:DIG-AGCACCAGCACCGGCGCCAGCTCCAGCACCAGCACCC-DIG
PR 探头:DIG-AGACCCCGTCCTCGTCCTCGTCCAAGACCAAGGCCGAGGC-DIG


探针设计将根据您的目标序列而有所不同。通常,寡核苷酸探针应在 18 到 50 个碱基之间以平衡产量和特异性。最佳 GC 含量在 40 – 50% 之间,建议进行计算分析以优化特异性。


食谱


除非另有说明,否则缓冲液和溶液应在室温下保存长达一年。
1. 基因组提取缓冲液
25 mM NaCl、10 mM Tris-HCl pH 8.2、1 mM EDTA 和蛋白酶 K 200 μg /mL
在室温下储存不含蛋白酶 K 的缓冲液;蛋白酶 K 应以 1 mL 等分试样在 –20°C 下储存,并在使用前立即加入适当体积的缓冲液中。
2. 阳性对照
野生型果蝇DNA 在每 1 µg 基因组 DNA 中加入 157 ng 含有目标重复序列的线性化质粒,以产生等效的单倍体 DNA。在 -20°C 下保持 10 ng/µL 的库存。
3. 阶梯混合
1:1 DIG 标记的 DNA 标记物 II:1 kb 加上 DNA 梯,加上 1 ×上样染料(在电泳后修剪凝胶时,可见梯用作 DNA 片段大小的参考)。将这种混合物储存在 -20°C。
4. 70% 乙醇(在无核酸酶水中配制)
在无核酸酶水中化妆
5. 1 × TBE
在超纯水中化妆
6. 脱嘌呤溶液
0.25 M 盐酸
7. 凝胶变性溶液
0.6 M NaCl,0.2 N NaOH
17.53 g NaCl,4 g NaOH 溶于 500 mL 水中
8. 凝胶中和溶液
1.5 M NaCl,0.5 M Tris-HCl pH 8.0
43.8 g NaCl,30.30 g Tris,在 500 mL 水中加 HCl 至 pH(约 30 mL)
9. 20 × SSC 库存
3 M NaCl,300 mM 柠檬酸钠 pH 7.4
每升蒸馏水 175.3 g NaCl,88.2 g 柠檬酸钠
10. 10% SDS 溶液
50 克十二烷基硫酸钠在 500 毫升水中的总体积
11. 2 × SSC,0.1% SDS
50 mL 20 × SSC,5 mL 10% SDS,总体积为 500 mL 蒸馏水
12. 0.5 × SSC,0.1% SDS
12.5 mL 20 × SSC,5 mL 10% SDS,总体积为 500 mL 蒸馏水
13. 0.1 × SSC,0.1% SDS
2.5 mL 20 × SSC,5 mL 10% SDS,总体积为 500 mL 蒸馏水
如果您的印迹有大量背景且信号较弱,这可用于最后 15 分钟的严格洗涤。
14. 1 × DIG块(当天准备)
×马来酸缓冲液中稀释 10 × DIG 块(DIG 洗涤和块缓冲液套装)
15. 1 ×马来酸缓冲液
用蒸馏水稀释 10 ×苹果酸缓冲液(DIG Wash and Block Buffer Set)
16. 1 ×马来酸洗涤缓冲液
用蒸馏水稀释 10 ×苹果酸洗涤缓冲液(DIG Wash and Block Buffer Set)
17. 1 ×检测缓冲液(当天配制)
用蒸馏水稀释 10 ×检测缓冲液(DIG Wash and Block Buffer Set)


致谢


我们感谢 Sara Rollinson 博士在优化此协议方面提供的帮助和专业知识。
这项工作得到了授予 RJHW (AS-JF-16b-004 (510))、皇家学会 (RGS\R2\212216)、授予JLS的医学研究委员会奖学金和 Jean Corsan基金会奖学金的阿尔茨海默氏症协会奖学金的支持授予NSH。该协议源自我们之前的工作(West等人,2020; doi :10.1186/s40478-020-01028-y)。  


利益争夺


作者声明没有竞争利益


参考


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引用:Sharpe, J. L., Harper, N. S. and West, R. J. H. (2022). Identification and Monitoring of Nucleotide Repeat Expansions Using Southern Blotting in Drosophila Models of C9orf72 Motor Neuron Disease and Frontotemporal Dementia. Bio-protocol 12(10): e4424. DOI: 10.21769/BioProtoc.4424.
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