144
GATA 10(6): 144-146, 1993
Amplification of Specific Gene
Products from Human Serum
S T U A R T L. E M A N U E L and
SIDNEY PESTKA
The polymerase chain reaction (PCR) is an in vitro
method f o r the primer-directed enzymatic amplification
o f specific D N A sequences. Ordinarily, sources with ob-
vious D N A content such as cells, viruses, and plasmids
serve as the origin o f templates f o r the PCR. Here we
report a simple and efficient method to obtain cellular
D N A f r o m serum suitable f o r use in PCR reactions or
gene analysis. This procedure should facilitate the de-
tection o f disease and provide a basis f o r the examina-
tion o f mutations in genes before the onset as well as
during the progression o f various diseases.
Introduction
The polymerase chain reaction (PCR) [1, 2] is an in
vitro method for the primer-directed enzymatic
amplification of specific DNA sequences. The se-
quence specificity of the amplification enables
DNA from a single gene to be amplified from a
heterogeneous mixture of DNAs. Ordinarily,
sources with obvious DNA content such as cells,
viruses, and plasmids serve as the origin of tem-
plates for the PCR. Blood serum has been an ef-
ficient source for the detection of viruses and
blood-borne pathogens [3] and for the detection of
viral RNA by reverse-transcriptase-PCR [4].
However, serum has not been routinely used as a
source of cellular DNA.
The standard PCR requires the presence of two
primers and a DNA template. The source of the
DNA can be cellular DNA (nuclear, cytoplasmic,
or mitochondrial) or DNA isolated from other
sources such as bacteria, plasmids, and viruses.
Sources, such as blood, saliva, and stools, that are
From the Department of Molecular Genetics and Microbi-
ology, University of Medicine and Dentistry of New Jersey,
Robert Wood Johnson, Medical School, Piscataway, New Jer-
sey, USA.
Address correspondence to Dr. S.L. Emanuel, Department
of Molecular Genetics and Microbiology, University of Medi-
cine and Dentistry of New Jersey, Robert Wood Johnson Med-
ical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA.
Received 4 June 1993; revised and accepted 30 September
1993.
© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
1050-3862/93/$6.00
expected to contain cells have also been a source
for PCR analyses [5, 6]. Serum, which is consid-
ered to be devoid of cells, was not expected to
contain DNA. We hypothesized, however, that
during the clotting process that results in serum
formation, there may be enough cell destruction
and debris remaining to provide sufficient DNA
for PCR analysis. Furthermore, as the blood
courses throughout the body, it may pick up cells,
cellular debris, and other components from tis-
sues. Thus, the serum would contain DNA from
peripheral blood cells as well as, possibly, from
other tissues. This report discusses the results of
this study demonstrating that indeed, sufficient
DNA can be obtained from serum for routine PCR
analysis.
Experimental Procedure
D N A Extraction
One-milliliter samples of crude serum were boiled
for 5 min in 1.5-ml polypropylene tubes and cen-
trifuged for 10 min at 12,000 g. About 100 p,l of
supernatant was removed and used directly as
template in PCR reactions. If no product was ob-
tained or when cleaner DNA was desired, the su-
pernatant was diluted up to 500 ~1 with water. This
was extracted with an equal volume of phenol-
chloroform-isoamyl alcohol (25:24:1) until no pro-
tein was seen at the interface.
The DNA was precipitated with 0.1 vol of 3 M
sodium acetate, pH 5.5, and 2.5 vol of ethanol.
The pellet was dried in a SpeedVac (Savant In-
struments, Farmingdale, NY, USA), and the DNA
was dissolved in 100 lxl of water. About 1-5 p.l of
this solution was used in each 50-p,l PCR reaction.
From absorption measurements at 260 nm, 1 ~1 of
this solution contained 200 ng of template DNA.
PCR Reaction
The PCR was carried out in 25-~1 or 50-~1 reac-
tions containing 1-3 p,l of serum supernatant or
1-5 p.l of precipitated serum DNA in 10 mM
Tris • HC1, pH 8.3, 50 mM KCI, 1.5 mM MgCI2,
100 ng/ml gelatin, 200 nM each primer, 0.4 mM
dNTPs (dATP, dCTP, dGTP, and dTTP), and 2.5
units of Taq DNA polymerase (Boehringer Mann-
heim, Indianapolis, IN, USA). Samples were pre-
pared in MicroAmp thin-wall tubes (Perkin-Elmer,
Norwalk, CT, USA) and subjected to 35 cycles of
PCR amplification with a Perkin-Elmer model
9600 Thermal Cycler. One c y c l e c o n s i s t e d

Gene Products from Serum
of 15 s denaturing at 94°C, 30 s annealing at 58°C,
and 1 min extension at 72°C. After amplification,
the DNA was resolved by electrophoresis through
1.2% agarose or 4% NuSieve (FMC, Rockland,
ME, USA), gels containing 0.8 v.g/ml ethidium
bromide.
Primers
Primers were designed to amplify a single-copy
human gene: the gene for human interferon oL-2
(also called o~-A; Hu-IFN-ot2 and Hu-IFN-otA)
[7-I 1] and a gene for the human interferon ot re-
ceptor [12, 13]. The specific primers used are
given in the Figure 1 legend.
Results and Discussion
To determine whether serum could be used for
PCR analysis of cellular DNA, PCR products were
1 2 3 4 5 6 7 8 9
~17bp
396
344
.~98
.~20
-~01
Figure 1. Polymerase chain reaction (PCR) products amplified
t~-~])'T~IA derived from serum. The amplified samples (15 wl)
were electrophoresed in a 4% NuSieve gel containing 0.8 o,g/ml
ethidium bromide. Primers YH1 and YH2 representing the 5'
end (5'-AGCCACCTCCAGCAGAGCAC-3')and the 3' end (5'-
GTTTACCAAAGGTCCTAAGG-3'of ) the human interferon a (Hu-
IFN-c0 receptor, respectively, were designed to amplify a 240-
base-pair (bp) segment (nucleotides 24,654-24,894) [12, 13] at
the junction of exon 7 of the Hu-IFN-ct receptor: Lane 1,200 ng
of serum DNA; lane 2, no DNA template; and lane 3, 10 ng of
cosmid 8-2 [13], which contained the 3' end of the Hu-IFN-a
receptor in cosmid pCV-108 [15], served as template. Primers
3683 and 4369 specific for the 5' end (5'-AACCCACAGCCTGGG-
TAG-3') and the 3' end (5'-CATGATTTCTGCTCTGACAACC-3')of
the Hu-IFN-a2 gene, respectively, amplified a 430-hp fragment
(nucleotides 594-1023) [7-11] spanning amino acids 6-148: lane
4, 200 ng of serum DNA; lane 5, no DNA template; and lane 6,
10 ng of plasmid pBR325-a2 containing the Hu-IFN-a2 gene
[9]. Primer 4603 specific for the 5' end of the Hu-IFN-a2 gene
(5'-GCTCCTGCsCACAGATGAGGAG-3'was ) used with primer 4369
above to amplify a 397-hp fragment (nucleotides 62%1023) of
the Hu-IFN-a2 gene from amino acids 1%148: lane 7, 200 ng
serum DNA; lane 8, no DNA template; and lane 9, 10 rig of
plasmid pBR325-a2.
© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
145
GATA 10(6): 144-146, 1993
amplified from the human interferon ot (Hu-IFN-tx)
receptor gene and from the human interferon or-2
(otA) gene. DNA was isolated from serum and am-
plified by the PCR with the use of three sets of
primer pairs (Figure 1). It can be seen that in all
three cases the DNA amplified from genomic
DNA in the serum (Figure l, lanes l, 4, and 7) was
identical to the DNA amplified from control plas-
mids containing the same genomic sequence (Fig-
ure l, lanes 3, 6, and 9). Reactions without DNA
showed no amplification.
Thus, we demonstrated that genetic analysis
can be performed on DNA obtained from serum.
This DNA may be released from healthy cells that
remain in the serum or from cells that have rup-
tured and released their DNA. The DNA is of suf-
ficient quality and quantity for digestion with re-
striction endonucleases, electrophoresis, hybrid-
ization, and PCR amplification.
Template DNA was rapidly obtained from hu-
man serum samples by using 1 ~l of supernatant
after simply boiling and centrifuging the serum in a
microfuge to remove excess protein. We have se-
lectively amplified a 430-base-pair (bp) fragment
of Hu-IFN-ot2 and a 240-bp fragment of the Hu-
IFN-o~ receptor as described under Experimental
Procedures. All amplification reactions produced
a single PCR product of the expected size (Figure
1). The identity of each product was confirmed by
restriction enzyme analysis.
While we were writing this report and searched
the literature for similar studies, we discovered a
report by Martin et al. [14] describing the use of
DNA isolated from serum for use in H L A typing
by PCR. The method described here is simpler and
yields DNA from serum in one-tenth the time. Our
results and theirs [14] demonstrate the ability to
examine DNA sequences from serum samples.
This observation provides a new source of DNA
for the study of genes by using molecular genetic
techniques and should simplify the process of di-
agnosis in clinical laboratories for these purposes.
Serum samples need not be refrigerated and can be
transported at ambient temperatures to diagnostic
laboratories, which would reduce shipping costs.
The availability of large banks of serum will facil-
itate the analysis of changes in gene sequences
over time and in various disease states, because
the DNA in these frozen specimens should be sta-
ble for years.
We thank Dr. Karel Raska for samples of serum. This study
was supported in part by US Public Health Service grants ROI-
146
GATA 10(6): 144-146, 1993
CA46465 and RO1-CA52363 to S.P.; and a training grant T32-
AI07403 awarded to S.P. with stipend support to S.L.E.
References
1. Saiki RK, Scharf S, Faloona F, MuUis KB, Horn CT, Er-
lich HA, Arnheim N: Science 230:1350-1354, 1985
2. Mullis KB, Faloona F: Methods Enzymol 155:335-350,
1987
3. Kwok S, Mack DH, Mullis KB, Poiesz B, Ehrlich G, Blair
D, Friedman-Kien A, Sninsky JJ: J Virol 61:1690-1694,
1987
4. Ravaggi A, Primi D, Cariani E: PCR Methods Appl 1:291-
292, 1992
5. Lench N, Stanier P, Williamson R: Lancet 1:1356-1358,
1988
6. Sidransky D, Tokino T, Hamilton SR, Kinzler KW, Levin
B, Frost P, Vogelstein B: Science 256:102-105, 1992
7. Maeda S, McCandliss R, Gross M, Sloma A, Familletti PC,
Tabor JM, Evinger M, Levy WP, Pestka S: Proc Nati Acad
Sci USA 77:7010-7013, 1980; and 78:4648, 1981
© 1993 Elsevier SciencePublishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
S.L. Emanuel and S. Pestka
8. Goeddel DV, Yelverton E, Ullrich A, Heyneker HL, Mi-
ozzari G, Holmes W, Seeburg PH, Dull T, May L, Steb-
bing N, Crea R, Maeda S, McCandliss R, Sloma A, Tabor
JM, Gross M, Familletti PC, Pestka S: Nature 287:411-
416, 1980
9. Lawn RM, Gross M, Houck CM, Franke AE, Gray PV,
Goeddel DV: Proc Natl Acad Sci USA 78:5435-5439, 1981
10. Henco K, Brosius J, Fujisawa A, Fujisawa J-I, Haynes JR,
Hochstadt J, Kovacic T, Pasek M, Schamb6ck A, Schmid
J, Todokoro K, W~ilchliM, Nagata S, Weissmann C: J Mol
Biol 185:227-260, 1985
11. Emanuel SL, Pestka S: J Biol Chem 268:12,565-12,569,
1993
12. Lutfalla G, Gardiner K, Proudhan D, Vielh E, Uze G: J
Biol Chem 267:2802-2809, 1992
13. Mariano T, Donnelly RJ, Soh J, Pestka S: In Baron S, et al.
(ed): Interferon: Principles and Medical Applications.
Galveston, TX, University of Texas Press, 1992, pp 129-
138
14. Martin M, Carrington M, Mann D: Hum Immunol 33:108--
113, 1992
15. Lau YF, Wai-Kan Y: Proc Natl Acad Sci USA 80:5225-
5229, 1983