Cloning Insertion Sites by Splinkerette PCR - Updated June 2005 protocol based on Devon et al. (1995) Nucleic Acids Research 23:1644-1645 and Mikkers et al. (2002) Nature Genetics 32:153-159 for questions/suggestions e-mail Anthony Uren a.uren@nki.nl Splinkerette Ligation Digest 1-3ug genomic DNA at 100ng/ul with enzyme of choice and heat inactivate. 6 base cutter average length = 4096bp, 1ug DNA = 0.4 pmole 5 base cutter average length = 1024bp, 1ug DNA = 1.6 pmole 4 base cutter average length = 256bp, 1ug DNA = 6.4 pmole We cut for 12+ hours with more than the recommended number of units to ensure complete digestion. We normally use SauIIIA and Tsp509I, giving GATC or AATT lower strand 5 overhangs (SauIIIA we inactivate for 20minutes at 65, Tsp509I for 20 minutes at 85). Doing two ligations/pcrs using two different enzymes (eg. SauIIIA and Tsp509I) yields more insertions from each tumour than using just one enzyme. The amount of redundancy seen between two enzymes varies from tumour to tumour. Tsp509I seems to give us better results than SauIIA. Have not tested other 4 base cutters. 6 or 5 base cutters usually give a better distribution of fragment sizes over a gel but a 4 base cutter but will usually give more insertion sites overall. Splinkerette adaptor Top strand primer Splinklong 5 cga aga gta acc gtt gct agg aga gac cgt ggc tga atg aga ctg gtg tcg aca cta gtg g Lower strand primer/hairpin Splinkshort-GATC (has GATC overhang in this case) 5 gatccc act agt gtc gac acc agt ctc taa ttt ttt ttt tca aaa aaa Dilute and mix splinkerette adaptor oligos at highest concentration possible in 1x Boehringer RE buffer M (10mM TrisHCl, 10mM MgCl2 50mM NaCl ph7.5) Use thermal cycler to denature at 95 o C for 3 min followed by cooling to room temp at 1 o C per 15 sec (4 o C per min). Dilute annealed splinkerette to 4pmol/ul.
Ligation Immediately before setting up ligation, heat DNA to 60 o C and put on ice. This is supposed to melt annealed sticky so they are free for ligation. I haven t checked whether it really helps. Use 300ng cut DNA 6bp cutter = 0.12 pmole 5bp cutter = 0.48 pmole 4bp cutter = 1.92 pmole Mix - 300 ng cut DNA - 10x molar excess of splinkerette - 4U T4 ligase (Roche) - 4ul 10x buffer - H 2 O to 40ul Incubate overnight at 16 degrees. EcoRV Digestion Inactivate ligase at 65 degrees C for 20 minutes. Cut 40ul ligation with 10U EcoRV in 100ul for 3+ hours. Inactivate 20 mins at 65 degrees. This step prevents amplification of internal Moloney MuLV fragments from the internal U3 LTR sequence by cutting an EcoRV site 100 bases in from the internal U3 LTR. If your primers are in a sequence that is repeated internally (in this case the U3LTR) you will need to find an appropriate enzyme to prevent amplification of internal fragments i.e. any enzyme that is closer to your internal primer than the enzyme you are using for the splinkerette, in this case EcoRV is closer to the internal LTR than SauIIIA or Tsp509I.
Ligation cleanup Desalt/concentrate in Microcon YM-30 Some samples spin down much slower than others. Usually to keep volumes similar between samples we spin everything dry and add 50ul water, wait 10mins and then collect sample. For individual columns add 400ul H2O, microfuge ~ 10min at 14000RPM/20,000g. For 96 column retentate assembly add 150ul H2O, centrifuge ~ 20min at 4000RPM/3000g You can quantiate the DNA in the ligation or you can just proceed assuming 100% recovery of DNA from column. PCRs usually work OK when you assume 100% recovery. If quantitating, Microcon columns leave you with a high 260/280 ratio. Hoechst or Pico green quantitation is probably better than A260/A280. Residual linker may also affect quantiation but doesn t seem to influence PCR.
Amplification 1 o PCR - 100ng ligated DNA (about 1/3 of total ligation) - 200nM each primer (Splink1 and LTR#5) - 200uM dntps - 1-4U Pfu Turbo hotstart (Stratagene) - 1x Pfu Turbo buffer (with 2.0mM MgCl2 included) - H2O to 50 ul 4U Pfu Turbo seems best but this is expensive. Can also use - 1U Pfu Turbo in 2mM MgCl2-1U Expand High Fidelity Plus (Roche) in 1.75mM MgCl2-1U of Finnzymes Phu Phusion with 1.5mM MgCl2. Volume of reaction can also be reduced to cut enzyme costs. Using PTC 100 Perkin Elmer 3min 94 o x 1 15sec 94 o, 30sec 68 o, 5min 72 o x 2 15sec 94 o, 30sec 66 o, 5min 72 o x 27 5min 72 o x 1 Can shorten extension time to 3min 30sec but 5 min is slightly better (at least for Pfu). Other higher processivity or less thermo stable enzymes might require shorter extensions times. 2o PCR - 2ul of 1 o PCR as template (0.5-2ul works just as well) - 200nM each primer (Splink2 and LTR#3 or LTR#1) - 25ul Qiagen Multiplex PCR kit mix - H2O to 50ul Qiagen Multiplex gives slightly better signal for larger fragments and for fainter bands but is more expensive. Can also lower the volume to 25ul or use 1U Taq (Invitrogen) with 1.75mM MgCl2, 200nM dntps. Using PTC 100 Perkin Elmer 15min* 94 o x 1 15sec 94 o, 30sec 60 o, 3min 72 o x 25 5min 72 o x 1 *15min start step is only required for Qiagen enzyme. For Taq use 3min.
To look at PCRs Running PCR on 3-4% agarose gel should (depending on the DNA) give you a ladder of fragments ranging from 50bp to 1500bp. Can use FAM labeled 2 o PCR primers and run 2ul on capillary sequencer along with size marker to give a better estimate of fragment sizes. Bands can be cut from a 4% agarose gel and in some cases sequenced directly or in other cases reamplified/subcloned and then sequenced. For better separation of bands use radiolabelled PCRs run on a polyacrylamide gel. Radioactive amplification (taken from Harald Mikkers protocol) 1 o PCR - Label primer with 32P-gamma using PNK. Use 10pmol of radioactively labeled primer. Fragments are separated on a 3.5% denaturing acrylamide gel (separation of 100bps-1000 bps). Dry gel, expose to a film and excise radioactive fragments. Put the samples into 100 ul TE and heat the excised fragments for 15 at 94 o C. Use 1:100 of this mixture for a nested cold reaction or if a tumor contains a lot of integrations a second radioactive PCR might be required. 2 o PCR - Similar as previously described except for the number of cycles. Use 1 pmol of radioactively labeled primer. Fragments are separated on a 3.5% denaturing acrylamide gel (separation of 100bps- 1000 bps). Dry gel, expose to a film and excise radioactive fragments. Put the excised fragments into 100 ul TE and heat for 15 at 94 o C. Use 1:100 of this mixture for a cold reaction.
Shotgun subcloning (less effort but more expensive) Prior to TA cloning PCR products can be cleaned up using Microspin S-300 HR columns (Amersham) for individual samples or GFX-96 PCR Purification columns (Amersham) for a 96 well plate full of samples. Freeze until ready to ligate Topo TA vector shotgun subcloning (Invitrogen) (Method from Invitrogen/Sanger) Put 1ul of PCR product in each tube. Dilute the salt solution provided with topo kit 1 in 4. Note: The use of ¼ strength salt solution allows the ligation to be incubated longer than without salt, but is dilute enough for electroporation of 1ul of ligation without arcing. Mix up the following: Per reaction: 1ul of dilute salt solution 3.9ul of H2O (from kit) 0.14ul Topo TA vector (i.e. 1/7 of recommended amount) Per 48 reactions: 50ul of dilute salt solution 195ul of H2O (from kit) 7ul of Topo TA vector (i.e. 1/7 of recommended amount) Add 5ul of master mix to each tube containing 1ul PCR product. Incubate 20 min at room temperature and stop reaction by freezing at -80 degrees. Note: Using only 1/10-1/7 of the recommended amount of vector means that the vector is limiting in the ligation and the PCR product is in excess. This means most ligations will yield similar numbers of transformants regardless of variability in the amount of DNA in the PCR product. Method used to transform the ligation will determine how much of it should be used. Using 1ul for electroporation (Invitrogen electromax DH10Bs or Topo10s) yields over 500 colonies. The number of clones which need to be sequenced to identify the majority of prominent bands in the PCR will depend on the complexity of the PCR. Doing a second splinkerette ligation/pcr using another restriction enzyme will usually yield more new sequences than just increasing numbers of clones sequenced for one PCR.
Primer sequences 1 o PCRs - Splink1 cga aga gta acc gtt gct agg aga gac c 2 o PCRs - Splink2 gtg gct gaa tga gac tgg tgt cga c Vector/virus primers will vary depending on what vector/virus you are retrieving. Test primers on infected and uninfected DNA to find ones which give the most acceptable combination of good signal vs. minimum amplification of endogenous virus like sequences (large amounts of the mouse genome consists of retrovirus/ltr like sequences). If you want to sequence from the PCR product itself it is good to have primers set back from the insertion site so you can see the exact base of integration. We find quality of the oligos can affect the results. Start by ordering a few separate batches from different suppliers. Each new batch of primer should be tested against an older batch which you know works. HPLC puried oligos aren t necessarily better than desalted ones, we just see large amounts of batch to batch variation.
Moloney U3LTR primers Have tried a range of Splinkerette and U3 LTR primers and changing these doesn t seem to matter too much so long as there is sufficient nesting (6bp or more). We use LTR#5 for the primary and LTR#3 or LTR#1 for the secondary. U3LTR#1 might be giving us the most signal for a secondary PCR. U3 LTR sequence EcoRV SauIIIA GATATCCTGTTTGGCCCATATTCAGCTGTTCCATCTGTTCCTGACCTTGATCTGAACTTCTCTATTCTCAGTTATG U3 LTR#8 TTCCTGACCTTGATCTGAACTTCTCTATTCTC U3 LTR#7 CCTGACCTTGATCTGAACTTCTCTATTCTCA U3 LTR#6 CTGACCTTGATCTGAACTTCTCTATTCTCAGTTAT TATTTTTCCATGCCTTGCAAAATGGCGTTACTTAAGCTAGCTTGCCAAACCTACAGGTGGGGTCTTTCATT -LTR end- U3 LTR#5 GCGTTACTTAAGCTAGCTTGCCAAACCTAC U3 LTR#4 CGTTACTTAAGCTAGCTTGCCAAACCTACA U3 LTR#3/AB949 GCTAGCTTGCCAAACCTACAGGTGG U3 LTR#2 TTGCCAAACCTACAGGTGGGGTCT HM001 GCCAAACCTACAGGTGGGGTCTTT U3 LTR#1 CCAAACCTACAGGTGGGGTCTTTC