Exonuclease III digestion produces multiple sets of nested deletion mutants
Exonuclease III digestion produces multiple sets of nested deletion mutants
The nested deletion mutagenesis method, in which multiple oligonucleotides are progressively deleted from one or the other end of the target DNA, has been used to determine the boundaries of functional cis-regulatory elements, and was earlier used as a template to guide DNA sequencing. The method relies on nucleases that all digest DNA in a predictable manner, with the exonuclease HI being the best available. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Exonuclease III digestion produces multiple sets of nested deletion mutants
Materials and Instruments
dNTP solution Ethanol Exonuclease III buffer Nuclease S1 stop reaction mixture Phenol Chloroform Sodium acetate Exonuclease III Klenow mixture Ligation mixture Nuclease S1 reaction mixture Restriction endonuclease Gel Target DNA Move makings For more product details, please visit Aladdin Scientific website.
Microcentrifuge tubes or microtitre plates with U-wells Water baths
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the stock solution to the appropriate concentration.
The dNTP solution contains dATP, dGTP, and dTTP, each at a concentration of 0.5 mmol/L in 25 mmol/L Tris-Cl (pH 8.0).
Ethanol (100%, ice-cold; 70%, room temperature).
10x Exonuclease III Buffer
660 mmol/L Tris-Cl (pH 8.0)
66 mmol/LMgCl2
100 mmol/L β-mercaptoethanol
Nuclease S1 stop reaction mixture
0.3 mmol/L Tris
50 mmol/L EDTA (pH 8-0)
Phenol: chloroform
Sodium acetate (3 mol/L,pH5.2)
Enzyme and buffer
Exonuclease III
The quality of exonuclease III varies from vendor to vendor, and analytical digestion should be performed to check the quality of exonuclease III before preparing nested deletion templates in large batches.
Klenow Blend (enough for 30 samples)
Water 20ul
1mol/LMgCl2 6ul
0.1mol/LTris-Cl 3ul
Klenow fragments 3 units
Prepare the above mixture on ice before use.
Connection Mixture (24 samples)
Water 550ul
10x Phage T4 Ligase Buffer 100ul
5 mmol/L rATP 100ul
Polyethylene glycol (30% m/V PEG8000) 250ul
Phage T4DNA splicase 5 units
Prepare the above mixtures on ice before use.
Nuclease S1 reaction mixture
Water 172ul
10XS1 buffer 27ul
Nuclease S1 60 units
Prepare the mixture before use
Mung bean enzyme can be used as a substitute for nuclease S1 in the above reaction.
Restriction endonucleases (two types)
For a discussion of the selection of restriction enzymes and the appropriate introduction of their recognition sites into the target DNA, see the column on exonuclease III in the preface section and information column of this protocol.
Gel
1% (m/v) agarose gels (two-component)
Nucleic acids and oligonucleotides
Target DNA
Analyze the target DNA sequence for the presence of appropriate restriction enzyme sites. Clone the DNA to be digested into a plasmid or phage vector that contains as few non-essential sequences as possible. Dissolve a portion of the recombinant plasmid DNA to be used as a mutagenic substrate in TAE buffer (see Scheme 1 in Chapter 5). Analyze by agarose gel electrophoresis. The plasmid should contain more than 90% superhelical molecules. If the plasmid is prepared with a linear fraction or more than 10% nicked or relaxed plasmid, it should be repurified.
Because exonuclease III digestion begins at the single-stranded nick, closed-loop molecules must make up the majority of the template DNA product. Purification of the template has the added benefit of removing small fragments of DNA and RNA from the closed-loop DNA product that interfere with exonuclease III digestion.
Specialized Equipment
Microcentrifuge tubes (0.5 ml) or microtitre plates with U-wells (e.g., Boxter B1190-17). Pre-adjustable 30°C, 37°C and 70°C water baths.
Other reagents
The reagents required for Step 14 of this protocol are listed in Chapter 1, Protocols 24, 25, or 26 (for transformation).
The reagents needed for step 15 of this protocol are listed in Chapter 1, Options 1 or 4 (for microplasmid DNA preparation) or
Chapter 3, Scheme 3 (for preparation of replicative M13DNA).
The reagents needed for Step 16 of this protocol are listed in Chapter 12, Options 3, 4, or 5.
Methods
1. Digest 10ug of target DNA (recombinant phage M13 replicative DNA, phage DNA, or plasmid DNA) with two restriction enzymes capable of digesting the polyclonal site between the primer-binding region on the vector and the target DNA.
For optimal digestion, avoid using restriction enzyme sites in close proximity to each other in the polyclonal site. Restriction enzyme digestion reactions should be performed in a large reaction volume with a low DNA concentration and a suitable digestion buffer recommended by the manufacturer. Digestion with enzymes that produce blunt or 3' concave ends should be performed first. When gel electrophoresis confirms that all closed loop DNA has been converted to linear DNA, adjust the buffer and add the second enzyme. Alternatively, perform the usual phenol:chloroform extraction and ethanol precipitation, and perform the second enzyme digestion in a suitable buffer. Any DNA that escapes the second enzyme digestion will be digested in both directions by exonuclease III, and therefore it is unlikely that a viable clone will be produced. The efficiency of the second digest can be determined by labeling the first cut site at the end of 32P and checking for a 50% loss of radioactivity after the second digest.
If there is difficulty in digesting the template completely with two restriction enzymes, we recommend the use of a commercially available kit based on the method developed by Henikoff (1990). In this procedure, single-stranded cyclic phage DNA is used as the template for an oligonucleotide primer extension reaction catalyzed by phage T4DNA polymerase. The reaction produces nicked double-stranded circular molecules immediately at the 5' end of the primer. Exonuclease III is used to cleave the 3' end produced by the above reaction, exposing the single-stranded DNA through digestion by a single-stranded DNA-specific endonuclease (nuclease S1 or mung bean nuclease.) The above process produces linearly nested DNA fragments with an identical set of ends that correspond to the 5' end of the primer. These missing molecules are recirculated for use in transformed cells. The advantage of this method is that nested deletions can be generated from any point of the target DNA. The enzymatic reaction can be carried out sequentially in the same reaction tube. Therefore, no organic solvent extraction or precipitation of DNA is required.
Several commercially available kits for the construction of deletion mutants based on the method of Henikoff (1990) are available (see the information section on commercially available kits for targeted mutagenesis).
2. Purify the DNA by conventional phenol/chloroform extraction and ethanol precipitation, carefully removing the supernatant and adding 0.5 ml of 70% ethanol to clear the DNA precipitate.
Lavage of the precipitate with ethanol is an important step because sodium ions inhibit the exonuclease III reaction (Hoheisel 1993) (see the information section on exonuclease III).
3. Centrifuge the DNA precipitate in a microcentrifuge for 2 min at maximum speed and 4°C. Recover the DNA precipitate washed with 70% ethanol and carefully remove the top. Place the reaction tube on a benchtop with the lid open and allow all the ethanol to evaporate. Finally, dissolve the DNA in 60ul1x Exonuclease Buffer and place on ice.
4. Set up 25 0.5 ml microcentrifuge tubes with 7.5ul of Nuclease S1 reaction mixture in each tube or use 25 wells of a 96-well microtitre plate with U-shaped wells and add 7.5ul of Nuclease S1 reaction mixture to each well and place on ice.
5. Incubate the DNA solution prepared in step 3 for 5 min at 37°C. Transfer 2.5ul of the solution to the first microcentrifuge tube or U-well of the microtitre plate containing the Nuclease S1 reaction mixture.
6. Add exonuclease III to the remaining DNA solution at a rate of 150 units of exonuclease III per pmole of recessed 3' end (1 unit of exonuclease III incubated at 37°C for 30 min will yield 1 nmole of acid-soluble total nucleotides). Tap the walls of the tube to mix the reaction material in the tube and immediately return the tube to the 37°C water bath.
Under these conditions, the amount of exonuclease III is saturated. Approximately 200 nucleotides per minute can be removed from the blunt or 3' concave end of each DNA molecule. The amount of nucleotides removed can be controlled by varying the sampling interval between successive reactions. In general, the efficiency of DNA digestion by exonuclease depends on the molar ratio of enzyme to template. To ensure that all DNA templates are degraded at the same rate. To maintain synchronized cleavage of DNA fragments of several thousand bases, exonuclease III must be present in excess in the digestion reaction.
The digestion rate of exonuclease can also be controlled by varying the reaction temperature; Henikoff (1987) estimated that a 2°C difference in temperature changes the digestion rate by 100 bases/min at 30-40C.
7. At 30-s intervals, 2.5 ul of sample from the DNA solution is added to the wells of a microcentrifuge tube or microtitre plate containing the nuclease S1 reaction mixture.
8. When all samples have been added, place the microcentrifuge tube or microtitre plate containing Nuclease S1 and the digested DNA at 30°C for 30 min.
9. Add lul of Nuclease S1 Reaction Stop Mix to each tube or well and hold at 70°C for 10 minutes.
Heat at 70°C to inactivate Nuclease S1 and any residual Exonuclease III, see Chapter 7 on Nuclease S1 and later in this chapter on Exonuclease III for further information on these enzymes.
10. Transfer microcentrifuge tubes or microtitre plates to a bed of ice and analyze each sample by gel electrophoresis at a gel concentration chosen to maximize discrimination between the starting DNA and DNA fragments smaller than 2 kb of the starting DNA.
11. Collect the samples containing the desired size of DNA, add 1ul of Klenow's mixture to every 10ul of sample, and incubate the reaction mixture at 37°C for 5 min.
12. Add 1ul of 0.5 mmol/L dNTP per 10ul of collected sample and continue incubation at room temperature for 15 min.
Incubate briefly with Klenow enzyme, initially in the absence of dNTP in step 11, to allow Klenow enzyme to exert 3' exonuclease activity to remove any 3' protruding ends retained in the digested DNA.
13. Add 40ul of T4 Phage Ligase mixture per 10ul of collected sample and mix well, then continue to incubate at room temperature for 2 hours.
14. Take the ligated DNA and transform it into a suitable E. coli host bacterium.
15. Prepare small quantities of phage M13 replicative DNA, plasmid or phage DNA from at least 24 randomly selected phage spots or clones.
16. Digest the DNA into linear molecules with a suitable restriction endonuclease and analyze their molecular weight size on a 1% agarose gel. Use the original plasmid or phage DNA, which has also been digested into linear molecules by restriction enzymes, as a standard reference construct. Select clones of appropriate size for further sequence analysis (Chapter 12) or restriction enzyme mapping.
Approximately 80% to 90% of the deletions retain a universal or reverse primer binding site, which can be sequenced using appropriate oligos. To ensure that a complete set of overlapping DNAs can be analyzed, it is best to sequence clones with a molecular weight difference of about 400 bases. See Chapter 12 for protocols on how to sequence rejoined and transformed DNA.