【精华】vol 687 Chapter 3 采用Splinkerette-PCR技术对前病毒基因组插入位点进行分离
丁香园
PART II ：Cloning and Sequencing
Isolation of Genomic Insertion Sites of Proviruses Using Splinkerette-PCR-Based Procedures
Bin Yin
Abstract
The availability of whole genomic sequences provides a great framework for biologists to address a broad range of scientific questions. However, functions of most mammalian genes remain obscure. The forward genetics strategy of insertional mutagenesis uses DNA mutagens such as retroviruses and transposableelements; this strategy represents a powerful approach to functional genomics. A variety of methods to uncover insertion sites have been described. This chapter details SplinkTA-PCR and SplinkBlunt-PCR,modified from splinkerette-PCR, for mapping chromosomally the insertion sites of a murine leukemiavirus that causes leukemia in the BXH-2 strain of mice. These protocols are easy to use, reliable, and efficient.
Key words: Functional genomics, Insertion mutagenesis, Murine leukemia virus, PCR, Oncogene,Tumor suppressor gene, Leukemia
1. Introduction
With the whole genome sequences of human and major modelorganisms available, biological processes can be explored in anunprecedented way – questions with significantly increased broadnessand complexity are pursued on a large scale and at a systemiclevel. On the wave of interest in dissection of gene functions andbiological pathways, chromosome insertional strategies usingDNA elements such as retroviruses and transposons represent apowerful forward genetics approach to functional genomics,among other induced or engineered mutagenesis (1–5). TheseDNA mutagens integrate into host genome DNA and can altergene function. Through the identification of the chromosomalinsertion sites of the mutagens, a gene can be assigned a certain phenotype-related function. Recently, insertional mutagenesis hasbeen demonstrated to be fruitful in genome-wide analysis of cancergenes (6–13). The BXH-2 strain of mice constitutes a model ofhuman acute myeloid leukemia (AML), which arises from infectionby a murine leukemia virus (MuLV) (14). The BXH-2 MuLVnot only acts as an insertional DNA mutagen to cause leukemiabut also serves as a tag for leukemia-associated genes.
A variety of methods used to uncover insertion sites have previouslybeen described, including genomic DNA library screening(15), ligation-mediated PCR (6), inverse PCR (16), VISAtechnique (17), T-linker PCR (18), and single nucleotidepolymorphism-based mapping (19). These methods have beenadopted to generate a large amount of insertion sites; however,their inherent limitations are posed either by excessive laboriouswork, low cloning efficiency, restriction site-related cloning bias,or nonspecific amplification. To facilitate functional genomicstudies and maximize information that can be delivered from theuse of insertional mutagenesis strategy, it is crucial to capture ascomplete a genome-wide profile of insertion sites as possible. Theprotocols that will be elaborated in this chapter provide a timesavingtool allowing for less laborious, less biased, and more efficientmapping of insertion sites.
1.1. Splinkerette-PCR
The splinkerette-PCR is derived from vectorette-PCR (20), alsosimilar in principle to cassette ligation-anchored PCR (21),single-specific-primer PCR (22), rapid amplification of cDNA ends(23, 24), and rapid amplification of genomic DNA ends (25). ThisPCR begins with the digestion of genomic DNA with a restrictionenzyme, followed by ligation of a double-stranded DNA linker,namely splinkerette, to the digested DNA. The insertion flankingsequences are then PCR amplified using a pair of primers specific tothe integrated DNA and the splinkerette, respectively (26). Splinkerette-PCR features a hairpin structure present in thesplinkerette, which helps overcome the undesired amplification,so-called end-repair priming phenomenon that decreases the specificityof conventional linker-mediated PCR. Another advantage ofthe approach is the elimination of the requirement for circularizationthat can often be a problematic step in some types of PCR. Splinkerette-PCR has been used to characterize genomic integrationof provirus, transposon, and gene trap vector (6, 13, 27, 28).However, there is still a need for the development of more sophisticatedand streamlined protocols that better accommodate theemerging large-scale insertion site mapping efforts. In line with thistrend, a Web-based automated analysis and mapping of insertionalmutagenesis sequence data has recently become available (29).
1.2. SplinkTA-PCR
In our experience of cloning retroviral insertion sites, splinkerette-PCR worked better than inverse PCR; however, splinkerette-PCR mostly produced smears – there were rarely discernible discretePCR bands for recovery and cloning. This is likely due to excessivenonspecific amplification, which is consistent with other investigators’ observations (18). We also tried to clone the resulting smearPCR products for sequencing, only to find a very low percentageof positive colonies and an even lower number of specific PCRamplicons. With inverse-PCR, we observed even fewer PCR bands. In order to achieve saturation recovery of insertion sites, a moreefficient approach is needed. SplinkTA-PCR (STA-PCR) is developedfrom splinkerette-PCR, and its strategy is illustrated in Fig. 1a(30). This PCR approach starts from digestion of genomic DNAwith a cocktail of restriction enzymes generating 5? overhang ends. The digested DNA is purified and modified at the 3? end withaddition of an adenosine, by taking advantage of the terminal nontemplatedextension capacity of conventional Taq DNA polymerases(31, 32). This Addition step is followed by ligation toSplinkTA, a modified splinkerette linker, which is made up ofSplinkerette (the hairpin oligo) and PrimerLongTA (the oligowith an extra thymidine at 3? end). Primary PCR is then performedon the ligation products as templates using primers specific toBXH-2 MuLV and SplinkTA, respectively. In secondary PCR,templates are switched to primary PCR products, and primers to anested pair of primers specific to the long-terminal repeats ofBXH-2 MuLV and complementary to PrimerLongTA, respectively. Individual secondary PCR bands are resolved on agarose geland subsequently recovered for sequencing.
1.3. SplinkBlunt-PCR
SplinkBlunt-PCR is also modified from splinkerette-PCR (30). Inthis PCR method, as illustrated in Fig. 1b, genomic DNA is firstdigested with a cocktail of restriction enzymes. Next, a biotinylatedprimer is annealed to the digests to drive the primer extension reaction. Pfu DNA polymerase is used to produce blunt-end doublestrandedDNA fragments. The extension products are subsequentlypurified with streptavidin magnetic particles, prior to ligation toSplinkBlunt, which is made by mixing Splinkerette with PrimerLong(the oligo without extra thymidine at the 3' end), using the sameprocedure as in the STA-PCR protocol. Following ligation, thebiotin-bearing DNA fragments are captured magnetically, denatured,and separated from other unbound DNA fragments. Thesupernatant is collected and used as templates for subsequentprimary and secondary PCRs, which are performed similarly towhat was described in STA-PCR Protocol. The downstream procedurefollowing amplification is the same as that in STA-PCR.
Fig. 1. Schematic of strategies for cloning genomic DNA sequence flanking a proviral insertion. (a) STA-PCR protocol. Genomic DNA (horizontal lines) is digested with a cocktail of restriction enzymes (noted as E, F, and G), which have norecognition site downstream to the primers (horizontal arrow heads) used for primary and secondary PCR within proviralgenome sequence (black boxes). The digestion produces 5? overhang ends that are subsequently filled in and added anadenosine at the ends with Taq DNA polymerase. The filled-in products are ligated to SplinkTA, a modified Splinkerettewith a thymidine (empty star) at the end, which can exactly pair to the adenosine (filled star) at the end of extensionproducts. Then two consecutive rounds of PCR are performed followed by PCR products cloning and sequencing. (b) SplinkBlunt-PCR protocol. Genomic DNA (horizontal lines) is digested with restriction enzymes (X, Y, and Z in this case)in a similar way described in (a). After the digested double-stranded DNA is denatured, proviral genome sequence-specificprimer, which is biotinylated at its 5' end (filled circle), is annealed to its cognate region on single-stranded DNAfragments and immediately extended with Pfu DNA polymerase to produce blunt ends that are subsequently ligated toSplinkBlunt. Those biotin-bearing DNA fragments are then purified with streptavidin magnetic particles prior to tworounds of PCR and products cloning as performed in (a) (Taken from Yin, B. et al., BioTechniques 2007. . 2008BioTechniques. Used with permission).
STA-PCR and SplinkBlunt-PCR can consistently recovermore insertion sites of BXH-2 MuLV with high specificity (foran example, see Fig. 2). The high efficiency of SplinkTA-PCRmay be attributed to the integration of the following improvements: (1) Use of multiple enzymes rather than a single enzyme for one digestion reaction gives rise to more readily amplifiableDNA fragments, reduces insertion cloning bias introduced by single restriction enzyme-dependent digestion, and saves timeand effort by alleviating the requirements of more reactionsand linkers which are necessary for single enzyme-based protocols;(2) Adoption of splinkerette eliminates the end-repairpriming phenomenon and thereby results in greater efficiency in ligation-mediated PCR (26); (3) Formation of the SplinkTAin STA-PCR with an extended thymidine at the end increasesligation efficiency, maximizes its compatibility with a variety ofdigestion fragments generated by different restriction enzymes,and decreases the likelihood of forming tandem blunt ligationproducts; (4) Inclusion of primer extension and purificationsteps in SplinkBlunt-PCR eliminates the majority of nontemplateDNA that would adversely affect efficiency of downstreamPCR, since the magnetic beads used in the purification onlybind and retain double-stranded DNA fragments formed viastreptavidin-labeled primers.
Fig. 2. Application of PCR-based protocols to large-scale screen of proviral insertionpatterns in BXH-2 leukemias. Lanes 1–17 are leukemia samples. The white dots denotethe PCR products representing variant somatic acquired proviral insertions. M: 100-bpDNA ladder (Taken from Yin, B. et al., BioTechniques 2007. . 2008 BioTechniques. Usedwith permission).
When comparing between STA-PCR and SplinkBlunt-PCR,the former has fewer steps and reduced likelihood of formation oftandem ligation. SplinkBlunt-PCR is characterized by cleanerPCR gel background and no limitation in choice of restrictionenzymes to 5? overhang-producing enzymes because both 3'overhang and blunt DNA fragments can be primer extended. Inaddition, although comparable efficiency was observed for STAPCRand SplinkBlunt-PCR in cloning of BXH-2 insertion, thereare slight differences between these two methods in the recoveryprofile of insertions (see Fig. 3). The ability of STA-PCR and SplinkBlunt-PCR to recoverinsertion sites could be enhanced by digesting genomic DNAwith more than one combination of restriction enzymes. We havesuccessfully tested various combinations of restriction enzymes,including: (1) Aat II, Mfe I, Nde I, (2) Hind III, Pvu II, Xho I,or (3) Ase I, Bgl I, Eag I, with different resultant PIS patterns. Further improvement of PIS recovery can be made by runningthrough the cloning procedures for both upstream and downstreamof the DNA integrated. We expect that with minor modification, these protocols can be readily adapted to identifyinsertion sites of other types of insertional mutagens, such astransposons, retrotransposons, etc. The techniques can also beapplied to determine endpoints of genomic DNA fragments,chromosomal breakpoints involved in deletion or translocation,intron–exon junctions and gene regulatory regions.
Fig. 3. Comparison of PCR band patterns amplified from leukemias using SplinkBlunt-PCR and STA-PCR protocols. B112, B68, and B75 are BXH-2-derived leukemia samples. NC: healthy BXH-2 mice tail DNA used as a control. M: 100-bp DNA marker. The whitearrows indicate the differential bands. The size of DNA marker in base-pair is noted tothe left (Taken from Yin, B. et al., BioTechniques 2007. . 2008 BioTechniques. Used withpermission).
2. Materials
2.1. Extraction of Genomic DNA from Cell Culture or Tissues
1. TE buffer: 10 mM Tris–HCl (pH 8.0) and 5 mM EDTA (pH8.0). Stored at 4°C.
2. STE buffer: 1 mM EDTA, 10 mM Tris–HCl (pH 8.0) and0.5 M NaCl. Stored at room temperature.
3. Proteinase K: dissolved in TE buffer at 10 mg/mL. Storealiquots at -20°C.
4. RNase A: dissolved in TE buffer at 2 mg/mL. Store aliquotsat -20°C.
5. Lysis buffer: mix 4.6 mL of STE, 100 mL of 0.5 M EDTA,and 200 mL of 10% sodium dodecyl sulfate (SDS). Add100 mL of 10 mg/mL proteinase K and 20 mL of 2 mg/mLRNase A prior to use (see Note 1).
6. Saturated phenol (pH ~7.9): Stored at 4°C. Avoid lightexposure.
7. Chloroform: Stored in hazardous chemical carbine.
8. Phenol:chloroform (1:1): Stored at 4°C. Avoid light exposure.
9. Isopropanol: Stored in hazardous chemical carbine.
10. 70% Ethanol: Stored at 4°C.
2.2. STA-PCR (Including DNA Digestion, Extension, Ligation, PCR Amplification, and Examination)
1. Restriction enzyme and digestion buffer: BspLU11 I, Bcl I,and Taq I (New England Biolabs). Stored at -20°C exceptfor BspLU11 I that is stored at ?80°C (see Note 2).
2. Silicon oil.
3. QIAquick Nucleotide Purification Kit (Qiagen).
4. QIAquick PCR Purification Kit (Qiagen).
5. Splinkerette oligonucleotides (see Table 1 for sequences). Resuspended in TE buffer to the concentration of 100 mM. Store at -20°C.
6. T4 DNA ligase and ligation reaction buffer (Promega). Storeat ?20°C.
7. PCR grade water or Millipore purified water.
8. Biolase Taq DNA polymerase (Bioline USA Inc.). Store at?20°C.
9. 10× PCR reaction buffer and 50 mM MgCl2, supplied withBiolase (Bioline USA Inc., Taunton, MA).
10. dNTPs: Resuspended in PCR grade water at 10 mM for eachdeoxynucleotide. Store aliquots at -20°C.
11. PCR primers. Resuspended in TE buffer at 100 mM anddiluted further to 20 mM as working solutions (see Table 1). Store at -20°C.
12. Agarose powder.
13. Ethidium bromide at 10.0 mg/mL (Invitrogen).
Table 1
Sequence (5‘→3') of oligonucleotides used in STA-PCR and SplinkBlunt-PCR
14. 50× TAE stock solution: 0.242 g/L Tris Base, 5.7% glacialacetic acid, 50 mM EDTA (pH 8.0).
15. 100-bp DNA ladder (Invitrogen). Store at -20°C.
16. GeneAmp PCR system 9700 (PE Applied Biosystem).
2.3. SplinkBlunt-PCR (Including DNA Digestion, Extension, Ligation, PCR Amplification, and Examination)
1. Restriction enzyme and digestion buffer: BspLU11 I, Bcl I andTaq I, or other enzymes of your choice (New England Biolabs).
Stored at -20°C except for BspLU11 I that is stored at -80°C.
2. Silicon oil.
3. QIAquick PCR Purification Kit (Qiagen).
4. Pfu DNA polymerase (5 U/mL) and 10× PCR Buffer (Stratagene).
5. dNTPs: Prepared and stored as described in Subheading 2.2.
6. Biotinylated primer Bio-AKVp7711 (see Table 1 for sequences).
7. Microcon YM-30 column (Millipore).
8. Streptavidin-coupled Magnetic Particles (Roche).
9. Magnetic Particle Concentrator (MPC) (Roche).
10. 2× BW Buffer: containing 10 mM Tris–HCl (pH 7.5), 1 mMEDTA, and 2 M NaCl.
11. 0.1 N NaOH.
Other reagents required are the same as listed in Subheading 2.2.
2.4. PCR Product Cloning and Sequencing
1. In-gel DNA purification kit (Qiagen) (see Note 3).
2. TOPO-TA cloning vector kit. Store at -20°C (see Note 4).
3. TOP10 competent bacteria (Invitrogen). Store at ?80°C (seeNote 5).
4. Luria Bertani (LB) medium: 10 g/L of tryptone, 5 g/L ofyeast extract, and 10 g/L of NaCl (Note: For bacteria culture)(see Note 6).
5. S-gal LB powder (Sigma, St. Louis, MO) (see Note 7).
6. Solution I: 50 mM glucose, 25 mM Tris–HCl (pH 8.0), and10 mM EDTA (pH 8.0). Filter sterilized. Store at 4°C.
7. Solution II: 0.1 M NaOH and 1% SDS. Store at 4°C.
8. Solution III: 3 M potassium acetate and 5 M glacial aceticacid. Store at 4°C.
3. Methods
3.1. Extraction of Genomic DNA from Cell Culture or Tissues
1. Collect tissues or pellet cells into 15-mL conical polypropylenetubes (see Note 1). Add appropriate amount of lysis buffer forDNA extraction and incubate at 56°C overnight with shaking.
2. Add an equal amount of Tris-buffered phenol to samples. Mixthoroughly by inversion. Centrifuge at 2,500 × g for 5–10 min.
3. Transfer aqueous phase (top layer) to new 15-mL conicalpolypropylene tubes using pipet tips cut at the end to avoidgenomic DNA shearing. Take care not to take any undissolvedtissue or white precipitates between layers.
4. Add equal volume of phenol:chloroform (1:1) and mix thoroughlyby inversion for 5–10 min.
5. Centrifuge at 2,500 × g for 5–10 min and transfer the aqueousphase to new 15-mL conical polypropylene tubes.
6. Add equal volume of chloroform and mix thoroughly byinversion for 5–10 min.
7. Centrifuge at 2,500 × g for 5–10 min and transfer the aqueousphase to new 15-mL conical polypropylene tubes. If theaqueous phase is not clear, repeat step 2.
8. Add 0.7 volume of isopropanol and mix thoroughly by gentleinversion.
9. Genomic DNA is usually visible at this step as fluffy or silkywhite material. Proceed to transfer the DNA into a new 1.5-mLcentrifuge tube with a pipet tip and allow to air dry. If DNApellet is not visible, centrifuge at 2,500 × g for 10 min at 4°C topellet genomic DNA.
10. Discard supernatant and wash with 70% ethanol.
11. Centrifuge at 2,500 × g for 10 min at 4°C to pellet genomicDNA again.
12. Discard supernatant and add 100–300 mL of TE buffer.
13. Incubate at 56°C for 30 min to let genomic DNA dissolveinto the TE buffer. For a more complete dissolve, incubate at37°C overnight.
14. Determine genomic DNA concentration using a spectrophotometer. DNA can then be aliquoted and stored for long termat -20°C.
3.2. STA-PCR (Including DNA Digestion, Addition, Ligation, PCR Amplification, and Examination) (see Notes 8–10)
1. Digestion: Digest 2 mg of genomic DNA with 7.5 U of Taq Iat 65°C for 4 h in a final volume of 50 mL, followed by another4-h digestion with 7.5 U BspLU11 I and 10 U Bcl I at 48°C(see Notes 11–13).
2. Purification: Purify the digestion products (from step 1) withQIAquick PCR Purification Kit. According to the directionsprovided by the kit, add 250 mL Buffer PB to each sampleand apply the mixture to the mini columns. Centrifuge at11,000 × g for 30 s. Wash with 750-mL Buffer PE at 11,000 × gfor 30 s and centrifuge the column for an additional 60 s atmaximum speed. Add 30-mL H2O to the columns and spin toelute the DNA bound to the membrane.
3. Addition: To each purified digestion (from step 2), add thefollowing:
Incubate at 72°C for 20 min
(see Notes 14 and 15).
4. Purification: purify the extension reaction with QIAquickPCR Purification Kit as described in step 2.
5. Making SplinkTA: mix 20 mL of 100 mM Splinkerette and20 mL of 100 mM PrimeretteLongTA. Incubate at 80°C for5 min. Cool to room temperature (20–25°C) (see Note 16).
6. Ligation: To 5 mL of each purified sample (from step 4), addthe following:
Incubate at 16°C overnight.
Note. For multiple samples, prepare a master mix.
7. Purification: purify the ligation reaction with QIAquick PCRPurification Kit as described in step 2.
8. Primary PCR: In 0.2 mL thin-wall PCR tubes, compose thefollowing components for one reaction:
The primary PCR cycling conditions are as follows:
(a) 95°C for 1 min 30 s
(b) 10 cycles of
●● 95°C for 5 s
●● 70°C for 3 min 10 s with decreasing 0.5°C each cycle(c) 20 more cycles of
●● 95°C for 5 s
●● 65°C for 3 min 10 s
(d) Final extension at 70°C for 10 min
(e) 4°C hold
9. Secondary PCR: all components in each reaction are the sameas those in primary PCR (step 8) except for using primaryPCR products as templates and replacing the pair of primerswith nested primers AKVp8712 and P-nest (see Note 17).
The amplification parameters in secondary PCR are changed to:
(a) 95°C for 1 min
(b) 11 cycles of
●● 94°C for 15 s
●● 70°C for 2 min 40 s with decreasing 0.6°C each cycle(c) 25 more cycles of
●● 94°C for 15 s
●● 64°C for 30 s
●● 70°C for 2 min 10 s
(d) Final extension at 70°C for 10 min
(e) 4°C hold
10. Check secondary PCR results by resolving 10 mL of eachsample in 1% agarose gel and examining on a UV-light box.
3.3. SplinkBlunt-PCR (Including DNA Digestion, Extension, Ligation, PCR Amplification, and Examination) (SeeNotes 9 and 18)
1. Digestion: 2 mg of genomic DNA are incubated with Taq I at65°C for 4 h followed by another 4-h digestion with BspLU11I and Bcl I at 48°C (see Notes 11, 13, 19, and 20).
2. Purification: Purify the extension reaction with QIAquick PCRPurification Kit as described earlier in STA-PCR (step 2).
3. Extension: To each purified digestion (from step 2), add thefollowing:
Incubate at 95°C for 5 min, 64°C for 30 min, and 72°C for20 min.
Note. For multiple reactions, making mastermix is highlyrecommended.
4. Purification: Purify the extension reaction withMicroconYM-30 column according to the manufacturer’sinstructions. Briefly, add 450 mL of H2O to each sample, andtransfer to the column. Centrifuge at 14,000 × g for 10 min. Place the column upside down and centrifuge for 3 min.
5. DNA capture: To recover the Bio-AKVp7711-derived extensionproducts, wash 10 mL of Streptavidin-coupled MagneticParticles per sample with 50 mL of 2× BW Buffer. Resuspendthe particles in 40 mL of 2× BW Buffer, then mix with thepurified extension products (from step 4), and incubate atroom temperature (20–25°C) for 1–3 h. Capture the biotinbearingDNA fragments using Magnetic Particle Concentrator,and wash twice with H2O.
6. Making SplinkerBlunt: Mix 20 mL of 100 mM Splinkerette and20 mL of 100 mM PrimeretteLong. Incubate at 80°C for 5 min. Cool down to room temperature (20–25°C) (see Note 16).
7. Ligation: The captured DNA was ligated to the SplinkerBlunt.To 5 mL of each purified sample (from step 5), add thefollowing:
Incubate at 16°C overnight.
Note. For multiple samples, prepare a master mix.
8. Recapturing DNA: After ligation, the biotin-bearing DNAfragments (from step 7) are captured again in MPC andwashed with 100 mL of H2O. Denature the recaptured DNAin 5 mL of 0.1 N NaOH at room temperature for 10 min. Separate the magnetic particles from released DNA fragments with MPC. Save the supernatant as templates for subsequentprimary and secondary PCR.
9. Primary PCR: In 0.2 mL thin-wall PCR tubes, compose thefollowing components for one reaction:
The primary PCR cycling conditions are as follows:
(a) 95°C for 1 min 30 s
(b) 10 cycles of
●● 94°C for 15 s
●● 70°C for 3 min 10 s with decreasing 0.6°C each cycle(c) 20 more cycles of
●● 94°C for 15 s
●● 64°C for 30 s
●● 70°C for 2 min 30 s
(d) Final extension at 70°C for 10 min
(e) 4°C hold
10. Secondary PCR: Performed as described in STA-PCRProtocol (step 9).
11. Check secondary PCR results by resolving 10 mL of eachsample in 1% agarose gel and examining on a UV-light box.
12. An example of large-scale cloning PCR results produced isshown in Fig. 2 (see Note 21).
13. A comparison of STA-PCR and SplinkBlunt-PCR results isshown in Fig. 3 (see Note 22).
14. A comparison of SplinkBlunt-PCR and Southern blottingresult is shown in Fig. 4 (see Note 23).
Fig. 4. Comparison between SplinkBlunt-PCR and Southern blot assay in detectingdifferential proviral insertions. The genomic DNA from Ara-C-sensitive cell line B117P(lane S ) as a passage control, or Ara-C-resistant cell line B117H (lane R ), was subjectedto proviral insertion cloning PCR protocol (a) or Southern blotting analysis (b). Arrowsindicate the differential insertion (Taken from Yin, B. et al., BioTechniques 2007. . 2008BioTechniques. Used with permission).
3.4. PCR Product Cloning and Sequencing
1. Run secondary PCR products on preparative 1% agarose gelcontaining ethidium bromide (see Note 24).
2. Cut out bands with sterile blades and recover DNA usingQIAquick Gel Purification kit following the manufacturer’sinstructions.
3. Use TOPO TA Cloning Kits according to the manual exactly. Ligate 4 mL of recovered PCR bands with 1 mL of TOPO-TA cloning vector in the presence of 1 mL of 6× ligation reactionbuffer, transform the TOP10 competent cells, and spreadbacteria culture onto S-gal LB plates containing the appropriateantibiotic for the cloning vector used. For each PCR band,3–5 white colonies are typically picked to determine positivetransformants (see Notes 25–27).
4. Plasmid DNA is prepared from positive colonies for sequencingusing primer AKVp8753.
5. DNA sequences are aligned with Ensembl Mouse GenomeDatabase to map the chromosomal position of insertion sites.
4. Notes
1. Recipe is given here for 2–10 × 107 cells or 100–200-mg tissues;it can be scaled up or down for more or less cells/tissues.
2. Any restriction enzymes that produce 5? overhang should, inprinciple, work equally well. Indeed, we tried other enzymesgenerating 5? overhang and obtained different PCR bandpatterns. We therefore recommend comparing various combinationsof enzymes before deciding which enzymes to usein a particular insertion site cloning work.
3. Other economic kits, such as glass binding-based methods,also work very well. We found that the Qiagen kit designedfor purification of DNA from agarose gel is relatively convenient,giving rise to high yield and clean PCR products.
4. For cloning of large PCR products, TOPO-TA-XL worksbetter for us.
5. For transformation of ligated insert/vector DNA: In our hands,the TOP10 competent cells from Invitrogen give rise to efficienttransformation; however, they are relatively expensive. To reducethe cost, the use of smaller than recommended amount of cells,as low as 25 mL per transformation, works equally well.
6. Use of the S.O.C. medium is optional here, which contains 2%tryptone, 0.5% yeast extract, 2.5 mM KCl, 10 mM NaCl, 10 mMMgCl2, 10 mM MgSO4, and 20 mM glucose with pH adjustedto 7.0 ± 0.2. The S.O.C. bacteria culture medium generallyworks better, producing more colonies after transformation.
7. Making LB plates: Add the appropriate antibiotics for selectionfor transformed colonies.
8. The STA-PCR procedure typically requires less than 7 daysfor completion of mapping insertions.
9. Take great care to prevent PCR cross-contamination. Common solutions include using aerosol-resistant filter pipettips, dedicated PCR working area and reagents, and theirUV-light exposure, etc.
10. When compared with the T-linker PCR, STA-PCR featuresthe combined advantages of adoption of splinkerette, use ofan enzyme cocktail, and fewer experimental steps. In addition,since STA-PCR involves complete digestion with 5? overhangenzymes, there is no limitation by the need to perform theincomplete digestion – which can often go out of control –with 3' overhang enzymes required by T-linker PCR.
11. Digestion with multiple restriction enzymes (Taq I, Bcl I, andBspLU11 I) not only gives rise to shorter DNA fragments onaverage than digestion with a single enzyme and renders thefragments more readily PCR amplified, but also reduces cloningbias introduced by single restriction enzyme-based methods aswere used in previous reports. This is an important feature thatsaves time and effort by alleviating the need for extra reactionsand types of linkers necessary for single enzyme approaches. This is also likely to increase the specificity of PCR.
12. Choice of restriction enzyme cocktails can be tailored to theparticular interest of individual studies.
13. Use of silicon oil to cover the top of digestion solution is recommendedto prevent reaction loss arising from liquid evaporation.
14. For multiple samples, making master mix is highly recommendedfor easier and consistent handling.
15. Taq DNA polymerases are used here in order to form stickydouble-stranded DNA ends with an extruding adenosinethrough the terminal nontemplated adenosine additionreaction. To test whether this approach can increase ligation efficiency, the Taq-treated digests were ligated to the pCR2.1-TOPO vector (Invitrogen) that has a 3? thymidine, followedby transformation of competent cells. We found that theTaq-treated digests produced a higher number of transformantsthan did the untreated digests as a control.
16. To do this, we use a heating block preheated to 80°C, followedby turning it off and allowing it to slowly cool down to roomtemperature. Alternatively, this can be done using a PCRamplifier with a decreasing 0.5°C per min over 2 h.
17. If no discrete bands are visible on gel, we recommend tryingto amplify 1:20–200 dilutions of primary PCR products insecondary PCR.
18. The procedure described here typically requires less than10 days for completion of mapping insertions.
19. In principle, any other combination of restriction enzymescan be applied which do not cut the integrant DNA downstreamto the primer used in the extension reaction (also seethe description of extension reaction).
20. For a similar reason as with digestion in STA-PCR, it is recommendedthat comparison of different combinations of enzymes be made to select a favorable enzyme cocktail, priorto a large-scale insertion site cloning project.
21. The presence of two germline proviral insertions in BXH-2tumors poses a heavy interference with isolation of somaticacquired proviral insertions, which made the proviral insertioncloning very challenging.
22. Because STA-PCR and SplinkBlunt-PCR give rise to differentPIS patterns in some samples (for an example, see Fig. 3),use of both procedures would improve PIS recovery rate. Inour experience, nonoverlapping PISs account for approximately10% of total PISs isolated. Therefore, combination ofthe two protocols is highly recommended in some cases wherea full or nearly full recovery is desired.
23. When comparing our PCR methods with Southern blottinganalysis, we found a close-to-full recovery of insertions byPCR, with 197 insertions amplified out of 80 tumors, versus68 insertions detected by Southern from 29 BXH2 tumors.
Given the proviral insertion recovery rate achieved, thesePCR protocols represent an improved method for proviralinsertion site cloning in BXH-2 tumors.
24. To increase gel resolution for smaller PCR bands, up to 2%agarose gel can be readily used here.
25. Alternatively, sequence directly the DNA samples that arerecovered from strong and pure bands with internal sequencingprimer AKVp8753.
26. If the PCR bands are too faint, it is sometimes helpful to PCRreamplify the recovered DNA prior to TA cloning of the PCRproducts or direct sequencing.
27. The use of pCR畗4-TOPO vector (Invitrogen) may significantlyreduce transformation background because self-ligatedvectors are lethal to competent cells.
Acknowledgments
The author would like to express thanks to Ms. Marianna L. Wongand Mr. John W. Myers III for their help with grammatical editing. This work is supported by the National Basic Research Program ofChina (973 Program, No. 2011CB933501, to Dr. Bin Yin) andthe NSFC Project (No. 81070417, to Dr. Bin Yin).
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