hydroxyapatite column chromatography in procedures

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ANALYTICAL BIOCHEM ISTRY 59, 555-563 (1974)

Hydroxyapatite Column Chromatography in Proceduresfor Isolation of Purified DNA

Institute of Biochemistry, Bulgarian Acutlemy of Scierlc es,

13 Sofirc, Bulgaria

Received. August. 2 1, 1973; accepted Piovember 16, 1973

Two procedures are described for the isolation of purified DNA from

mamm alian tissue s hy using hydroxyapatite chromatography. They avoid

enzyme treatment and are eas ily carrird out in one day. The first one is

a mo difica tion of the MFP (8 M urea-O.24 M sod ium phosp hate buffer)

method of Britkn et al. (2) in which some techn ical diffic? llties (clogging

of the colum n) are overcome and the yield and the purity of the DNA are

improved. The second procedure represent s a comb inniion of the cla ssi cal

rn:thods of lys is of nu c*!r i with hydroxy:tpatite chromatography of the DNA

and may be espec ially convenient for the isolation of high-molecular-weightDXA.

Most of the currelIt methods for the isolation of purified DXA require

enzyme treatment with ribonuclease and pronase, combined with many-

fold deproteinization, precipitation and redissolving. Such methods last

several days and lead to considerable loss of DNA.

The hydroxyapatitc (HA) chromatography offers new possibilit’ies

for the isolation of purified DNA. Double-stranded DNA has a much

higher affinity to HA t.han R’NA, prot’cins, carbohydrates and various

low-molecular-weight substances (1 I. In principle, this would permit

the isolation of DNA, free of contaminants, simply by loading the tissue

lysate onto a HA column and then cluting by phosphate buffers of appro-

priate concentrations.

Recently, Britten et nl. (2) developed a method based on this principle

for the isolation of purified DIVA from both prokaryotic and eukaryotic

cells. In their method, the starting material (fresh tissue, tissue frozen

in liquid nitrogen or dry ice, or isolated nuclei) is lyscd in 8 M urea-O.24 M

sodium phosphate (Nap), pH 6.8 COUP) ,I containing 1% sodium dodecyl

’ Abb reviations used : MUP. 8 M urcaa-0.24 M sodiu m phosp hate buffer; NaP ,

sodiu m phosp hate buffer (an equim olar mixture of iSaH?P O, and Na2HPO I, pH 6.8) ;

HA, hydrosyapatite; EDT A, ethylenediaminetetraacctic acid, sodium salt; SSC,

0.15 M NaCl-0.015 M sodium citrate; SDS, sodium dodecyl sulphate.

555

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sulphatc (SDS) and 0.01 RI EDTA. The lysate is loaded onto a HA

column, the column is washed with MUP, and urea is removed by washingwith 0.014 M KaP, pH 6.8. DNA is eluted with 0.40 &XINaP, pH 6.8. Sev-

eral tnodifications have been proposed to improve the method: lysis of

previously isolated nuclei instead of whole tissue (31 ; batch procedures

(2,3) ; treatment of the lysate with an equal volume of chloroform-octanol

(20: 1) before loading (2).

The m&hod is simple, fast, permits processing of small amounts of

tissue and gives a high yield of DNA. However, in our hands, this pro-

cedure shon-ed some shoitcomings and technical difficulties. The firstone is the clogging of the column which strongly decreases the flow rate

and makes it necessary to apply vacuum or pressure. This leads to

channeling and incomplete retention of DNA. The difliculties caused

by clogging become critical when large amounts of material are loaded.

Secondly, the lysing medium decreases the ability of HA to adsorb

DNA. This effect is more pronounced in the presence of proteins in the

lgsate.

Thirdly, even the extensive washing of the column with MUP maynot e1ut.e completely the proteins and other substances, which may con-

taminate DKA in the 0.40 M fraction.

Al l these disadvantages prompted us to study in more details the

factors affecting H-4 chromatography as applied to the isolation and

purification of DNA from mammalian tissues. As a result, we worked

out two technically convenient procedures, which gave best results in

respect to yield and purity of DNA.

MATERIALS

Hydroxyapatite was prepared according t.o Miyazawa and Thomas

(4) under very slow stirring to obtain large crystals (1). Hydroxyapatite

Bio Gel HTP (Bio-Rad Labs, Richmond, CA) was also used with

similar results. The cellulose powder 123~ was a product of Schleicher and

Schuell, West Germany. The hemoglobin, crystalized four times, was

purchased from Reanal, Hungary, and the bovine serum albumin fromKoch-Light Laboratories, England. In some experiments DNA was iso-

lated from tissue labelled in viva for proteins with [l%]valine (95 mCi/

mmole), production of the Institute for research, production and uses of

isotopes, Prague, Czechoslovakia, and for RNA with [YJ]orotic acid

(15 mCi/mmole), production of the NAEC, Institute for isotopes, Buda-

pest. Hungary. Al l other reagents and chemicals were of analytica

grade.



ISOLATION OF DNA BY HA CHROXATO!:RAI’HT :xJ 1

Proteins were determined by the LolT-ry proccdurc (5) and RNA by

the orcinol reactio:l (6). Radioactivity was measured in :L model 33’20

Tricarb Packard scintil lation spectrometer and absorbnnry in a Zeiss

spectrophotometer VSU 2. Melting curves were recorded in an rnicam

SP 1800 spectrophotometer.

lSOLATION PROCEDURES

Procehe I: Ml;P Procedure

Lysis. The lysis was performed as described by Brittcn et nl. (2) and

Hell et al. (3). The starting material was suspendecl in 10 vol of lysing

medium (8 M urea-O.24 M NaP, pH 6.8-0.01 &I EDTA-1% SDS) and

blended in a filled sealed container to avoid foaming, using a MSE

homogenizer run at 14000 rpm. For complete lysis the blending was re-

peated 4-6 times for 30 set each with intervals of 1 min to cool the

lysate in an ice bath.Preliminary Pwification of the Lysate. The lysate was mixed with an

equal volume of chloroform-isoamyl alcohol-phenol (24: 1: 25)) vigor-

ously shaken for 15 min at room temperature and centrifuged for 10 min

at 5000 rpm. The supernatant was removed, treated once more in the

same way and then extracted three times with ether to remove the

phenol. The crude DNA preparation thus obtained was passed through

a column of cellulose powder whose volume was about three times the

volume of the initial tissue. The cellulose n-as previously boiled forseveral minutes in distilled water, poured into the column and equili-

brated with MUP. In some cases the material retained on the cellulose

decreased considerably the flow rate, which made it necessary to appIy

a peristaltic pump. After passing the liquid, the column was washed with

two bed volumes of MUP. The eflluents were combined and loaded on

a HA column.

Hydrozyapatite Chromutog~aph~. The dry HA was suspended in

0.01 M NaP, pH 6.8, heated to boiling, poured into the column andequilibrated with MUP. One gram of dry HA was sufficient, for 1.5-2 mg

of DNA. To achieve an optimal flow rate (0.5 ml/min by grarity) the

height of the HA layer should be 3-5 cm. After loading, the column was

washed with MUP until no absorbance at 260 and 280 nm rould be

recorded. In practice this was achieved after passing about 59-100 ml

of MUP for every 2-5 g of tissue.

The urea was removed next by washing the column with 0.014 M Nap,



which was monitored by refractive index mtasurcmc~nt of the effluents.

DNA was eluted with 0.48 M NaP, pH 6.8 and collected in 5 ml fractions.”It was found that under these conditions a certain amount of DNA

was not adsorbed on the column. To recover this portion of DNA, the

Iysate should be passed through a second HA column with a bed volume

about half that of the first one.

Collection of DNA. To precipitate the total DXA, al l fractions eluted

with 0.48 M NaP were combined and dialyzed against distilled wat’er for

18 hr at 4°C. The solution was made 0.2 M in respect to NaCl by adding

solid NaCl and DNA was precipit,at,cd with 2 vol of ethanol at -20°Cfor 18 hr.

If necessary, the precipitation of DNA from fractions with very low

concentration (0.5-0.05 A,,,/ml) could be achieved using the procedure

of Dessev and Grantcharov (7) in the following modification: the frac-

tions were diluted with an equal volume of distilled water to reduce

the concentration of the NaP, thus preventing further precipitation of

the sodium phosphates by ethanol and 0.1 vol of 0.5 M CaCl, was added

dropwise under continuous stirring. A voluminous precipitate of calciumphosphate was formed. An equal volume of ethanol was then added, the

suspension was shaken and centrifuged for 5 min at 5000 rpm. The pellet

containing the whole amount of DYA was dissolved in 1 M EDTA (half

the volume of CaCl,), salts were removed by dialysis and DXA was pre-

cipitated with two vol of ethanol after adding solid XaCl to 0.2 M.

Procedure II: Lysis in NaCMDS-EDTA Solution

Isolation of Nuclei and Lysis. The tissue was homogenized in 10 volof ice-cold 0.14 M NaCl-0.02 M EDTA, pH 7.0, and nuclei were pelleted

by centrifugation for 6 min at 8009 at 4°C. The nuclear pellet was lysed

by adding 1 M NaCl-0.1 M EDTA-2% SDS, pH 8.0, final volume 50%

of that of the homogenate. The lysing medium was warmed to dissolve

SDS, then cooled to about 30°C and added to the pellet with vigorous

stirring. The lysis was conducted with shaking at 60°C for 10 min. The

sample was then cooled quickly in an ice bath.

Preliminary Purification. An equal volume of chloroform-isoamylalcohol (24: 1) was added and shaking continued at 4°C for 10 min.

After centrifugation for 5 min at 5000 rpm the aqueous phase was col-

lected, treated with an equal volume of water saturated phenol, pH 8.0

a Whe n working with large amou nt of materia l the flow rate of the 0.48 M NaP

effluent decreases considerably due to the viscosity of DNA. In such case s the

elution can be performed as a hatch procedure. Hpdrosyapatite with adsorbed DNA

is transferred into a beaker, susp ende d in 0.48 M p\‘aP and centrifuged. Th is is

repeated3-4 timesand the supernatantscontainingDNA are combined.



ISOL ATIO T\’ OF DSA BP HA CHROh3ATOGRAPHT 559

in the same way and then extracted three times with ether to eliminate

the phenol. EDTA was neutralized by adding 0.5 M CaCl, to equimolaramount and the solution was butiered at pH 6.8 by adding 0.48 M NaP

to a final concentration of 0.18 31. Before loading onto the H-4 column,

the solution was passed through n cellulose column as described in pro-

cedure I.

Hydrozyapatite Chro?natoyraphy. The HA column was prepared as

in procedure I except that the HA was equilibrated with 0.18 M NaP.

After loading, the column was washed with 0.18 IVI NaP until no absorb-

ance at 260 and 280 nm was recorded in the effluent. Al l procedures wereperformed at 4°C. The adsorbed DXA was then eluted with 0.48 M

NaP at room temperature and precipitated as in procedure I.

Other Isolation Procedures

For comparison, DSA was isolated also by the original method of

Britten et al. (2) and by a modified procedure of Marmur (8) including

additional deproteinization with phenol and treatment with ribonuclease

(100 p.g/ml) and pronase (200 kLg/ml).

RESULTS AND DISCUSSION

The characteristics of the DSA preparations, isolated by the different

procedures, are given in Table 1. As judged from the absorbancy ratios

(Az6”/Azg0 and A,,,/A,,,I and the content of RNA and proteins, the

purity of the DNA obtained by the two HA procedures is at least equal

to that of the highly purified DNA, isolated by the classical procedure

of ;19armur (8) using enzymatic treatment and rareful dcprotcinizations.In addition, the yield is severalfold higher than in the case of the Mar-

mur’s procedure or the original i\ll’P method. On thermal dcnaturation

in 1 X SW the DNA isolated by bot#hprocedures showed a hyperchromic

effect of 32-33%, t.he melting rurycs being t’ypicnl for native doublc-

stranded DNA.

It should be mentioned t,hat due to the low concentration of proteins

and RNA in the DNA preparations, an accurate determination of their

content by the Lowry procedure and by the orcinol reaction was impossi-ble even when samples of DNA with a concentration above I mg/ml

were used. The content of proteins was found to be always below 1%,

i.e., it was below the detection limits of the method. The data for RNA

are overestimated due to the interference with the orcinol reaction of

different substances, including DNA (6). For these reasons the purity

in respect to proteins and RXA was checked also by radioactivity

measurements. DNA was isolated by procedure I from rat liver labelled

in viva with [14C]orotic acid for 40 min (30 pCi/lOO g body weight).



560 MARKOV AXD IVAXOV

TABLE 1

Characteristirs of TIN.4 Prevnrations Isolated bv Ijifferent Procedllres

Yield mg RNA Protein content

DNA/g con-

Isolat.ictn fresh organ A26nj A26,,/ tent. Radioactivity

procedrlre Starting mab erial weigh t -4 280 A130 (‘2) (%I,, (cpm/mgDNA)

Procedure I

Britten pf al.

(2)Procedure II

1llarmiir

procedure

Procedure I

Britten ct al.

(2)

Procedure I

Bri: t,en ct al.

(2,

Rat liver 3.5

(whole tissue)

Rat liver 1.1

(whole tissue)Rat liver 1.5

(nuclei)

Rat liver 0.6

(nuclei)

Moirse spleen 8.55

(whole tissue)

Mouse spleen 3.3c

(whole tissue)

Rat liver

chromatin1~at, liver -

chromatin

2.20 2.30 1.3 <1 -

2.06 2.12 1.3 <l -

2.20 2.30 2.2 <l -

2.20 2.30 1.5 <1 -

3.05 2.34 - - 94 (-o.lyo)

1.90 1.44 - - 8100 (-10%)

2.20 2.30 - - 25 (o.l%)a

1.92 1.72 - - 2140 (8.7%)”

a The spe cific radioactivity of the total chromatin proteins was 24500 cpm/mg DNA.

Total nuclear RNA was isolated from a part of the same liver and its

specific radioactivity was determined. According to the radioactivity

present in the DSA preparation, the concentration of RNA was esti-

mated to be about 0.1%. The same approach was applied to estimate thequantity of the protein rontaminants. Rat proteins were labelled in viva

with [W]valine (100 PCi introduced in three portions at 12 hr intervals).

As seen in Table 1, in the DNA isolated from spleen by our modification

of the MUP procedure, only traces of radioactivity are present cor-

responding to less than 0.1% of protein.

The modification of the MUP procedure proposed in this paper avoids

the three main disadvantages of the original method of Britten et al. (2) :

a. the technical difficulties connected with clogging of the column; b. theloss of DNA due to incomplete retention on HA; c. the presence of con-

taminants in the DNA preparations,

In our modifiration clogging is overcome by a preliminary passing of

the lysatc through a cellulose column. Experiments with 3H-labelled

DNA showed that the amount of DNA retained on the cellulose was

negligible.

The most important problem in the MUP procedure is tile purity of

the DNA in particular when whole tissue or crude nuclear preparations



562 ;MARI~ov AND 1vm0v

TABLK 2

Factors Affecting the Recovery of DNA in Hydroxyapatite Isolation Procedllres

Loading mixture

I )ist.ribut.ion of recovered

Total I)NA (taken as 100) among

recovery subsequent HA columns

(y;, of

t,he DNA Column Column Column

loaded) I II III-

A. Artificial mixtures

DNAa in 0.18 M NaPDNAa in MUP

__-.

DNAa in MUP-l’j$ SDS-O. 01 M EI>TA

DNA6 f hemoglobinc in MUP-170 SDS-

0.01 M EDTADNA* + bovine serume albumind in MUP-

1% SDS-O. 01 M EDTA

B. Tissue lysateseDirectly loaded onto HA

Lysate deproteinieed with chloroform and the

water phase passed through celluloseThe same procedure, deproteinization with

chloroform-phenol

8S 98.194 96.5

94 72.672 44.8

87 42.8

46.0

61.6

85.0

1.9 -3.5 -

27.4 -

48.2 7.0

45.2 12.0

54.0

38.4

15.0

u Twelve &,,-units of mouse mammary gland DNA isolated by procedure I were

dissolved in 10 ml of the corresponding medium and loaded onto a HA column containing

1 g dry hydroxyapatite.b 50A2~units of bovine thymus DNA (Koch-Light) and 100 mg hemoglobinc or

300 mg bovine serume albumind were dissolved in 25 ml of the lysing medium of Britten

et al. (2), treated with an equal volume of chloroform and the water phases were loaded

onto HA columns containing 2 g of dry HA each.c Rat liver nuclei isolated as described in procedure II were lysed in the lysing medium

of Britten et al. (2) as in procedure I.

was demonstrated also in experiments with artificial DNA-protein mix-

tures (Table 2). The preliminary deproteinization with chloroform or

chloroform-phenol improved the retention of DNA on HA, the latter

mixture giving better results. Nevertheless, even under such conditions

no full adsorption of DNA was achieved. As shown in Table 2, theamount of DNA not retained on the first column (15-2074 in the case

of phenol pretreatment) could be recovered by recycling the lysate on a

second column. Therefore when a maximum yield of DXA is desirable,

the HA chromatography in procedure I should be repeated twice. This is

not necessary for the procedure II where the retention of DNA on the

first column is practicaIly compIete (Table 2).

Comparing both procedures it should be mentioned that they give

equally good results in respect to the purity of DNA. However, concern-



ISOLATIO N OF DK.4 BY HA CHROMATOGRAPHY 563

ing other characteristics, there are some important differences which

should be taken into consideration. Procedure I has some technicaladvantages. It is carried out at room tempcraturc (except the blending).

Whole tissue (fresh or preliminary frozen) can bc used as starting ma-

terial, which improves the yield (up to 807% of the DNA). These ad-

vantages may be of importance especially when labelled DNA should

be isolated from a small amount of tissue or from tissues where the pre-

liminary isolation of nuclei is difficult. However, it should be emphasized

that the DNA obtained by this procedure is slightly sheared due to the

blending, so that it can not be used in experiments where high-molecular-weight DNA is needed. In such cases procedure II is preferable. Both

procedures are easily carried out in one day.

REFERENCES

1. BER XAR DI, G. (1971) in Methods in Enzymology (Colowick, S. P., and Kap lan,

N. O., eds.), Vol. 21, p. 95, Acad emic Press, New York.

2. BRITT EN, R. J., PAVICH, M., .&ND SMITH, J. (1969) Ca?xegie Inst. Wash ington

Yea&. 68, 400.

3. HEL L, A., BIRNIE , G. D., SLIMMIKG. T. K., AND PAU L, J. (1972) Ana l. Bioc hem .

48, 369.

4. MIYAZAWA, Y., AND THOMAS, C. A. (1965) 1. Mol. Bio l. 11, 223.

5. LOWRY, 0. H. (1857) in Methods in Enzymology (Colowick, S. P., and Kaplan,N. O., eds.). Vol. 4, p. 366, Acad emic Press, New York.

6. SCHN EIDER, W. C. (1957) in Methods in Enzymology (Colowick, S. P., and &plan,N. O., eds.), Vol. 3 , p. 680, Acad emic Press, New York.

7. DES SEV, G. N., AND GR.ANCHAROV, K. (1973) Anal. &o&em . 53, 269.

8. MARMUR, J. (lQ63) in. Methods in Enzymology (Colowick, S. P., and Kaplan, N. O.,eds.), Vol. 6, p. 726, Acad emic Press, New York.

Q. OIsHI, M. (1971) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O.,eds.), Vo l. 21, p. 140, Acad emic Press, New York.

hydroxyapatite column chromatography in procedures

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