EXONUCLEASE III DELETIONS FOR SEQUENCING

1. The following will be needed

   a) A good restriction map of the target DNA sequence (the DNA to be sequenced).

   b) Some of the target DNA purified.

   c) Purified SK+ and SK- DNA. These phagemids give better color response to X-gal than does the KS phagemids.

   d) Restriction enzymes Sac I, Bst XI, Apa I, and Kpn I. The target DNA needs to be mapped for these enzymes (hopefully the target DNA does not contain the appropriate two sites required for deletions from either side of the polylinker).

   e) Other restriction enzymes as needed.

   f) Thiotriphosphates of the 4 deoxynucleotides. This is just in case the target DNA has the restriction sites listed in d), therefore, making it impossible to protect the priming site from Exonuclease III with the 3' overhang left from these enzymes.

   g) Exonuclease III (Exo III) bought from Stratagene. High quality samples of one of the following single-stranded endonucleases: S1 (Sigma), Neurospora crassa (Pharmacia), or Mung Bean (Stratagene) nuclease. T4 DNA ligase. Klenow fragment. The E. coli strain XL1-Blu. Low melting point agarose and the priming oligo for sequencing might be needed if problems are encountered.

2. Clone the target DNA into the cloning site polylinker of the SK phagemids. The following are considerations.

   a) Remember that an unique restriction site with a 3' overhang is needed to protect the priming site. The overhang needs to be at least 4 bases.

   b) Also, an unique restriction site for an enzyme which leaves a 5' overhang is needed next to the cloned target DNA sequence so that the protecting 3' overhang can be removed.

   c) If these requirements are impossible an unique 5' site is usable for protecting the priming site if filled in with the thiotriphosphates (using Klenow). Indeed, I have heard that this works better. Remember that another 5' site to make the target DNA susceptible to Exo III nuclease digestion is still required.

   d) To sequence both strands the target DNA must be cloned in both directions or the target DNA cloned into both SK+ and SK-. Only one of the strands gets rescued; therefore, only one deletion direction works for a particular cloning. Work it out before hand. Think about it.

3. Purify the cloned DNA.
4. Do the appropriate restriction digests. The amount of DNA which needs digesting depends on the number of time points taken (see below). Do the restriction digests which leave a 3' overhang first and analyze the digest on a gel for completeness. Be sure and do at least an ethanol precipitation between the two digests, because the first enzyme may inhibit the second. Use a 5-10 fold excess of the second enzyme, which leaves a 5' overhang, since there is no way to measure the completeness of this reaction. Precipitate the DNA.
5. Resuspend the DNA into Exo III buffer.

   50 mM TrisHCL pH 8.0

   0.6 mM MgCl2

   10 mM 2-mercaptoethanol

   I have found that 3' overhangs are protected in low Mg++ concentrations, whereas, they are not in high concentrations (10mM). The 2-mercaptoethanol stabilizes the enzyme for the longer digestions. Note that 6.8ul of 2-mercaptoethanol into 1mL of H2O is 100mM 2-mercaptoethanol.

6. The following are considerations for the Exo III digests.

   a) A single reaction should be set up and aliquots removed from it to be stopped.

   b) The extent of deletion depends on the temperature.

   37 C=400 bp/minute

   34 C=375 bp/minute

   30 C=230 bp/minute

   23 C=125 bp/minute

   c) The amount that can be sequenced easily on a single gel is about 300 bases. I, therefore, have performed my digestions at 30 C and taken 1 minute aliquots. Exo III tends to delete more quickly early in the reaction, therefore, it is a good idea to take 30 second aliquots for the first two minutes.

   d) Aliquots should contain 1ug of DNA. The total amount of DNA for the reaction depends on the number of aliquots. This obviously depends on the how many aliquots it takes to delete across the entire expanse of the target DNA.

   e) 20 units (Stratagene units) of Exo III are required per ug. The glycerol the enzyme is stored with needs to be diluted 10X (to 5%). I try to keep the reaction volume as small as possible with this caveat in mind. This dictates the size of the reaction and, therefore, the size of the aliquot (which is usually about 10-20 ul).

7. For S1 nuclease, aliquot the Exo III samples into EDTA to stop the reaction and immediately place it on dry ice. The final concentration of EDTA should be approximately 1mM. The EDTA concentration should not be so high that dilution of the sample into 100-200ul of S1 buffer will interfere with the S1 digest (by chelation of Zn++ which is at only 1mM in that buffer).
8. When all the aliquots have been taken, place the samples at 68 C for 15 minutes.
9. S1 should be diluted to 67 Vogt units/mL in S1 buffer.

   10mL 5X S1 buffer

   3.15 gm glycerol

   0.73 gm NaCl

   0.75 mL 2M KAcetate pH 4.6 (made by dissolving 2M KAC and pHing with glacial acetic acid)

   50 ul 1M ZnSO4

   (Final concentration of 1X is 0.25 M NaCl, 30mM KAC, 1mM ZnSO4, 5% glycerol)

10. Add 100-200 ul of diluted S1 to the aliquots (do at least a 5X dilution of the Exo III digest). Incubate 30 minutes at room temperature.
11. Add per 100 ul

    5 ul TrisHCL pH 9.5

    2 ul 20% SDS

    100 ul 9M NH4AC

    500-600 ul Ethanol

12. After precipitation resuspend into 18 ul of H2O. Add 2 ul of 10X Klenow buffer.

    10X Klenow buffer

    0.2 M TrisHCL pH 8.0

    70 mM MgCl2

13. Add Klenow (10 U/mL) and incubate at 37 C for 2 minutes. Add 1.25 ul of a solution containing 2.5mM each of the 4 deoxynucleotides. Incubate for another 2 minutes at 37 C. These samples can be run on a low melting point agarose gel if there is concern about the quality of the size deletions. Typically I have, but this is unnecessary if the deletions went well (they tend to if you use the low Mg++ buffer), the buffers are uncontaminated, and the restriction digests were complete.
14. Ligate the samples from agarose or dilute the Klenow reaction into 50-100 ul of T4 ligase buffer and ligate.
15. Transform XL1-Blu. Note that I have had difficulty in storing these transformants. These strains are easily lost so make plasmid DNA or prepare single-stranded DNA immediately.
16. Check the size of deletion by the quick screen using the phenol lysis as described by Stratagene.

ALTERNATE METHOD USING N. CRASSA NUCLEASE

7. This method was reported to me by Robert Donald. The nuclease comes as an NH4SO4 suspension. Prepare it by spinning it 1 minute in an eppendorf centrifuge and resuspending it in N. crassa buffer.
8. Aliquot the Exo III digestion into 1/10 volume of 10X Stop buffer (see below). Freeze on dry ice.
9. After collecting all samples incubate at 68 C for 15 minutes.
10. Add an equal volume (total DNA solution volume) of N. crassa nuclease in N. crassa buffer at 2.4 units/mL. Incubate 30'.

    1X N.crassa Buffer

    50 mM TrisHCL pH 8.0
    0.2 M NaCl
    10 mM MgCl2
    1 mM CoCl2
    0.5 mM ZnSO4
    0.1 mg/mL BSA
    (Stop solution is simply missing the Tris)
    

11. Add 1/2 volume of Klenow buffer. Add deoxynucleotides as described above for S1 preparations. (An example of volumes is 10 ul of Exo III stopped with 1.5 ul of 10X N. crassa buffer, 11.5 ul of N. crassa, 11.5 ul of Klenow reaction mix.) Incubate 30 minutes at room temperature.
12. Stop with EDTA, phenol extract, and precipitate or run on a gel as described above.

Method Using Mung Bean Nuclease

7. Stop the reaction by aliquoting into 200 ul of Mung Bean Buffer. Immediately freeze on dry ice.
   5X Mung Bean Buffer
   150 mM NaAC pH 5.0
   250 mM NaCl
   5 mM ZnCl2
   25% glycerol
8. After collecting samples incubate at 68 C for 15 minutes.
9. Add 15 units Mung Bean nuclease per 200 ul (1 ug DNA). Incubate 30 minutes at 30 C.
10. Add 4 ul 20% SDS, 10 ul 1M Tris, pH 9.5, 20 ul 8M LiCl to each. Phenol-CH3Cl extract.
11. Add 25 ul 3M NaAC, pH 7. Ethanol precipitate. Treat as above.