Exo III sequencing deletions

Reference: Henikoff, S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene (1984) 351-359.

MATERIALS

DNA insert cloned into a polylinker containing vector
Recommended vector: pBluescript or pTZ. These vectors are specifically designed for Exo III deletions because the restriction sites in the polylinker are arranged so that the 3'-overhang enzyme sites are located on the edges of the polylinker and the 5'-overhang restriction sites are clustered in the interior region.
CsCl purified plasmid DNA - miniprep DNA is not pure enough
Exonuclease III Recommended enzyme: BRL 8013SA
conc. 5000 units in 77ul
Nuclease S1 Recommended enzyme: Boehringer Mannheim 818 330
conc. 400 units/ul
Restriction site selections
Do test restriction digests of your plasmid with all restriction endonucleases that are candidates for this Exo III procedure. For deletion in each direction, you need a unique flanking 5'-overhang site and a unique 3'-overhang site which is on the outer edge of the polylinker on the same side of the insert as the 5'-overhanging site. Since exonuclease III requires a 5'-overhang to start deletions from, the sites need to be placed such that the doubly-digested plasmid will be digested into the insert DNA, but the 3'-overhanging cut prevents digestion in the other direction, into the vector DNA. Although the sites must be on the same side of the insert DNA, they should be at least a few bp apart so that both sites will be digested to completion.

Exonuclease III Buffer 10 ml from stock solutions

Volume	Stock	Final Conc.
0.66 ml	1M Tris-Cl pH 8	66 mM
6.6 ml	1M MgCl2	0.66 mM
9.3 ml	dH2O

S1 Salts Buffer 10 ml from stock solutions

Volume	Stock	Final Conc.
0.5 ml	5M NaCl	0.25 M
0.1 ml	3M K acetate pH 4.6	30 mM
0.01 ml	1M ZnSO4	1 mM
0.5 ml	glycerol	5%
8.9 ml	dH2O

Stop Buffer 10 ml from stock solutions

Volume	Stock	Final Conc.
5.0 ml	1M Tris pH 8	0.50 M
2.5 ml	500 mM EDTA	0.125 M
2.5 ml	dH2O

T4 DNA Ligase New England Biolabs #202 (use at 40 units/ｵl)
Ligase Buffer (10X)

0.2 M Tris pH 8

0.1 M MgCl2

0.5 M 2-mercaptoethanol
Competent cells Recommended strain: DH5a
TE : 10 mM Tris pH 8 / 1 mM EDTA

EXPERIMENTAL PROCEDURE

Doubly restrict 5 ug of plasmid DNA with the chosen restriction endonucleases for one direction. Do a separate restriction digest for the Exo III deletion of your plasmid in the other direction.
Phenol extract, add 1/9th volume 3M Na Acetate pH 5.2 and two volumes of 95% EtOH, precipitate on dry-ice EtOH for 10 min. Centrifuge 10 min, wash pellet in 70% EtOH and dry.
Resuspend the DNA pellet in 77 ul Exo III Buffer. Mix well and put on ice.
Label 35 eppendorf tubes (0.5 ml size tubes) with 30 second interval time points (for each reaction set). Aliquot 25 ul of S1 Salts Buffer into tubes and store on ice.
Add 7.6 ul of BRL Exonuclease III (493 units) to the DNA and immediately transfer the tube to a 37｡C waterbath.
This is time = 0 min.
At intervals of 30 seconds transfer 2.5 ｵl of the Exo III-plasmid reaction to the S1 Salts Buffer containing tubes, leave on ice.
Take time points until all the mixture is gone (about 17 min). A 3 kb insert will be completely digested in approximately 10 min.
Prepare an S1 nuclease solution. [1 ul (400 units) in 1103 ｵl S1 Salts Buffer]. Aliquot 25 ul to each time point, mix and incubate at room temp for 30 min.
Add 6 ul Stop Solution to each reaction tube.
At this point the time points can be stored at -20｡C (for several weeks, perhaps longer) or prepared for ligation and transformation. Usually it is sufficient to select time points of 1 min intervals (ie. t=1 min through t=10 min) to continue processing.
Phenol extract, EtOH ppt, wash and dry selected time points. Resuspend each in 20 ul TE pH 8.

Religate the digested plasmids at 14｡C overnight (or 16｡C for 4 hours).

10 ul	Exo III timepoint DNA
2 ul	10X ligase buffer
2 ul	10 mM ATP
1 ul	DNA ligase (40 units/ ul)
5 ul	dH2O

Transformation into competent DH5a cells:
50 ｵl competent DH5a cells
10 ｵl ligation
incubate 30 min on ice
heat shock for 2 min at 42｡C
add 1 ml LB, incubate at 37｡C for 1 hr, centrifuge 2 min and resuspend in 100 ul, saline or L broth
Plate entire transformation on one LB + 100 ug/ml ampicillin plate. Expect 50-300 colonies/plate.
Select several colonies from each transformation and start overnight cultures.
Prepare minipreps of the cultures and run the samples on a 1% agarose gel. It is important to have two controls on this gel: both your supercoiled original clone and the cloning vector (ideally in the outside wells of both sides of your gel). These controls will serve as markers to size your deletions.

Recommended miniprep procedure: Maniatis T. et al (1982) Molecular Cloning: A Laboratory Manual, Alkaline Lysis Method p. 368.
Ethidium bromide stain and photograph the gel. Use a ruler to line up your controls and make a list of your deletions in order of increaseing or decreasing sizes. The Exo III deletions may not show perfect progression in the comparison of size to the length of time exposed to the Exo III enzyme. This is not unusual because Exo III is a processive enzyme which will take a single molecule of DNA through a complete digestion before it is released and free to bind another DNA molecule. This should not affect your ability to select clones of all sizes, it may just require screening more colonies to find the appropriate deletion sizes.
Sequence the minipreps using the universal and reverse primers and Sequenase 2.0 (see related protocal). Use the primer which is on the same side of the polylinker as your Exo restriction sites. If you use the wrong primer you will sequence the same region of your insert no matter what size deletion you choose (ie. using the vector pBluescript: use the universal primer for Sac I/ Bam HI deletions and the reverse primer for Kpn I/ Sal I deletions).