Received

16th

Septem.b:+r

A SIMPLE, EFFICIENT
PURIFICATION

METHQD FOR

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
-2OC/-9OC.
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
ratio was greater than
followed by ethanol precipitation. The yield was 20-40% and the A,,/A,,
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.
tion of 25-40 bp nuckotides, I5 % polyacrylamidef3OR

urea gels

are commody

For the purifica-

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

primers are then precnpitated in ethanol (Sambrook et al., 1989). QoIyacrylamide

extraction.

The

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 -20C or -90C 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.

1.. ~CETIILXIE~~ address:

Institute

of Biophysics,

Scmm&veis

Medical

479

University,

P. 0. Box

263,

Budapest,

H-1444,

&ngary.

MATERIALS AND METZZODS

Agarose Gel Electronhoresis
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 (-20C) ethanol. The precipitate 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 Thermocycler. 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 denatured at 105C 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: denaturation, 94C for 1.5 min, primer annealing, 60C for 1 min, and chain elongation, 72C for 1
min, these temperatures are solution temperatures. Fifteen ~1 of the reaction mixture was electrophoresed 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 primers, 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

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 polyacrylamide 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 electrophoretic mobility seemed to be dependent on the base composition: the CC content is 50 % and
68% for primer #l and #2, respectively.
synthesized, unpurified primers.

A similar electrophoretic pattern was obtained with newly

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.

FIGURE

This event can and

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,

DNA as a positive control 5: 123 bp DNA ladder as a size standard 6: I ng S19 DNA,

primsrs only 4: Cztus

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

481

5: primer #2 after purification.

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 perfect 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 from 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
and precipitated with ethanol. The yield (20%) and the oligonucleotide purity,

phenol/chloroform

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

polyacrylamide

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

lanes 6-8 show the electrophoretic

15 % yield with

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

analysis of the PCR reaction with the purified primers.

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

The

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 -90C. 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 manuscript.
United States Department of Agriculture-ARS

Cooperative

This work was supported by a

Agreement Number 58-5114-9-1008.

REFERENCES
Fekete, A., Bantle, LA.,

Hailing, S.M., Sanbom, M.R. (IPPOa). J. Appl. Bacteriology

Feketc, A., Bantle, IA.,

Halling, KM.,

Manns, A., Grosse, F. (1991). Biotechniqucs

Sambmok,

I., Ftitsch,

Harbor Laboratory,

10, 158-160.

E.F., Maniatis, T. (1989). Molecular

Cloning - A Laboratory

Cold Spring Harbor, NY.

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

Tollis, R.H., Fetberoff,

69, 216-227.

Sanbom, M.R. (1990b). Biotechnol. Techniques 4, 31-34.

P., OHara, B. (1989). Amplifications

3, 17-18.

482

Manual,

2nd Edition.

Cold Spring