DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols^©

non-radioactive Protein Truncation Test (PTT)

(last modified November 22, 2004)

Protocol

for a non-radioactive Protein Truncation Test (kindly provided by Elsa Bik and Els Voorhoeve)

• target group
• responsibilities
• definitions
• description
• durables
• consumables
• chemicals
• solutions
• quality control
• PROTOCOL
  • in vitro transcription / translation
  • SDS-PAGE analysis of translation products
  • electro blotting
  • detection of proteins
  • interpretation
  • protein marker
  • estimation of the part of the PTT fragment to be sequenced
  • troubleshooting
• references

Target group

The technicians, students, guests and staff members working at the Clinical Molecular Genetics Laboratory.

Responsibilities

Personnel within the target group who use PTT in DNA diagnostic procedures are responsible to perform the diagnostic procedures according to this protocol.

Definitions

• (M): available at the "magazijn" of the Sylvius Laboratory
• PTT: Protein Truncation Test
• RT: room temperature

Description

The PTT-assay has been developed to detect (point) mutations, which lead to premature translation termination. The assay is useful for mutation detection in any genetic disease where such mutations contribute to a significant fraction of all mutations found in patients. PTT detects prematurely terminated translation products by in vitro transcription and translation of the coding sequences amplified from patients’ samples by PCR. 

In a standard PTT-assay, RNA isolated according to ALG014, is used as a starting template. However, when a gene contains large exons, DNA can also be used (see MOC002, FAP002 [available on request; Elsa Bik and Els Voorhoeve]). The majority of genes do not contain large exons; they are split into many small exons, leaving RNA as the only workable template (see DMD007). When DNA is used as a template, a nested PCR is not necessary and the T7 tailed forward primer can be used directly. After PCR the amplified fragments are analysed on agarose gel. Subsequently the fragments are analysed using a coupled one-tube in vitro transcription/translation system. By means of SDS-PAGE analysis and luminescence or autoradiography the protein size of the translation products is determined and compared to fully translated control samples to reveal mutations, which affect the reading frame. 
Detection of the translation products is facilitated by incorporation of biotin-labelled amino acids; biotin-labelled lysin is used in this protocol. The biotin-labelled translation products are detected by chemiluminescence. 

Durables

• Autoclave, Prestige Medical
• Heat block, Unitek HB 130, Biozym B.V.
• Maxima Ultra Pure Water System, ELGA, Salm & Kipp
• Mini-Protean II Electrophoresis Cell, BioRad
• Mini Trans-Blot Module, BioRad
• Freezer -80°C, Ultrafreezer, Denley, Beun de Ronde
• Freezer -20°C, ER 1530 T, Electrolux
• Fridge +4°C, ‘Koel/vries combinatie’ KGK 330, Zanker
• Centrifuge, 5417C, Eppendorf, VWR International
• Power supply, Power pac 200, BioRad
• Shaker, Ika-vibrax-vxr, Typ vx7, Janke & Kunkel
  Stirrer, Beun-de Ronde B.V.
• Glass box 9*9 cm (M)
• Storage box, Roche Diagnostics
• Spacers, 0.75 mm, Mini-Protean II cellsystem, BioRad
• Pipetman, micropipettes P20, P200 en P1000, Gilson (M)
• Inner plate mini PII, BioRad
• Outer plate mini PII, BioRad
• 15 wells comb, 0.75 mm, Mini-Protean II cellsystem, BioRad
• 10 wells comb, 0.75 mm, Mini-Protean II cellsystem, BioRad
• Cassette 20*40 cm, Auto-G 1190 1050, Konica
• Lumi-ImagerTM, Roche Diagnostics

NOTE: equivalent durables may be used as well

Consumables

• Blue cap tubes, 50 ml, Greiner (M)
• Blue cap tubes, 14 ml, Greiner (M)
• refill puntjes 101-1000 microliter, Isogen LifeScience 
• Filter paper minitrans blot cell, BioRad
• Nitrocellulose 0.2u 7 * 8.4 cm, BioRad
• Yellow tips, Corning
• Pasteur pipettes plastic, 3 ml, Greiner (M)
• Safe-Lock tubes, 1.5 ml, Eppendorf (M)
• Gelsaver tips, 1-200 ul, Biozym
• Photogene Development Folders, VWR International
• Plastic pipettes, 10 ml, Greiner (M)
• Plastic petridish, 120*120*17 mm, Greiner (M)
• X-Ray films, AX, 35*43 cm, Konica (M)
• Paper towels (M)
• Sterile syringe, Becton Dickinson

NOTE: equivalent consumables may be used as well

Chemicals

• Tris Ultrapure, USB (M)
• NaCl, Baker (M)
• KCl, Baker (M)
• Na₂HPO₄×2H₂O, Merck (M)
• KH₂PO₄, Baker (M)
• NaOH, Baker (M)
• Glycine, Fluka
• Hydrogen peroxide (H₂O₂), 30%, ICN
• Maleic acid, Sigma
• Blocking reagens, Roche Diagnostics
• Tween-20, ICN
• Hydrochloric Acid (HCl) 36-38% p.a. (M)
• Laemmli Sample Buffer, BioRad
• beta-mercapto-ethanol, BioRad
• TEMED, tetramethylethylenediamine, Amersham Biosciences
• Ammonium persulfate, Promega
• Methanol, >99.5%, Baker (M)
• 30% Acryl amide/Bis Solution, 37.5:1 (2.6% C), BioRad
• 10* Tris/glycine/SDS Buffer, BioRad
• Developer, Roentogen, Konica
• X-Ray Fixing Solution, Tetanal Roentogen Superfix, Konica
• Ethanol, >99.5%, c/z (M)

NOTE: equivalent consumables may be used as well

• Streptavidine-HRP, ITK Diagnostics
• Lumi-Light Plus Western Blotting Substrate, Roche Diagnostics
• nT T7 Quick for PCR DNA, Promega
• tRNAnscend tRNA, Promega
• Prestained Protein Marker, Broad Range, Westburg
• Biotinylated SDS-PAGE Standard, low Range 14.4-97.4 kDa, Biorad

Solutions

The solutions are prepared according to the protocols below. The solutions marked with an * are prepared according to the "werkformulier".

• 10% Ammonium persulfate (APS), for 10 ml: see WFA09
• 10% Blocking reagents, for 100 ml: see WFB04
• 2% Blocking reagent, for 40 ml (2 membranes):
  add 8 ml 10% blocking reagent (defrost slowly at RT) to 32 ml 1 * Maleic acid
  Prepare freshly each time
• Conjugate, for 2 membranes:
  add 4 ml Steptavidine-HRP to 40 ml 1 * PBS-T
  Prepare freshly each time
• 3% Hydrogen Peroxide (H₂O₂), for 500 ml:
  dilute 50 ml 30% Hydrogen Peroxide to 500 ml with Elga water in a brown bottle. Store at 4°C for a maximum of 1 month.
• Loading buffer
  • for 1.0 ml (for 60 samples):
    Preparation has to be carried out in the fume hood. Add 50 ml b-mercapto-ethanol to 950 ml Laemmli Sample Buffer in a 1.5 ml Safe-Lock tube. Prepare freshly each time.
  • for 1.5 ml (for 90 samples):
    Preparation has to be carried out in the fume hood. Add 75 ml b-mercapto-ethanol to 1425 ml Laemmli Sample Buffer in a 2.0 ml Safe-Lock tube. Prepare freshly each time
• Lumi-Light Plus substrate solution, for 2 membranes: 
  mix 3 ml of component I with 3 ml of component II. Prepare freshly each time
• 10* Maleic acid, for 1 liter: see WFM02
• 1* Maleic acid, for 500 ml:
  dilute 50 ml 10 * Maleic acid to 500 ml with Elga water. Store at 4°C for a maximum of one month
• 10* PBS, for 2 litres: see WFP06
• 1* PBS-T, for 1 litre:
  add 100 ml 10 * PBS and 500 ml Tween-20 to 900 ml Elga water. Store at RT
• 1* Running buffer, for 2 litres:
  dilute 200 ml 10 * Tris/glycine/SDS Buffer to 2 litres with Elga water. Store at RT
• 10% SDS, 15553-035, Life Technologies.
• 10 * Transfer buffer, for 2 litres: see WFT08
• 1 * Transfer buffer, for 1 litre (for 2 gels):
  preparation has to be carried out in the fume hood. Add 100 ml 10 * Transfer buffer (4°C), 700 ml Elga water (4°C) and 200 ml Methanol (4°C) in a 1 litre bottle. Add in this order to avoid precipitation. Prepare freshly each time
• 1.5 M Tris-HCl (pH 8.8), for 100 ml: see WFT04
• 1.0 M Tris-HCl (pH 6.8), for 100 ml: see WFT04.

Quality control

• 1st line: PTT reaction without template DNA, prestained and biotinylated size markers, 
• 2nd line: see disease specific protocols
• 3rd line: see disease specific protocols
  

In vitro transcription / translation

The procedure has to be carried out in the post-PCR, with exception of steps 4 and 5, which are carried out in the pre-PCR lab.

1. Print a worksheet for PTT reactions using the Oracle LIMS system (see LP004)
2. Put a heat block at 30°C and a heat block at 100°C.
3. Pipet 1 ul of PCR product in a labelled autoclaved Safe-Lock tube, unless a weak PCR product is seen pipet 2 or more ml of PCR product. Pipet 1 ul autoclaved Elga water for the blank of each mixture.
4. The components needed for the PTT reaction are stored at the pre-PCR lab in -80°C freezer B. New kits are stored in -80°C freezer A. When you have to start with a new kit, transfer a complete new kit to box 20C in the freezer. The stock of tRNA is stored in a box "Stock PTT tRNA" in -80°C freezer A.
5. Prepare a mixture as defined in Table 1 (pre-PCR lab). After thawing (thaw the TnT T7 Quick Mix in your hand), store all components on ice! The lysate can be thawed only twice, write down on the tube the amount of lysate left.

Table 1

NOTE: mix carefully by pipetting up and down! Air bubbles may inactivate the lysate.

6. Add 5.5 ul mixture to each tube (post-PCR lab). Be aware that air bubbles may inactivate the lysate. For low concentrated PCR products 2 ul of PCR product and 5.5 ul mixture can be used.
7. Incubate for 60 - 90 min. at 30°C in a heat incubator.
8. Stop the reaction by adding 15 ul loading buffer. Perform this step in the fume hood!
   NOTE: beta-Mercapto-ethanol is toxic, gloves should be worn while handling the solution.
9. Prepare a mastermixture of 140 ul Prestained Protein Marker (visible, to check the transfer efficiency of electro blotting) and 1 ul of the Biotinylated SDS-PAGE Standard, Low Range. Store 70 ul of the mastermix in the freezer for the next time. Denature the samples and the other 70 ul of the markermix for 2 minutes at 100°C, in the fume hood.
10. Samples may be stored at -20°C for a maximum of 2 months.

SDS-PAGE analysis of translation products

NOTE: acrylamide monomer is neurotoxic, gloves should be worn while handling the solution.

For SDS-PAGE analysis of the samples, the Miniprotean II gel system is used. The system requires only a short electrophoresis time (about 1 hr). The gels described can be used to analyse peptides from 10-70 kDa depending on the percentage acrylamide (see table 2). 

Pouring the separating gel

1. Start with clean glass plates.
2. Assemble cassettes and place in casting stand (level the casting stand).
3. Prepare gel mixture for the separating gel as indicated in Table 2 and use immediately. Add TEMED in the fume hood. See disease specific protocols for the percentage of the gel.
4. Pour the gel mixtures between the glass plates with a plastic Pasteur pipet until approx. 1 cm below the top of the smallest glass plate. Gently overlay the gel with Elga water across the top of the gel mixture. Allow the gel to polymerize for at least 30 minutes at RT.

Table 2

Pouring the stacking gel

5. Remove the water by inverting the gel and dry with blotting paper.
6. Prepare gel mixture for the stacking gel as indicated in Table 3.
7. Completely fill the cassette with stacking gel and insert the comb. Be aware of air bubbles trapped under the teeth. Allow to polymerize for 15-30 minutes.

Table 3

Loading the SDS-PAGE gels

8. Remove comb from stacking gel and rinse wells with water or 1* running buffer using a syringe. Place the cassette in the electrophoresis tank and fill the inner chamber with 1* running buffer. Check for leakage. Fill the tank (outer chamber) till just above the bottom of the glass plates.
9. Centrifuge the samples and the marker for 5 minutes at 14.000 rpm.
10. oad max. 10 ml sample into each well when a 15 wells comb is used and load max. 15 ml sample into each well when a 10 wells comb is used. This loading procedure has to be carried out in the fume hood. Use a Gelsaver tip and be careful not to disrupt the pellet. 
    Load 5 ml of the standard mastermix (see 11.9) when a 15 wells comb is used and 7 ml of the standard mastermix when a 10 wells comb is used. It is advisable to apply the standard in different lanes to the gels to be able to easily identify the gels.

Running the gel

11. Run the stacking gel at 60 Volts (± 30 minutes, until the bromophenol blue tracking dye is in the separating gel) and the separating gel at 200 Volts.
12. Disconnect the power supply, just after the bromophenol blue tracking dye has left the bottom of the separating gel.

Electro blotting

This part of the protocol has to be carried out in the fume hood!. Wear powder-free gloves when handling gels, membranes and filter papers.
Methanol increases binding from the protein to the membrane.

1. Prepare 1 litre cold (4°C) 1* transfer buffer for 2 gels. (Don’t forget the methanol!) Number the membrane in a corner with a pen. Optional: cut about 0.5 cm from the membrane.
2. Pre-wet the membrane and blotting paper in the transfer buffer and immerse for 5 minutes.
3. Put the electrophoresis tank in a box with ice on the stirrer.
4. Set up buffer tank with cool element and small magnetic stirrer.

Disassembling the gel

5. Remove and open gel cassettes.
6. Take off the glass plates from the cassette.
7. Remove the spacers and the top plate using a spacer or forceps.
8. Remove the stacking gel.
9. Carefully remove the gel from the lower plate. The gel is electroblotted onto a nitrocellulose membrane filter for subsequent protein analysis.
10. Assemble blotting ‘sandwich’ as follows on black (negative side) of cassette in transfer buffer:
    NOTE: be aware of air bubbles, trapped in the sandwich.
    • soaked sponge
    • soaked sheet of blotting paper
    • gel, in mirror face
    • soaked membrane, with the number right below towards the gel
    • soaked sheet of blotting paper
    • soaked sponge
11. Close the clamp. Insert cassette into gel holder with the black side to black (negative) marker.
12. Place gel holder in the buffer tank in a box with ice on the magnetic stirrer.
13. Add 1* transfer buffer to the buffer tank till white clamp.
14. Blot for 1 hr at 100 Volts and stir slowly.
15. Clean the glass plates with detergent and water.
16. Meanwhile defrost the 10% blocking reagents at RT and prepare 2% blocking reagents (20 ml for each membrane).
    After blotting the cool element is filled with distilled water and kept in the -20°C freezer. Clean the equipment with water, dry afterwards. Store the dried sponges between paper towels.

Detection of proteins

Before incubation with conjugate the membranes are treated with hydrogen peroxide to reduce endogenous peroxidase activity, followed by pre-incubation with blocking agents to block unoccupied protein binding sites on the membrane.

1. Add 50-100 ml 3% H2O2 to a storage box. Immerse the membranes in the box with the protein side facing upwards. Carefully shake for 5 minutes.
2. Rinse the membranes in 50-100 ml 1* maleic acid.
3. Transfer each membrane to a glass box, protein side up (use forceps). Immerse the membrane in 2% blocking reagents (20 ml). Shake slowly for 40-50 minutes at RT.

After this step one proceeds with step 4 or store the (covered) boxes overnight at 4°C.

4. Rinse the membranes with 1* PBS-T.
5. Incubate each membrane in 20 ml conjugate (freshly made) for 40-60 minutes. Shake carefully.
6. Rinse the membranes with 1* PBS-T and then wash 6 * 5 minutes in ± 20 ml PBS-T. Shake carefully.
7. Prepare the Lumi-Light substrate solution: mix 3 ml of component I with 3 ml of component II in a blue capped tube (for 2 gels).
8. Drain the excess buffer from the washed membranes and place them in a plastic petridish or box.
9. Pipet 3 ml of the substrate solution on each membrane, protein side up; make sure that the entire membrane is soaked with substrate solution and does not dry out during the detection procedure.
10. Incubate for 1-5 minutes at RT.
11. Remove the membrane from the Lumi-Light substrate solution (using forceps). Let most of the solution drip off the membrane and place the membrane, with protein side up into a plastic folder. Gently smooth out air bubbles
12. Place the plastic folder, protein side up, in the Lumi-Imager on the glass plates. Reserve the Lumi-Imager by writing your name in the notebook from the Lumi-Imager.
13. Switch on the computer and start program LumiAnalyst by double clicking the icon. Click on LumiCapt and choose LumiAnalyst Capture; the window LumiAnalyst Acquisition will appear on the screen.
14. The temperature of the camera has to be -36°C. Check the temperature of the camera, choose Lumicapt, Temparature, Current temperature, Update.
15. Select Set and fill out the exposure time (10 – 30 seconds exposure gives good results).
    Save the file names automatically on D:\DNA-Diagnostiek\PTT-W[year]_[workformnumber]_[exposure time]. Click on Save. Save the files also on a Zip disk.
    At Binning select None, at Illumination select Chemiluminescence and for Auto save select Enabled.
16. After selecting the correct options click on Start, to start the exposure.
17. Selecting Adjust image Display Range under Edit can change the intensity.
18. To print the results, click on Print under File. The HP DeskJet 895C Series Printer on LPT1 has to be selected. Or make a printscreen.

Optional
Place the membranes, protein side up, in a film cassette and place a X-ray film on top of the membranes, close the cassette and make different exposures (30 seconds or 1 minute). Develop the films. If necessary, expose for a shorter or longer period (see ALG005)

Interpretation

1. Write the WPTTnr. and the exposure time on the printouts or the films.
2. Check the blank: one should see one intense band and two weaker bands originating from the lysate. Rabbit Reticulocyte Lysate contains one biotinylated protein, which migrates as a faint band at 100 kDa and, in some lots, an additional very faint band at 47 kDa [8].
3. Fill in the results in Oracle LIMS in the submenu ''invoeren testuitslagen''.
4. Store the WPTT form with the film and or the prints of the Lumi-Imager in the folder WPTT.

Protein marker

Table 4

Details of protein markers used in the Western Blot procedure.

Estimation of the part of the PTT fragment to be sequenced

1. Estimate with the ladder the size of the truncated PTT fragment in kDa.
2. In the Biolabs catalogue of Westburg it says that 1.0 kb DNA = coding capacity for 333 amino acids » 37000 Dalton protein
3. With the formula the part of the PTT fragment to be sequenced can be calculated. protein [Da]/factor 111= number of amino acids (1 amino acid = 3 bp).

Troubleshooting

1. Leakage of the electrophoreses cassette;
   • use parafilm under the cassettes and check cassettes for leaking, with water
   • Use silicone grease.
2. Fuzzy bands;
   Prepare the reaction with a 1.5 mM MgCl₂ concentration;

3. No bands;
   • use luciferase-encoding control plasmid as a control for the PTT reaction. The luciferase-encoding control plasmid will yield a 62 kDa protein after subjection to in vitro transcription/translation
   • pipet 0.7 ul luciferase-encoding control plasmid in a tube. Load 5 ul luciferase-encoding control plasmid on gel after the reaction is done (see In vitro transcription / translation step 8).

References

1.  Current Protocols in Human Genetics, unit 9.11, den Dunnen J.T.
2. Current Protocols in Molecular Biology, volume 2, section II, unit 10.2
3. Roest PAM, Roberts RG, Sugino S, Van Ommen GJB, Den Dunnen JT. Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum.Mol.Genet 2:1719-1721 (1993).
4. Hogervorst FBL, Cornelis RS, Bout M, Van Vliet M, Oosterwijk JC, Olmer R, Bakker E, Klijn JGM, Vasen HFA, Meijers-Heijboer H, Menko FH, Den Dunnen JT, Devilee P, Van Ommen GJB. Rapid detection of BRCA1 mutations by the protein truncation test. Nature Genetics 10:208-212 (1995).
5. Lumi-Light^PLUS Western Blotting Substrate protocol/ Roche Diagnostics
6. Lab FAQS/ Roche Molecular Biochemicals
7. Technical Bulletin, Transcend™ Non-Radioactive Translation Detection Systems/ Promega

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