a simple efficient method for purification of degraded pcr primers

8/17/2019 A Simple Efficient Method for Purification of Degraded PCR Primers

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Received 16th Septem.b:+r

A SIMPLE, EFFICIENT METHQD FOR

PURIFICATION OF DEGRADED PCR PRIMERS

Andrea Fekete” and John A. Bantle*

Department of Zoology, Oklahoma State University, Stillwater,

Oklahoma, 74078.

SUMMPARY

A simple, efficient method was used to purify QCR primers which had degraded during storage at

-2O”C/-9O’C. The primers were electrophoresed ou 3 A (w/v) agarose gel, the main band was

electroeluted via a trough cut in the gel. The primers were recovered by isobutanol extraction

followed by ethanol precipitation. The yield was 20-40% and the A,,/A,, ratio was greater than

1.8. The purification resulted in good amplification.

Many applicatioras of QCR technology including diagnostic research require routine amplification

of a large number of samples and high reproducibility.

To achieve this, we found that it is often

necessary to use purified primers and carefully control Lhe quality of the primers. For the purifica-

tion of 25-40 bp nuckotides, I5 % polyacrylamidef3OR urea gels

are commody used

to obtain good

band separation followed by cutting the band of interest, eluting *with appropriate buffer and then

purifying by reversed-phase chromatography on silica gei or phenolfchloroform extraction. The

primers are then precnpitated in ethanol (Sambrook et al.,

1989). QoIyacrylamide gels have the

disadvantage of being made from very toxic monomer, which must be handled with care.

The

procedure is also very time consuming.

Recently, we have experienced that even purified oligonu-

cleotides can be degraded during storage at -20°C or -90°C causing complete failure to amplify the

intended target. Here we describe a reliable, simple method for purification of degraded QCR

primers by elecctrophorcsis on 3 %

(w/v)

agarose gel and electroelution.

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. ~CETIILXIE~~ address: Institute of Biophysics, Scmm&veis Medical University, P. 0. Box 263, Budapest, H-1444, &ngary.

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Agarose Gel Electronhoresis

MATERIALS AND METZZODS

Oligonucleotide primers (28 bp long each) were dissolved in O.OlM Tris-HCl buffer (pH 7). The

concentration was determined by absorbance measurement and adjusted to 0.1 pg/pl.

15-15 pl was

analyzed on a 3 % (w/v) agarose gel (Bethesda Research Laboratories, BRL, Gaithersburg, MD)

containing 0.5 pg/ml ethidium bromide (Sigma, USA) in TBE buffer (89 mM boric acid, 89 mM

Tris-HCI, 2 mM EDTA, pH 8). @X174 DNA/HaeIII fragments were used as size standards (BRL).

The samples were electrophoresed at 100 V for 30 min using a BRL “baby gel” apparatus.

Purification of primers

The bands were detected by a UV transilluminator. A sharp scalpel was used to cut a trough directly

in front of the leading edge of the desired band and about 2 mm wider than the band of each side.

The gel was returned to the base plate, the trough and the buffer reservoir was filled with buffer,

but the gel was not covered. The same current was applied and the electrophoresis was continued

and the progress was monitored every two minutes by a long-wavelength hand-held UV lamp.

When all the primers in the main band had moved from the gel, they were recovered in the fluid

taken from the trough (total volume approximately 25 ~1). The whole electrophoresis/electroelution

proceduie lasted only 45 min. From the pooled primers, the ethidium bromide was removed by two

extractions of equal volumes of isobutanol and precipitation by cold (-20°C) ethanol. The precipi-

tate was resuspended in distilled water and the concentration and purity determined by absorbance

measurements.

Polvmerase Chain Reaction

Polymerase chain reaction was performed using a Coy Model 60 Tempcycler or a Teclme Thermo-

cycler. Brucella abortus S19 DNA (1 rig/test) or freeze-thawed Brucella cells (IO rig/test) were

amplified by use of Perkin-Elmer Cetus GeneAmp amplification reagent kit. The reagents, stock

solutrons, as well as the primers used were the same as described earlier (Fekete et al. I 1990a).

Briefly, a master mixture of reagents was prepared (water, buffer, 1 mM magnesium, 70 pmol of

each deoxynucleotide, 200 pglml bovine serum albumin, 0.35 pmol of each primers and 1 U of Taq

polymerase) and irradiated for 5 min with a 300 run transilluminator. The template DNA was dena-

tured at 105°C before adding to the reaction mixture. Then the mixture was overlayed with 35 ~1

of mineral oil. Forty temperature cycles were performed.

The times and temperatures were: denat-

uration, 94°C for 1.5 min, primer annealing, 60°C for 1 min, and chain elongation, 72°C for 1

min, these temperatures are solution temperatures.

Fifteen ~1 of the reaction mixture was electro-

phoresed on 1% (w/v) agarose gel containing 0.5 pgiml ethidium bromide at 75 V for 1.5 hr using a

BRL “baby gel” apparatus.

RESULTS AND DISCUSSION

Earlier we developed a test to detect Bruceila DNA based on PCR (Fekete et ai.) 1990 a,b).

Figure 1A shows the results of the amplification cf a 607 bp fragment of Brucella abortus S19 DNA

with degraded and newly synthesized, unpurified primers: lanes 1: 1 ng S19 DNA, degraded prim-

ers, 2: 10 ng freeze-thawed Brucella cells, unpurified primers, 3: no template DNA, primers only

4: 500 bp Cetus DNA from the kit as a positive control 5: 123 bp DNA ladder size standard. It can

be seen that only a smear, mostly of high molecular weight material, was produced instead of the

expected 607 bp fragment. Thus result was obtained even though the previously optunized protocol

was used (Fekete et al., 1990a). Thus protocol had worked well for more than a year as long as the

primers were not degraded. The same smear was produced when template DNA was not added to

the reactron mixture (lane 3). The reason for the smear could only be the primers as the possible


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DNA contaminants were eliminated by UV irradiation (Sarkar and Sommer, 1990). The Perkin-

Elmer Cetus positive control also worked well during the same run, indicating that the reaction

components were good. We then purified the primers in order to prove that primer degradation

caused the smearing effect. We found that oligonucleotides can be separated not only on polyacryl-

amide gels but on 3% (w/v) agarose gels as well. Fig. 1B shows the electrophoretic pattern of the

28 bp oligonucleotide primers: lanes 2 and 3, primer l and 2, respectively. Lane 1 contained the

OX174 HaeIII fragments as size standards. The primers were dissolved in 0.01 M Tris-HC1 buffer

(pH 7) and were stored in aliquots at -90” C for more than six months.

Instead of the expected

single band, a number of other bands were observed, especially in the case of primer 2.

The elec-

trophoretic mobility seemed to be dependent on the base composition: the CC content is 50 % and

68% for primer l and 2, respectively.

A similar electrophoretic pattern was obtained with newly

synthesized, unpurified primers.

We obtained the same separation of bands on denaturing polya-

crylamide gels (data not shown). If these undesired sequences contained complementary bases to

each other, they would anneal with each other rather than to the template DNA. This event can and

FIGURE 1.

A: Electropboretic analysw of thz amplification product of Brucella on a 1 X (w/v) agarose gel. Lanes: 1: 1 ng S19 DNA,

degraded primers 2: 10 ng freeze-thawed

Bnrcello

cells, unpuntied primers 3: no template DNA, primsrs only 4: Cztus

DNA as a positive control 5: 123 bp DNA ladder as a size standard 6: I ng S19 DNA, purified primers 7: 10 ng freeze-

thawed Brucek~ cells, punfied primers 8. no template DNA, purified primers only.

B: Electrophoretis analysis of the 28 bp oligonucleotide primers on a 3X (w/v) agarose gel. Lanes: 1’ OX174 DNA HaelI

fragments as size standards 2, primer 1 3: primer 2 4. primer l aRer purification 5: primer 2 after purification .

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does result in the extension of each primer. As each primer serves both as primer and template, a

sequence complementary to each primer can be formed.

This product, upon denaturation is a per-

fect template for further primer binding and extension.

As can be seen on Fig. lA, identical rcac-

tions with and without added template DNA resulted in primer artifacts or smearing.

A variety of methods exist to remove DNA fragments f rom agarose gels (Sambrook et al., 1989).

We eluted the primers from 3 96 agarose or 3 96 low melting point agarose gels, then extracted with

phenol/chloroform and precipitated with ethanol.

The yield (20%) and the oligonucleotide purity,

as judged by absorbance measurement at 260 and 280 nm, was acceptable.

However, they may

have contained contaminants of agarose which inhibited the Taq polymerase activity: the reaction

still did not work but the smear was less.

We found that oligonucleotide primers can be electroeluted best from the horizontal slab gel via a

trough cut in the gel. There was no need to use a dialysis bag or other electroelution device (Manns

and Grosse, 1991). The electrophoresis/electroelution procedure lasted only 45 min and we could

use the same apparatus as for the analysis of the PCR samples. We found that was not necessary to

further purify the primers and we could simply remove the ethidium bromide by extracting with

isobutanol. The yield was about 20%-40%) which was superior to the typical 15 % yield with

polyacrylamide gels (Tullis et al., 1989). The A,,IA,,

ratio was greater than 1.8. On Fig. 1A,

lanes 6-8 show the electrophoretic analysis of the PCR reaction with the purified primers.

The

conditions are the same as in the case of lanes l-3.

The expected single band (607 bp) appeared in

each lane, indicating the effectiveness of purification.

The reason for the occurrence of degraded primers has not been resolved but our experience has

shown that it can happen during storage in unopened tubes at -90°C.

We found that the best way to

remove the degraded fragments was by agarose gel electrophoresis and electroelution.

ACKNOWLEDGEMENT

The authors wish to thank Mendi Hull for assistance in the preparation of this manuscrip t. This work was supported by a

United States Department of Agriculture-ARS Cooperative Agreement Number 58-5114-9-1008.

REFERENCES

Fekete, A., Bantle, LA., Hailing, S.M., Sanbom, M.R. (IPPOa). J. Appl. Bacteriology 69, 216-227.

Feketc, A., Bantle, IA., Halling, KM., Sanbom, M.R. (1990b). Biotechnol. Techniques 4, 31-34.

Manns, A., Grosse, F. (1991). Biotechniqucs 10, 158-160.

Sambmok, I., Ftitsch, E.F., Maniatis, T. (1989). Molecular Cloning - A Laboratory Manual, 2nd Edition. Cold Spring

Harbor Laboratory, Cold Spring Harbor, NY.

Sarkar, G., Sommer, S.S. (1990). Nature 343, 27.

Tollis, R.H., Fetberoff, P., O’Hara, B. (1989). Amplifications 3, 17-18.

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a simple efficient method for purification of degraded pcr primers

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