ANALYTICAL BIOCHEMISTRY 38,8&89 (1970)

Deproteinization with Phenol of Alternating

Polydeoxyadenylate-Deoxythymidylate

and Other DNA-like Polymers

DAVID A. SMITH, AARON M. MARTINEZ, AND ROBERT L. RATLIFF

Biomedical Research Group, Los Alamos Scientific Laboratory,

University of California, Los Alamos, New Mexico 87644

Received April 3, 1970

Skinner and Triplett (1) used a phenol extraction method based on the procedure suggested by Massie and Zimm (2) and reported selective loss into the phenol phase of the deoxyadenylate-deoxythymidylate [d(A - T)]l-rich satellite DNA of the land crab. Morgan and Wells (3) also used the procedure of Massie and Zimm (2) and found that poly d (A - T) ad (A - T) , enzymically prepared with Escherichia coli DNA polymerase was quantitatively lost into the phenol layer. In addition to loss of poly d(A-T)-d(A-T), we have obtained very low recoveries in the aqueous phase of other repeating polymers which had been syn- thesized with E. coli DNA polymerase. To prevent such losses, others have deproteinized with isoamyl alcohol/chloroform instead of phenol (1, 3). Because phenol extraction has certain advantages over extraction with isoamyl alcohol/chloroform (2, 4), we have investigated this phe- nomenon and developed a phenol extraction method that allows quanti- tative recovery in the aqueous phase of poly d (A - T) *d (A - T) and all other enzymically ‘synthesized polymers on which it has been tried.

METHODS

Poly d(A - T) *d(A - T)-14C was synt.hesized in poly d(A - T) * d (A - T) primed reactions essentially as described by Schachman et al. (5). Reactions were terminated by the addition of 0.5 volume lo/O sodium dodecyl sulfate (SDS). Acid-inlsoluble radioactivity was determined as previously described (6). Since phenol is a potent liquid ,scintillation quencher, it is important to wash well those discs taken from aqueous phases saturated with phenol. Liquefied phenol from Fisher Scientific Company was used as obtained.

’ Abbreviations used for the polynucleotides are those given in the Revised Tenta- tive Rules (1965) of IUPAC-IUB in J. Bill. Chem. 241, 527 (1966).

85

86 SMITH, MARTINEZ, AND RATLIFF

RESULTS

The results of Skinner and Triplett (1) #and of Morgan ,and Wells (3) indicated that pH and temperature were not the major factors causing poly d(A - T) *d(A - T) to remain in the phenol phase. Our early ex- periments indicated that perhaps high NaCl concentrations were an im- portant factor. To further investigate this possibility, an experiment was designed to determine the importance of NaCI concentration at two dif- ferent temperatures (Table 1). The data show that temperature has little effect upon the recovery of poly d(A - T) .d(A - T) in the aqueous phase. In 1 M NaCl the polymer is lost into the phenol layer, while it is quantitatively recovered in the aqueous phase at 0.2 M NaCl.

To investigate the variables involved ,and to determine if poly d(A - T) *d(A - T) could be removed from the phenol phase, the ex- periment in Table 2 was done. The data show that the polymer can be extracted from the phenol phase. The difference between extractions C and D, in which 0.1 M NaC1/05M Tris (pH 7.0) did not remove poly d(A - T) *d(A - T) from the phenol phase while 0.1 M NaCb’0.5 M Tris (pH 9.0) did, was rather surprising. Other experiments had shown that 0.01 M T&,/O.1 M NaCl at both pH 7.0 and pH 9.0 gave good re- covery of poly d (A - T) *d (A - T) in the aqueous phase.

On the basis of these and other experiments, we are using the following phenol procedure for the deproteinization of enzymically prepared poly-

deoxynucleotides. A concentrated solution is added to the reaction so that

TABLE 1 Phenol Extraction of poly d(A - T) . d(A - T) - 1% in 1 &f

and 0.2 M N&l at 55” and 24”

Tube

Recovery of NaCl Temperature poly d(A - T) *

(M) 0 d(A - T) - r4C (o/o)

A 1.0 55 4.8 B 1.0 24 0 C 0.2 55 97.5 D 0.2 24 98.7

A poly d(A - T) . d(A - T) - 1°C reaction was diluted to 8.5 ml, and an aliquot was removed for determination of acid-insoluble radioactivity. Four 2 ml aliquots were re- moved and placed in separate tubes. Tubes A and B were made 1 M in NaCI, and tubes

C and D were made 0.2 M in NaCl. Tubes A and C were heated to 55’ and extracted with an equal volume of phenol, as described by Massie and Zimm (2). Tubes B and D were extracted at 24’ with an equal volume of phenol. The aqueous phases were removed. To ensure complete recovery of aqueous phase material, l-2 ml of appropriate NaCl solu- tion was layered over the phenol layer and then removed and added to the original

aqueous phase. Acid-insoluble 1% was then determined in the aqueous phases. A total of 23,500 cpm poly d(A - T) . d(A - T) i 1% was placed in each tube originally.

PHENOL DEPROTEINIZATION 87

TABLE 2

Extraction of poly d(A - T) . d(A - T) - 1% from the Phenol Phase

Tube Extraction solution Recovery

(%)

A

B

C D

0.1 M NaCl 96.5 0.1 M NaCl/l% SDS 98.7

O.lM NaCl/O.SM Tris (pH 7.0) 0 O.lMNaCl/O.SMTris(pH9.0) 100.0

Poly d(A - T) . d(A - T) - 1% was extracted into the phenol layer (Table 1). The phenol layer then was divided into 4 portions, each containing 22,200 cpm poly d(A

-T) . d(A - T) - W. The phenol layers were then extracted with an equal volume of solutions described above, and acid-insoluble radioactivity was determined in the aqueous

phases as described in Table 1.

it contains 0.2 M NaCl, 0.5% SDS, and 0.1 M Tris (pH 9.0). The phases are separated, and phenol extraction is repeated twice on the aqueous phase. The phenol layers are then extracted once with buffer, and this aqueous layer is combined with the first aqueous phase. Phenol may then be removed from the combined aqueous phases by several extractions with ether. We routinely do phenol extracti’ons at room temperature, but in some cases heating to 55” may give better results (1, 7). This pro- cedure has given near-quantitative recovery of all DNA-like polymers on which it has been tested. These include other repeating polymers syn- thesized with E. coli DNA polymerase (8)) as well as a variety of polymers ‘synthesized with terminal deoxynucleotidyltransferase and DNA polymerase from calf thymus (9, 10). Of course, there may be other polydeoxynucleotides which are not recovered in the aqueous phase under these conditions.

DISCUSSIOX

The data presented above show that the loss into the phenol phase of ,d (A - T) -rich DNA observed by Skinner and Triplett (1) and by Mor- gan and Wells (3) is due to the high concentrati80ns (1 M or higher) of NaCl which they used. At 0.1 or 0.2 M NaCl, poly d(A - T) *d(A - T) is quantitatively recovered in the aqueous phase. Some published pro- cedures which utilize phenol extraction for the preparation of DNA also utilize high NaCl concentrations (2, 11). As was found by Skinner and Triplett (1)) these procedures may result in the loss ‘of some types of DNA. The reason for selective salting-out of some DNA’s from the phenol-saturated aqueous phase is not clear. A possible practical benefit of this phenomenon might be the removal of contaminating poly d(A - T) *d(A - T) from repeating polymers synthesized with E. coli DNA

88 SMITH, MARTINEZ, AND RATLJFF

polymerase. Such contamination sometimes occurs during amplification reactions performed with this enzyme (12).

In Table 2 it is seen that in 0.1 M NaC1/0.5M Tris (pH 7.0) poly d(A - T) *d(A - T) will remain in the phenol layer, while in 0.1 M NaC1/0.5 M Tris (pH 9.0) it is recovered in the aqueous phase. This could be due to the salt form of the polymer. The pK of Tris is 8.3 at 20” (13). Thus, at pH 7.0, the cationic form ‘of Tris predominates and poly d(A-T).d(A- T) may exist largely as the Tris salt, At pH 9.0, the Tris ion concentration is considerably lower and the polymer may be more in the Na salt form, which might be less soluble in the phenol phase. As mentioned above, other experiments have shown that 0.1 M NaCl/ 0.01 M Tris at either pH 7.0 or pH 9.0 allows recovery at poly d (A - T) . d(A - T) in the aqueous phase. In the procedure we have adopted in this laboratory (see “Results”), pH 9.0 is utilized. This is because we have sometimes obtained better recoveries at pH 9.0 than at pH 7.0 and because of reports in the literature in which similar results have been obtained (14, 15).

SUMMARY

It has been reported that the deoxythymidylate-deoxyadenylate-rich satellite DNA from the land crab is lost into the pheaol phase during phenol extraction of tissues from that organism. Others report that alter- nating poly deoxyadenylate-deoxythymidylate is lost into the phenol phase during deproteinization. The reasons for this selective I,oss were investi- gated, and it ,was shown that the high NaCl concentrations used during extraction are responsible. A phenol extraction method that gives quanti- tative recovery of alternating poly deoxyadenylate-deoxythymidylate and other enzymically synthesized polymers is described.

ACKNOWLEDGMENTS

The authors would like to thank Dr. F. N. Hayes for helpful suggestions during the course of this work.

This research was performed under the auspices of the U. S. Atomic Energy Commission.

REFERENCES

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5. SCHACHMAN, H. K., ADLER, J., RADDING, C. M., LEHMAN, I, R., AND KORNBERG. A., J. Bid. Chem. 235, 3242 (1960).

PHENOL DEPROTEINIZATION 89

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