Lot: ___ Exp.: ___
Quantity: for 20 reactions
1 One unit of Exonuclease III catalyzes release of 1 nanomole of acid soluble reaction products from E.coli [3H]-DNA in 30min at 37°C.
2 One unit of S1 nuclease produces 1µg of acid soluble deoxyribonucleotides in 1min at 37°C.
3 One unit of Klenow fragment catalyzes the incorporation of 10 nanomoles of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30min at 37°C, using poly(dA-dT)·poly(dA-dT) as a template·primer.
4 One unit of T4 DNA ligase catalyses conversion of 1 nanomole of [32PPi] into a Norit®-adsorbable form in 20min at 37°C (Weiss unit).
5 A SalI+PaeI digest of pBR322 DNA yields the following 2 discrete fragments (in base pairs): 4272, 89. Since PaeI restriction endonuclease forms ExoIII resistant 3' overhangs, meanwhile SalI produces ExoIII available ends, the degradation of both fragments will follow at one side.
ExoIII/S1 Deletion Kit is used for rapid construction of plasmid or M13 subclones with nested unidirectional deletions. The method is applicable for DNA sequencing, mapping of boundaries of regions involved in genetic control, DNA/protein interaction sites, or protein domains [1-3]. This strategy, for example, allows sequencing of large DNA fragments using a single universal priming site instead of synthesizing a new primer for each few hundred nucleotides.
The method is based on the controlled unidirectional degradation of one strand of double-stranded DNA with Exonuclease III (ExoIII) followed by removal of the remaining single-stranded overhang with S1 nuclease. ExoIII catalyzes the stepwise removal of mono-nucleotides from double-stranded DNA with free 3'-recessed (5'-overhang) or blunt ends as well as nicks in the DNA template, whereas 3'-protruding DNA termini of four or more bases remain immune to the enzyme. Therefore, unidirectional nested degradation of a DNA fragment is achieved by selectively formulating its termini to have 3'- and 5'- extensions on different ends of the molecule.
Alternatively, one of the DNA strands can be protected by filling-in the 3'-recessed ends with alfa-phosphorothioate deoxynucleotides using the Klenow fragment of DNA polymerase I. The phosphorothioate nucleotide linkage is resistant to degradation by ExoIII, but hinders neither ligation of such DNA fragments, nor in vivo replication of plasmids . Since the strand degradation with ExoIII proceeds at a constant rate, deletions in the desired size range can readily be obtained by assaying aliquots of the reaction mixture at appropriate periods of time.
The method normally involves the following 6 major steps (see picture below):
|Illustration of generation of unidirectional deletions using ExoIII/S1 Deletion Kit|
1. Linearizationof double stranded plasmid DNA by restriction endonucleases (REs) digestion. This procedure can be accomplished in two distinct ways depending on the availability of suitable restriction endonuclease sites and their arrangement:
2. Deletion.Controlled incubation of the linearized DNA with ExoIII to produce single-stranded deletions of a defined length followed by treatment of the timed aliquots of the ExoIII reaction with S1 nuclease to remove the 5' single-stranded overhangs.
3. Deletion Analysis.Determination of the approximate size of the double-stranded linear fragments by assaying samples by agarose gel electrophoresis.
4. Ligation.Ligation of the linear molecules with T4 DNA ligase.
5. Transformation.Transformation of competent E.coli cells with the ligate.
6. Screeningof the obtained library.
An important point to consider before starting an experiment is the purity of initial plasmid DNA. ExoIII easily picks up nicks in double-stranded DNA molecules creating random single-stranded gaps. Therefore, for the better results one should use plasmid preparations with high content supercoiled DNA, i.e. purified by conventional equilibrium sedimentation in CsCl-ethidium bromide gradient. One might also use other methods for DNA purification that ensure efficient removal of nicked DNA from supercoiled DNA. Miniprep DNA treated with RNase and extracted with phenol/chloroform might be suitable as well, but the rate of strand degradation with ExoIII will be lower in this case. In general, purification of DNA by this method is not very efficient in minimizing the amount of nicked and linear molecules.
Circular DNA needs to be digested with restriction endonucleases at two distinct sites A and B. Sites A and B are defined for the remainder of this document as follows: site A is the one that is located in the vicinity of the primer binding site side and at which the ExoIII resistant DNA termini are created (see Table 1 below); site B is used to generate ExoIII-accessible termini in the vicinity of target sequence. Please note, that sites A and B should be unique in a given plasmid. Restriction enzymes with hexanucleotide recognition sites in pUC/M13 polylinker usually cleave infrequently enough allowing the choice of a proper enzyme pair. It is important to perform cleavage with both enzymes to completion in order to maximize the yield of colonies with required deletions. Incomplete DNA cleavage leads to anomalies that are detectable when assaying the deletion reaction products by gel electrophoresis.
Table 1. Restriction endonucleases generating ExoIII resistant 3'-protruding ends.
Enzyme Fermentas enzyme Isoschizomers Recognition sequence AatII AatII 5'...GACGT^C...3' AloI AloI 5'...^7/12-13(N)GAAC(N)6TCC(N)12-13/7^...3' ApaI ApaI 5'...GGGCC^C...3' AvaIII Mph1103I EcoT22I, NsiI 5'...ATGCA^T...3' BplI BplI 5'...8/13^(N)GAG(N)5CTC(N)13/8^...3' BseSI BseSI 5'...G(G/T)GC(A/C)^C...3' BstXI BstXI 5'...CCANNNNN^NTGG...3' HaeII Bsp143II 5'...PuGCGC^Py...3' HgiAI Alw21I AspHI 5'...G(A/T)GC(A/T)^C...3' HgiJII Eco24I BanII 5'...GPuGCPy^C...3' Hin4I Hin4I 5'...^8/13-14(N)GAPy(N)5(A/C/G)TC(N)13-14/8^...3' KpnI KpnI 5'...GGTAC^C...3' NspI XceI 5'...PuCATG^Py...3' PpiI PpiI 5'...^7/12(N)GAAC(N)5CTC(N)13/8^...3' PstI PstI 5'...CTGCA^G...3' SacI SacI SstI 5'...GAGCT^C...3' SduI SduI BmyI, Bsp1286I 5'...G(G/A/T)GC(C/A/T)^C...3' SphI PaeI BbuI 5'...GCATG^C...3' Sse8387I SdaI 5'...CCTGCA^GG...3' TaiI TaiI 5'...ACGT^...3'
Single letter code: N = G, A, T or C; Pu =A or G; Py =C or T.
If it is possible to select a suitable restriction enzyme (A) that produces 3'-overhang ends, follow procedure 1.1 (below).
Otherwise, follow procedure 1.2.
1.1. Restriction endonucleases digestion of plasmid DNA.
Cut DNA at preselected sites A and B. Add both enzymes simultaneously if both of them retain activity in the same reaction mixture and at the same temperature. Keep in mind, that the volume of a restriction endonuclease added should not exceed 1/10 of the total reaction mixture, since glycerol from the enzyme storage buffer may interfere with the reaction. Most enzymes are active in a few buffers, hence it is often possible to choose a buffer that ensures sufficient activity of both enzymes. However, if the reaction conditions are incompatible, first digestion should be performed using restriction enzyme that requires a low ionic strength buffer (B+, G+ or Y+/Tango™). When the reaction is complete, increase the salt concentration by adding 1/10 volume of the 10X concentrated high salt buffer (R+, O+ or BamHI+ buffers) that satisfies the buffer requirements for the second enzyme.
Another convenient way to perform double digest is the use of universal Y+/Tango™ buffer. For detailed recommendations, see "Double Digestion of DNA with Fermentas Enzymes".
Make sure that cleavage reaction is complete. If not, we recommend to purify the DNA with phenol/chloroform prior to precipitation with ethanol. Dissolve DNA in a suitable 1X buffer and digest it with the second restriction enzyme. Since ExoIII is inhibited by NaCl, DNA must be treated with phenol/chloroform and precipitated with ethanol prior to deletion reaction.
Reagents required for this procedure:
- The following restriction digestion reaction is provided as an example:Incubate the restriction digestion reaction mixture at an appropriate temperature for 2 hours.
plasmid DNA with insert 5µg 10X buffer for restriction endonuclease A 5µl restriction endonuclease A 25u deionized water to the final volume of 50µl
Perform RE digestion of plasmid DNA as described above (1.1). If you need to change reaction buffer afterwards, perform DNA extraction and precipitation as recommended in steps 3-6 (below) and then proceed with the second digestion following the example provided in step 1.
- Check the completeness of RE reaction assaying 3µl of sample by 1% agarose gel electrophoresis.
- If cleavage is complete, extract reaction mixture with 1 volume of phenol/chloroform. Vortex for 1min and centrifuge at a maximum speed for 5 minutes.
- Transfer the upper aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1). Vortex for 1min and centrifuge at a maximum speed for 5 minutes.
- Transfer the upper aqueous phase to a fresh tube. Add 0.1 volume of NaCl/glycogen and 2 volumes of 95% ethanol cooled to -20°C. Mix and centrifuge at maximum speed for 10min.
- Pour off the supernatant and carefully wash the pellet with 1ml of 75% ethanol cooled to -20°C. Dry the pellet and follow procedure 2 "Deletion".
If the ionic strength of the reaction mixture is 50mM NaCl (KCl) or lower, omit steps 3-6. For your convenience you can inactivate the restriction enzyme (if the enzyme is not thermostable) by heating at 65°C for 20min. See Table 2 for the ExoIII reaction rates in various buffers.
1.2. Protection of 3'-recessed ends with alfa-Phosphorothioate deoxynucleotides.
The linearization of plasmid DNA follows with a restriction endonuclease generating 3'-recessed ends is performed and then the 5'-overhangs are filled-in with alfa-phosphorothioate deoxynucleotide using the Klenow fragment polymerase. Since the Klenow fragment retains activity in all Fermentas Five Plus System buffers, a change of buffer after inactivation of the restriction endonuclease to the Klenow reaction is not necessary. Digestion with the second restriction enzyme at the site B is performed in order to generate an ExoIII accessible terminus on the insert side.
The following reagents required for this procedure:
- Perform the linearization of plasmid DNA with appropriate restriction endonuclease at site A to produce 5'-protruding ends.
- Check if the reaction is complete by assaying 3µl of the sample by agarose gel electrophoresis.
- To the test tube containing 47µl of linearized DNA add:Mix gently and incubate for 10min at 37°C.
dNTPalfaS 1µl (up to final 40µM concentration of each) Klenow fragment
- In order to inactivate the Klenow fragment heat the samples for 10min at 75°C. If the second restriction enzyme that cleaves at site B retains activity in the same buffer, add it directly to the reaction mixture. Otherwise, extract DNA with phenol/chloroform and precipitate with ethanol (see 1.1, steps 3-6). Then resuspend in appropriate reaction buffer. Perform phenol/chloroform extraction and DNA precipitation once again after treating DNA with another restriction enzyme. Then follow procedure 2 "Deletion".
If ionic strength of the second reaction mixture is 50mM NaCl (KCl) or lower, omit the extraction and precipitation of DNA with ethanol. Rather heat-inactivate the restriction enzyme (if the enzyme is not thermostable) for 20min at 65°C. See Table 2. for the ExoIII reaction rate in various buffers.
The rate of DNA degradation by ExoIII depends on the frequency of successive nucleotide deletions. It is therefore recommended to vary the reaction temperature for achieving the most suitable rate of exonucleolytic digestion. Table 2. will be helpful for selection of a proper temperature depending upon your reaction buffer. Nested deletions with 300-450 bp increments are the most convenient for sequencing runs that read 350-450 bp, since overlaps of 50-150 bp will then be allowed. We recommend to perform the deletion reaction at 30°C with 1min timed aliquots if the reaction is carried out in ExoIII buffer.
Table 2. Temperature and buffer influence on the rate of ExoIII digestion (bases per minute)
25 30 37 45 ExoIII 80 210 420 600 ExoIII + 25mM NaCl 65 200 380 570 ExoIII + 50mM NaCl 50 140 320 500 ExoIII + 75mM NaCl 33 100 230 300 ExoIII + 100mM NaCl 20 65 140 180 B+ 100 230 450 600 Y+/Tango™ 80 200 420 600 G+ 60 170 400 580
Mononucleotide release rate is base-dependent and varies in the order: C>A=T>G ; degradation of different termini occurs at different rates. Therefore, the above table can only be used for your orientation but not as a principal protocol.
Choose an appropriate temperature and time point interval from the table. The number of samples will depend upon the size of the total deletion required:
number of samples =((size of total deletion in bp) / (size of deletion at time point interval)) + 1
- Set a selected temperature in your thermostat (see Table 2).
- Prepare S1 nuclease mix (for 25 time points):
7.4X S1 buffer 27µl S1 nuclease (50u) 3µl deionized water up to 200µl
- Pour 7.5µl of the S1 nuclease mixture to each tube, number them starting with "0" and place on ice.
- Dissolve DNA obtained after procedures 1.1 or 1.2 in 50µl of 1X ExoIII buffer. (If the second RE digest was not subjected to extraction and precipitation with ethanol, bring the final reaction volume with an appropriate buffer (see Table 2) up to 50µl.) Incubate the sample for 3-5min at the selected temperature.
- Add 2µl of the obtained DNA solution to 7.5µl of S1 nuclease assay 0 and pipette up and down a few times.
- Add 2.5µl (500u) Exonuclease III to the DNA solution (obtained in step 4) and mix vigorously. The deletion reaction starts after 30sec.
- Remove 2µl aliquots from the ExoIII reaction mixture at set time points and transfer the contents to the tubes containing the S1 nuclease mixture on ice; pipette up and down a few times.
- After having assayed all samples, move the tubes to room temperature for 30min. Add 1µl of S1 STOP solution and heat for 10min at 70°C to inactivate the S1 nuclease.
3. Deletion Analysis
Use 7µl of each sample for gel electrophoresis analysis. Save the remaining portion for the self ligation with T4 DNA ligase (procedure 4 - "Ligation"). Procedures 3 and 4 are performed simultaneously. The samples suitable for transformation are selected on the basis of gel electrophoretic analysis. The samples are assayed in 1% agarose gel, loading 2µl of marker on the flanking lanes of the gel. Control samples, linearized DNA with and without an insert, should also be loaded.
Ligation is performed with T4 DNA ligase. S1 nuclease, which is used for the removal the remaining single-stranded DNA overhangs, is a highly specific single-stranded endonuclease; it forms blunt ended DNA fragments and it is not necessary to additionally blunt the DNA with Klenow fragment and dNTPs. Since numerous macromolecular compounds greatly increase the rate of blunt-end joining by T4 DNA ligase [6, 7 ], PEG 4000 is included into kit. Taking into account that T4 DNA ligase is not inhibited by NaCl concentrations up to 150mM, the recyclization needs no change of buffer and repurification of DNA prior to the addition of the ligation mix.
Reagents required for this procedure:
- Prepare ligation mix: