Tissire Anti& :ens 1995: 45: 41-48 Copyrighl 0 Munksauurd 1995

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TISSUE ANTIGENS
ISSN 0001-28l5

High resolution HLA-DRB1 SSP typing for

cadaveric donor transplantation
l? H. M. Savelkoul, D. F! de Bruyn-Geraets, E. M. van den Berg-Loonen. Paul H. M. Savelkoul,
High resolution HLA-DRB 1 SSP typing for cadaveric donor transplan- Daisy P. de Bruyn-Geraets and
tation. Ella M. van den Berg-Loonen
Tissue Antigens 1995: 45: 4148. 0 Munksgaard, 1995
Tissue Typing Department, University Hospital
Maastricht, Maastricht, The Netherlands
Abstract: An HLA-DRB 1 typing procedure by means of sequence-specific
' primer (SSP) amplification was developed for 65 different DRBl sub-
types. Subtyping is achieved by the performance of two subsequent PCR
assays (PCR-1 assay and PCR-2 assay) using a limited number of reactions.
The PCR-1 assay determined low-resolution HLA-DRB 1 typing, i.e. the
serologically defined specificities DRI, 2, 3, 4,11, 13, 6, 7, 8, 9 and 10. The
second exon of the DRBl gene is amplified also in this PCR-1 assay.
High-resolution subtyping for positively identified alleles was performed in
the PCR-2 assay with the exon-2 product from PCR-1 assay as DNA
template. PCR reactions were carried out using unpurified primers in reac-
tion volumes of 20 pl and 100 ng of chromosomal DNA. After 3 hours,
the results of the PCR-1 assay were analyzed and subsequently subtyping
results in the PCR-2 assay were obtained in another 1.5 hours. A total of
249 DNA samples was typed by this method. No false positive nor false
negative results were obtained in DRBl typing of 32 homozygous cell
lines, 56 serologically well-defined panel cells and 125 unrelated individ-
uals. Segregation of the amplification patterns was investigated in 36
members of 7 two-generation families. DRB 1 subtyping revealed codomi-
nant Mendelian segregation for all subtypes investigated. In conclusion,
LR-HR-PCR-SSP typing is a fast and reliable typing technique for routine Key words: allele specific amplification - class
DNA typing purposes which gives complete DRB 1 subtyping within 4.5 II - HLA - HLA-DRB1 typing - PCR-SSP -
h. Besides low-resolution DRB typing, also high-resolution DRB sub- transplantation
typing for prospective HLA-DR matching in cadaveric renal transplan- Received January 21, revised, accepted for
tation is possible by this method. publication 19 July 1994

In cadaveric renal transplantation, serology was
and still is the most widely used method due to the
speed with which information on the HLA class I
and I1 antigens of a given donor can be made
available to the clinic. Time management in a do-
nor procedure is of the utmost importance and
therefore other typing techniques that give more
accurate and/or complete typing of an organ do-
nor can not be used thus far. Most of these
methods are time-consuming and results cannot be
obtained in a time-period comparable to serology.
In the last decade the application of DNA typ-
ing techniques in particular has largely contributed
to a more complete and accurate typing and
understanding of the different HLA genes (1-5).
The polymorphism of the second exon of the DRB
genes codes for most of the HLA-DRB antigens
that are typed for in a number of different tech-
niques (6, 7). DNA typing techniques using se-
quence-specific primers have been developed that
allow rapid detection of a number of DRB 1 alleles
(4, 8). Olerup et al. described a rapid DNA typing
method in which the DRB genes are amplified and
identified in one PCR reaction from chromosomal
DNA (4). He achieved so-called low-resolution
typing for the DRB genes involved. Bein et al.
amplified the second exon of the DRB genes from
whole cells and used this as a template in a second
PCR reaction to define the different DRB alleles,
i.e. high-resolution typing (9). For complete DRB
41
Savelkoul et al.
subtyping, both methods need a large number of
PCR reactions (4) and therefore large amounts of
Taq polymerase (9).
The present investigation was carried out with
the aim of obtaining complete DRB subtyping
within a few hours and with a limited number of
PCR reactions. The method had to be suitable for
everyday laboratory practice including prospective
organ donor subtyping.
Material and methods
DNA samples
Thirty-two homozygous cell lines from the Euro-
pean Collection for Biomedical Research (ECBR)
were used as reference material. They represented
the following DRBl alleles: 0101, 0102, 0103,
1501, 1502, 1503, 1601, 1602, 0301, 0302, 0401,
0404, 0405, 0406, 0407, 1101, 1102, 1103, 1104,
1201, 1202, 1301, 1302, 1303, 1401, 1402, 1404,
0701, 0801, 0802, 0803 and 0901. Most of these
cells were analyzed for the 11th workshop by Ki-
mura et al. (10). Fifty-six selected panel cells as
well as 161 samples from patients, their family
members and unrelated individuals were investi-
gated. The 56 panel cells were typed by PCR-SSOP
using the Eurotransplant Typing kit (11, 12, 13).
Serological typing of the cells was performed for
HLA-DR 1-18, Drw51-53 and DQ1-9 antigens by
means of the TCF method (14).
DNA extraction
Genomic DNA was extracted from peripheral
blood in 30 min by a salting out procedure (4, 15,
16). Briefly 500 pl of blood was mixed with Triton
lysis buffer (0.32 M Sucrose, 1% Triton X-100, 5
mM MgCI2. H20, 10 mM Tris-HC1, pH 7.5).
Leukocytes and nuclei were spun down and
washed with H20. The pellet was incubated with
proteinase K at 56°C and subsequently salted out
at 4°C using a saturated NaCl solution. Precipi-
tated proteins were removed by centrifugation. The
DNA in the supernatant was precipitated with
96% ethanol and washed with 70% ethanol. The
DNA pellet was dissolved in 50 pl H 2 0 (4). Con-
centration and purity were measured at OD260
and OD260/280 (17). The average ratio is about
1.6. DNA was used in a standard concentration of
100 pg/ml and stored at 4°C.
PCR primers
Oligonucleotide primers (Table I) were synthesized
on an ABI 391 oligonucleotide synthesizer. Syn-
thesis of primers was carried out using small scale
columns (0.2 pm). No purification of the primers
42
was needed, concentration was determined at
OD260 (17), and stock solutions in H 2 0 were
stored at -30°C at a concentration of 7 pM. Melt-
ing temperatures of specific primers varied be-
tween 60°C and 66°C. A primer mix amplifying,the
human growth hormone gene was used as an inter-
nal control (9, 18).
PCR amplification
Two different PCR reactions constituted the DNA
subtyping for DRBl alleles. In the first PCR-1 as-
say the common HLA-DR alleles DRl, 2, 3,4, 11,
12, 6, 7 , 8, 9 and 10 were determined. On the basis
of the positive alleles identified, subtyping was per-
formed in a second PCR-2 assay using an exon-2
amplified template obtained in the PCR-1 assay.
PCR-1 assay
DNA was amplified by 30 three-temperature
cycles: denaturation for 20 s at 94"C, annealing for
50 s at 65"C, extension for 20 s at 72°C (6). A posi-
tive control was included in the PCR-1 assay by
amplification of the human growth hormone
(HGH) gene (9). The primer mixes consisted of 2
mM dNTP, 1 pM of each specific primer (DRBl),
0.2 pM of each control primer (HGH), PCR buffer
(50 mM KCI, 4 mM MgCI2, 0.06 mg/ml BSA, 10
mM Tris/HCl, pH 8.4). The exon 2 primer mix
contained 0.5 pM of each specific primer (E2) and
0.15 pM of each HGH primer. PCR amplification
was carried out by adding 100 ng DNA and 0.5
unit Taq polymerase (AmpliTaq, Perkin Elmer/
Cetus) to 18 pl of each mix.
PCR-2 assay
For these PCR reactions, 22 three-temperature
cycles were carried out: denaturation for 20 s at
94"C, annealing for 30 s at 66"C, extension for 20
s at 72°C (4). The primer mixes consisted of the
following components: 0.4 mM dNTF', 0.5 pM of
each specific primer, 0.15 pM of each HGH primer
and PCR buffer. PCR amplification was carried
out by adding 1X exon-2 product and 1 U
Taq polymerase (AmpliTaq, Perkin ElmerKetus)
to 13.5 p1 of each mix.
The total reaction volume for both PCR reac-
tions was 20 pl. Mixes were prepared in advance,
and stored at 4°C. A negative control (NC) mix
consisted of the same components excluding the
specific primers. To 10 p1 NC mix,' 0.5 U Taq poly-
merase (AmpliTaq, Perkin Elmer/Cetus) and 1 p1
of each primer mix is added. Amplification reac-
tions were carried out in a GeneAmp PCR System
9600 (Perkin-ElmerKetus Instrument).
 Low resolution-high resolution PCR-SSP HLA-DRB1 subtyping
Table 1.
PCR-1 assay: specificities, nucleotide sequences and lengths of the ORB1 primer pairs

Primer mixes 5' primer L Tm 3' primer L Tm Size bp Specificity

MSP0144 5'llGTGGCAGCllAAGlllGAAT3' 22 60 5'GCTGllCCAGTACTCGGCAT3' 20 62 168 DRBI 401

MSP0145 5'TCCTGTGGCAGCCTAAGAG3' 19 60 S'CGCTGCACTGTGAAGCTCTC3' 20 64 258 ORB1'02
MSP0146 5'GTllClTGGAGTACTCTACGTC3' 22 64 5'TGCAGTAGllGTCCACCCG3' 19 60 260 ORB1'03
MSP0147 5'GlllClTGGAGCAGGllAAACA3' 22 62 S'CGCTGCACTGTGAAGCTCTC3' 20 64 261 ORB~*O~'
MSP0168 S'CACGTllCllGGAGTACTCTAC3' 22 64 5'CTGGCTGllCCAGTACTCCT3' 20 62 177 DRB1'11*
MSP0149 5'AGTACTCTACGGGTGAGTGll3' 21 62 S'CTGTTCCAGGACTCGGCGA3' 19 62 163 DRBI '123
MSP0150 5'GlllCllGGAGTACTCTACGTC3' 22 64 5'CGTAGllGTGTCTGCA(GA)TAGG3' 21 64 233 ORB1'06/'114
MSP0151 S'CCTGTGGCAGGGTAAGTATA3' 20 60 5'CCCGTAGlTGTGTCTGCACAC3' 21 66 231 DRBl'O7
MSP0152 5'AGTACTCTACGGGTGAGTGll3' 21 62 S'CCCGTAGllGTGTCTGCAG3' 19 60 225 ORB1'08
MSP0153 5'GlllCllGAAGCAGGATAAGTll3' 23 62 S'CCCGTAGllGTGTCTGCACAC3' 21 66 236 DRBl '09
MSP0103 5'CGGllGCTGGAAAGACGCG3' 19 62 S'CTGCACTGTGAAGCTCTCAC3' 20 62 205 DRB1'10
MSPOOl2 5'llCGTGTCCCCACAGCACGlllC3' 23 72 5'GCCGCTGCACTGTGAAGCTCTC3' 22 72 295 ORB-Exon2

*
included specificities: DRB1'1410. ORB1'1411. DRBl '1404.
excluded specificities: DRBl "1404/10/11 tORBl'1105.
L=length of the primer. Trn=melting temperature in "C.

Amplification products were analyzed on 1.5%
intermediate melting agarose gel (Metaphore,
FMC, Rockland). To 10 p1 of PCR product, 2 pl of
6 X loading buffer (0.25% BFB, 0.25% Orange G,
0.5% SDS, 30% Glycerol, 5% Ficoll 400, 5 mM
EDTA, 25 mM Tris/HCI, pH 8.0) was added. The
gels were prestained with 0.5 pg/ml ethidium bro-
mide and run for 30 min at 10 V/cm in OSXTBE
(IOxstock solution: 0.9 M H3B03,2.5 mM EDTA,
0.9 M Tris/HCI, pH 8.3). In all gels (D X174 HaeIII
DNA (BRL) was used as a molecular weight
marker. After electrophoresis, gels were analyzed
and photographed under UV transillumination.
Results
Low resolution SSP
By means of direct amplification from genomic
DNA, a total of 249 cells was typed by a 12-reac-
tion PCR-assay. In 11 reactions the serologically
defined specificities DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9
and 10 were determined while in the last reaction
an exon-2 amplification was carried out. Amplifi-
cation patterns were easy to interpret. DNA from
most heterozygous individuals gave rise to two of
the 11 possible PCR reactions. Only DRll-posi-
tive cells showed amplification in both the D R l l
and the DR6 mixes. Three subtypes of DRB1*14,
namely 1410, 1411 and 1404, were amplified in the
DRB1*04, *11 and *12 mixes respectively. A
primer pair amplifying the HGH gene was used as
a positive control in the specific mixes as well as'in
the exon-2 amplification mix.
No false positive nor false negative results were
obtained in typing 32 homozygous cell lines from
the ECBR, a serologically well-defined panel of 56
cells, 36 family members and 125 unrelated DR
homozygous and heterozygous individuals. The
unrelated individuals were not randomly selected.
A number of DNA typings was performed for pa-
tients whose serological DR typing turned out to
be difficult or even impossible. An example of a
heterozygous LR-SSP result is given in Fig. 1.
For the 56 panel cells comparison of serology and
DNA typing showed no differences for the DRBI
alleles detected in the LR-SSP Serologically also
DR13, 14, 15, 16 and 17, 18 were detected, which
were not demonstrated by the LR-SSP method. All
panel cells were typed by LR-SSOP In one cell
SSOP-typing turned out to be impossible despite re-
peated hybridization on different samples. For the
remaining cells complete concordance was shown
between LR-SSP and LR-SSOP
The frequency of PCR amplification failure was
less than 1%. LR-SSP gave reliable results irrespec-
tive of the source of blood or anticoagulant used.
DNA purified from EDTA blood by the salting
out procedure showed slightly reduced amplifi-
cation signals. PCR failure was noticed with non-
human and degraded DNA only. Blind retyping of
20 DNA samples showed 100% reproducibility.
High-resolution SSP
High-resolution typing was obtained by using 67
different primers designed to detect 65 DRBl al-
leles (Table 2). The primer combinations for the
DRBl subtypes are given in Table 3, including the
DRBl alleles *1301-1308 as mentioned in Fig. 2.
Hornozygous cell lines
Thirty-two homozygous cell lines with defined spe-
cificities were typed by the LR-HR-SSP method.
No aspecific amplification signals were .noticed in
the primer mixes. Positive reactions were obtained
43
Savelkoul et al.
4 1 2 3 4 5 6 7 8 9101112NC
Illustration of a DR3 and DR4 positive heterozygous cell. Lanes 1 through 11 represent the DR specificities DR1, DR2, DR3,
DR4, DRlI, DR12, DR6, DR7, DR8, DR9 and DR10. Lane 12 shows the exon-2 amplification, NC is the negative control and
X174RF HaeIII DNA fragmehts are used as molecular weight marker. Positive amplification is shown only in lanes 3 and 4. Specific
product in lane 12 shows the amplification of the exon-2 product.
in some primer mixes as a result of identical DNA
sequences present in other alleles. These signals
could be attributed to the respective DNA se-
quences and did not lead to false identification of
a DRBl subtype.
Panel cells
By means of the exon-2 amplification product the
HR-SSP-subtyping of the 55 well-defined panel
cells was carried out. The cells were typed also by
PCR-SSOP. In 2 cells no reliable SSOP-subtyping
results could be obtained. The SSOP probes used
did not discriminate between DRB1*0101/04, *
1501/03, *1102/03/04, *1201/02, *1401/07 and *
1401/10. All typings were found concordant for
HR-SSP and SSOP results.
Family studies
Segregation of DRBl alleles was studied in 36
members of 7 two-generation families. Most DR
specificities (except DR10, 16 and 18) were present
in at least one of the families. All broad DRBl
specificities were demonstrated by LR-SSP. Sub-
sequent HR-SSP revealed codominant Mendelian
segregation for all subtypes investigated. In family
JVB, the LR-PCR-reaction of the mother of this
family was already shown (Fig. 1). Segregation of
DRB1*1301 in family JVB is shown in Fig. 2.
Unrelated individuals
LR-HR-SSP was performed for 125 DR homo-
zygous and heterozygous individuals. Among these
were samples of 30 leukemic patients for whom
44
Figure 1. Low-resolution DRBl typing in the PCR-I assay.
POSITIVE CONTROL
SPECIFIC PRODUCT
PRIMERS
serological typing was not possible. All of them
were typed by the LR-HR-SSP method without
specific difficulties. The results were concordant
with the serological data obtained in the family
members.
Discussion
DNA-typing for cadaveric renal transplantation at
this moment is only possible using PCR-SSP tech-
niques as described by Olerup & Bein (4, 9). Al-
though these methods are reliable and specific they
have some disadvantages with respect to donor-
typing. To obtain high-resolution subtyping, a sub-
stantial number of PCR reactions is required. The
necessity for chromosomal DNA and HPLC-puri-
fied primers (4) and the high number of PCR-reac-
tions with accompanying amount of Taq poly-
merase (9) make their methods less suitable for
HLA-DRB 1 subtyping for clinical transplan-
Table 2.
Description of all 65 different DRBl alleles within the LR-HR-SSP method.
Validated specificities are underlined
DRB1'0101 *0102 "0103 '0104
DRB1'1501 *I502 '1503 '1601 '1602 '1603
DRB1'0301 *0302 '0303
D R B I m '0402 '0403 *0404 '0405 '0406 '0407 '0408-'0416
D R B I ' W I '1102 '1103 '1104 'fiT5 '1106
~ ~ ~ 1 9 1 *izoz
201
DRB1'1301 '1302 '1303 '1304 '1305 '1306 '1307 '1308
DRB1'1401 '1402 '1403 '1404 '1405-'1411
D R B I X
DRB1'0801 '0802 '0803 '0804 '0805 '0806
-
DRB1'0901
DRB1'1001
 LOWresolution-high resolution PCR-SSP HLA-DRBl subtyping

4 A B C D E F G H I J K 4 A B C D E F G H I J K
mother father
a A1 B8 Cw7 DR3 DQ2 (DRB1'0301) c A3 844 Cw5 DR13 DQ6(DRB1*1301)
b A32 B51 C- DR4 DQ8 (DRB1'0401) d A l l 855 Cw3 DR15 DQ6 (DRB1'1501)

4 A B C D E F G H I J K 4 A B C D E F G H I J K
child 1 child 2
b A32 B51 C- DR4 DQ8 (DRB1*0401) a A1 88 Cw7 DR3 DQ2 (DRB1'0301)
d A l l 855 Cw3 DR15 DQ6 (DRBI*I501) c A3 844 Cw5 DR13 DQ6 (DRB1'1301)

Figure 2. High-resolution DRBl typing in the PCR-2 assay:

Segregation of DRB1*1301 in family JVB.

Segregation of DRBl* 1301 is illustrated. Primer combinations

are shown in Table 2. The father's haplotype c is inherited by
child 2 and 3. Both show identical patterns in lanes A, B, D
and H. Child 2 shows a stronger signal in lane D as a result of
the simultaneous presence of DRB 1*0301. The same positive
amplification is also shown in lane D of the mother who is
DRB1*0301 positive.

4 A B C D E F G H I J K
child 3
b A32 851 C- DR4 DQ8 (DRBl'O401)
c A3 844 Cw5 DR13 DQ6 (DRB1'1301)

45
 Savelkoul et al.
Table 3.
PCR-2 assay: Nucleotide sequences and lengths of primer pairs for the DRBl alleles

Primer mixes 5' primer L Tm 3' primer L Tm Size bp Specificity

~ ~~~ ~

DAB1'01
MSP0014 S'llGTGGCAGCllAAGlllGAAT3' 22 60 5'CTGCACTGTGAAGCTCTCAC3' 20 62 255 0101/03
MSP0015 S'llGTGGCAGCllAAGlllGAAT3' 22 60 5'CCGCGGCCCGCCTCTGC3' 17 66 202 0101/02/04
MSP0016 S'llGTGGCAGCllAAGlllGAAT3' 22 60 5'CACTGTGAAGCTCTCCACAG3' 20 62 252 0102
MSP0017 5'llGTGGCAGCllAAGlllGAAT3' 22 60 5'CCACCGCGGCCCGCTCGTCT3' 20 72 205 0103
MSP0018 5'llGTGGCAGCllAAGlllGAAT3' 22 60 YCGTAGllGTGTCTGCAGTAAT3' 21 60 229 0104
DRBl'O2
MSP0019 5'TCCTGTGGCAGCCTAAGAG3' 19 60 5'TCCACCGCGGCCCGCGC3' 17 64 204 1501/02/03
MSP0020 5'TCCTGTGGCAGCCTAAGAG3' 19 60 ki'CTGCACTGTGAAGCTCTCCA3' 20 62 257 1501/03
MSP0021 S'TCCTGTGGCAGCCTAAGAG3' 19 60 S'CTGCACTGTGAAGCTCTCAC3' 20 62 257 1502; 1601/02/03
MSP0022 5'GTGCGGllCCTGGACAGAC3' 19 62 5'TCCACCGCGGCCCGCGC3' 17 64 154 1503
MSP0023 5'GTGCGGlTCCTGGACAGAT3' 19 62 5'TCCACCGCGGCCCGCGC3' 17 64 154 1501/02
MSP0024 5'TCCTGTGGCAGCCTAAGAG3' 19 60 5'CGCGCCTGTCllCCAGGAA3' 19 62 199 1601/03
MSP0025 5'TCCTGTGGCAGCCTAAGAG3' 19 60 5'GCGCCTGTClTCCAGGAG3' 18 60 198 1602
MSP0026 5'TCCTGTGGCAGCCTAAGAG3' 19 60 5'GTGTCCACCGCGGCGGC3' 17 62 211 1603
MSP0167 S'TCCTGTGGCAGCCTAAGAG3' 19 60 5'CCGCGGCGCGCCTGTCT3' 17 62 204 1601/02
DRB1'03
MSP0092 S'GACGGAGCGGGTGCGGTA3' 18 62 S'CTGCACTGTGAAGCTCTCCA3' 20 62 217 0301
MSPO033 5'TACllCCATAACCAGGAGGAGA3' 22 64 5'CTGCACTGTGAAGCTCTCCA3' 20 62 187 0301/03'
MSP0031 5'6ACGGAGCGGGTGCGGll3' 18 62 5'TGCAGTAGllGTCCACCCG3' 19 60 216 0302/03
MSP0094 5'ACllCCATAACCAGGAGGAGAI' 21 62 S'CTGCACTGTGAAGCTCTCAC3' 20 62 187 0302'
DRBl '043
MSP0096 S'GlllCllGGAGCAGGlTAAACA3' 22 62 5'GCTGllCCAGTACTCGGCATC3' 21 66 172 0401-04/0€-08/13-16
MSP0035 5'GlllCllGGAGCAGGlTAAACA3' 22 62 5'TGllCCAGTACTCGGCGCT3' 19 64 170 0405/09/10/11/12
MSP0036 5'GlllCTTGGAGCAGGTTAAACA3' 22 62 S'TGTCCACCGCGGCCCGCT3' 18 64 213 0401/02/09/13/14/16
MSP0038 5'ACllCTATCACCAAGAGGAGTCI' 22 64 5'TCTGCAGTAGGTGTCCACCT3' 20 62 151 0406
MSP0040 5'GlllCllGGAGCAGGTTAAACA3' 22 62 5'CTGCACTGTGAAGCTCTCAC3' 20 62 260 0401/05/07-09/14-16
MSPOO41 5'GlllCTTGGAGCAGGllAAACA3' 22 62 S'CTGCACTGTGAAGCTCTCCA3' 20 62 260 0402-04/06/10-13/1 53
MSP0042 5'GlllCllGGAGCAGGllAAACA3' 22 62 5'CCCGCTCGTCllCCAGGAT3' 19 62 201 0402/12/14
MSPO043 S'GlllCllGGAGCAGGllWCA3' 22 62 5'CTGCAGTAGGTGTCCACCAG3' 20 64 222 0412
MSP0044 5'GlllCllGGAGCAGGllAAACA3' 22 62 5'CTGGCTGllCCAGTACTCCT3' 20 64 177 0415
MSP0045 5'GlllCTTGGAGCAGGllAAACA3' 22 62 5'CllCTGGCTGllCCAGTACTG3' 21 64 180 0416
MSPOlOO 5'GlllCllGGAGCAGGllAAACA3' 22 62 YCTGCAGTAGGTGTCCACCT3' 19 62 223 0403/06/07/11
MSP0069 5'GlllCllGGAGCAGGllAAACA3' 22 62 S'CTGllCCAGTGCTCCGCAG3' 19 62 171 1410
DRBl*lls
MSP0047 5'GlllCllGGAGTACTCTACGTC3' 22 64 5'CCCGCTCGTCllCCAGGAT3' 19 62 200 11024
MSP0048 5'GlllCTTGGAGTACTCTACGTC3' 22 64 S'CTGCACTGTGAAGCTCTCAC3' 20 62 259 11015
MSP0050 5'lllClTGGAGTACTCTACGGG3' 21 62 5'CTGGCTGllCCAGTACTCCT3' 20 62 175 110!j6
MSP0107 5'lllCllGGAGTACTCTACGTCB' 21 60 5'CACTGTGAAGCTCTCCACAG3' 20 62 256 1106
MSPOlll 5'GlllCllGGAGTACTCTACGTC3' 22 64 5'TCCACCGCGGCCCGCTC3' 17 62 211 1102/03'
MSPOlO6 5'GlllCllGGAGTACTCTACGTC3' 22 64 5'CTGCACTGTGAAGCTCTCCA3' 20 62 259 1102/03/04/06*
MSPOl77 5'lllCllGGAGTACTCTACGTC3' 21 60 5'CCCGCCTGTCllCCAGGAA3' 19 62 200 1101/03/04/05/069
MSP0052 S'AGTACTCTACGGGTGAGTGll3' 21 62 5'CTGGCTGllCCAGTACTCCT3' 20 64 166 1411
DRB1'12'o
MSPOlO8 S'llCCATAACCAGGAGGAGC3' 19 60 5'CGCGCCTGTCllCCAGGAT3' 19 62 129 1201
MSP0109 5'TTCCATAACCAGGAGGAGC3' 19 60 5'GGCGCGCCTGTCTTCCAGGAA3' 21 62 127 1202
MSP0055 5'AGTACTCTACGGGTGAGTGll3' 21 62 5'CTGllCCAGTGCTCCGCAG3' 19 62 163 1404
DRB1'13
A MSP0056 5'ACllCCATAACCAGGAGGAGAS' 21 62 5'GTCCACCGCGGCCCGCTC3' 18 66 140 1301/02
8 MSPO111 5'lllCllGGAGTACTCTACGTC3' 22 64 S'TCCACCGCGGCCCGCTC3' 17 62 211 1301/02/04/0811
C MSP0057 5'GlllCllGGAGTACTCTACGTC3' 22 64 S'CTGllCCAGTACTCGGCGCT3' 20 64 172 1303/04
D MSP0033 S'ACllCCATAACCAGGAGGAGA3' 21 62 5'CTGCACTGTGAAGCTCTCCA3' 20 62 188 1301/06 1406"
E MSP0032 S'ACTTCCATAACCAGGAGGAGA3' 21 62 S'CTGCACTGTGAAGCTCTCAC3' 20 62 188 1302/05 1402/03/0913
F MSP0115 5'ACTTCCATAACCAGGAGGAGA3' 21 62 S'CCCGCTCGTCTTCCAGGAT3' 19 62 129 1304/0814
G MSP0166 S'CllCCATAACCAGGAGGAGll3' 21 62 5'GTCCACCGCGGCCCGCTC3' 18 66 139 1308
H MSP0047 S'GlllCllGGAGTACTCTACGTC3' 22 64 5'CCCGCTCGTCllCCAGGAT3' 19 62 201 1301/02/03/04/06/0815
I MSPOl77 5'lllCllGGAGTACTCTACGTCB' 21 60 5'CCCGCCTGTCllCCAGGAA3' 19 62 200 1305/0716
J MSP0175 5'ACllCCATAACCAGGAGGAGA3' 21 62 5'CCCGCCTGTCllCCAGGAA3' 19 62 129 1305
K MSP0178 5'ACAGCGACGTGGGGGAGTAJ' 19 62 5'CCCGCCTGTCTTCCAGGAA3' 19 62 97 1307"

46
 Low resolution-high resolution PCR-SSP HLA-DRB1 subtyping
Table 3
(continued)

Primer mixes 5' primer L Tm 3' primer L Tm Size bp Specificity

DRB1'14
MSP0063 5'GTilCllGGAGTACTCTACGTC3' 22 64 5'TCTGCAATAGGTGTCCACCTB' 20 60 223 1401/05/07/08
MSP0064 5'CGGGTGCGGTTCCTGGAG3' 18 62 5'CACCGCGGCCCGCCTCT3' 17 62 158 1402/06
MSP0065 5'CGGGTGCGGTTCCTGGAC3' 18 62 5'CACCGCGGCCCGCCTCT3' 17 62 158 140g7
MSPOlLO 5'GmCllGGAGTACTCTACGTC3' 22 64 5'CTGCAGTAGGTGTCCACCAG3' 20 64 222 1403
MSP0068 S'TACTCTACGTCTGAGTGTCAA3' 21 60 5'TCTGGCTGllCCAGTACTCA3' 20 60 166 1405
MSP0070 5IGAGCTGGGGCGGCCTGC3' 17 62 S'CTGCACTGTGAAGCTCTCCA3' 20 62 123 1401/04/10
MSP0071 5'GCGGCCTGCTGCGGAGC3' 17 62 5'CTGCACTGTGAAGCTCTCAC3' 20 62 115 1407
DRBl'O8
MSP0072 S'AGTACTCTACGGGTGAGTGTT3' 21 62 S'TGTTCCAGTACTCGGCGCT3' 19 64 162 0801/03/05/06
MSP0073 5'AGTACTCTACGGGTGAGTGll3' 21 62 5'CTGTTCCAGTACTCGGCATC3' 20 62 163 0802/0419
MSP0074 S'AGTACTCTACGGGTGAGTGll3' 21 62 S'CGCGCCTGTCTTCCAGGAT3' 19 62 191 080320
MSP0075 5' AGTACTCTACGGGTGAGTGTT3' 21 62 5'TGCAGTAGGTGTCCACCGC3' 19 62 213 080521
MSP0076 5'AGTACTCTACGGGTGAGTGll3' 21 62 5'CTGCACTGTGAAGCTCTCCA3' 20 62 248 0804/0622
MSP0123 5'AGTACTCTACGGGTGAGTGll3' 21 62 S'CTGCAGTAGGTGTCCACCAG3' 20 64 213 0801/02/03/04/06

included specificities: l 1301/06; 1406; 5'0201/02. 1302/05; 1402/03/09; 5'0101/02; 0203. 1410. 1301/02/03/04/06/08: 0302: 1302/03/05/07; 1402/03/
07/09. 1411. 1301/02/04/08. 0301/03; 1301/04/06/08; 1401/05/06/08. 1305/07. lo 1404. 111102/03. l 2 0301/03. l 3 0302. l4 0103; 0402/14; 1102/03.
1102. l6 1101/03/04/05/06. l 7 16; 0415; 08; 09; 5'01; 02. 04 (except 02/12/14/15). l9 1411. *O 1201. 1202/02; 1404/11. 22 1201/02; 1404/11.
L=length of the primer. Tm=melting temperature in "C.

tation. The present study was undertaken to de-
velop a fast DNA-SSP typing method resulting in
high-resolution DRBI subtyping with a limited
number of PCR reactions which would also be
suitable for cadaver donor typing.
The LR-HR-PCR-SSP method described con-
sists of two subsequent PCR assays. The PCR-1
assay is almost identical to the SSP method de-
scribed by Olerup et al. (4) except for the exon-2
amplification. DRB 1-subtyping is performed in
the subsequent PCR-2 assay. The exon-2 amplifi-
cation product is diluted and used as DNA tem-
plate. Some of the alleles are identified by one spe-
cific reaction but usually an allele is identified by a
specific pattern of amplified DNA. The patterns
are characteristic for the different subtypes. Each
pattern is validated by subtyping of homozygous
B-cell lines. The most complicated patterns are
found for the DRB1*04 specificity. Eleven reac-
tions are used to identify 16 DRB1*04 alleles. Not
all DRB1*04 alleles were present in the 249
samples in this study, but the validated alleles iden-
tified so far were clearly distinguishable from one
another.
The HR-SSP has a failure rate of less than 4%.
For HR-SSP the amount of exon-2 template is es-
sential. If the concentration is too low, no reliable
typing is possible. If the amount of template is too
high, background signals appear. Dilution of the
exon-2 product has to be carried out after analysis
of the LR-SSP results to avoid PCR failure. Back-
ground signals also appear when the annealing
temperature is lowered to 64°C or the number of
cycles is increased above 23.
All primers were designed based on the known
DNA sequences of the second exon of the DRB1
gene. Some of the primers had to be redesigned
during the study to obtain clear and reliable re-
sults. Since not all 65 alleles were presented by the
249 individuals studied it is possible that more
primers will need to be adjusted in the future. Fur-
thermore, the number of known sequences for
DRB 1 alleles increases rapidly and -therefore the
addition and redesignation of primers will remain
necessary. Optimization of the method is planned.
Alteration of the PCR amplification programs, the
use of different Taq buffers, increased amounts of
exon-2 product may lead to reduction of the
amount of Taq polymerase.
In summary we developed a DNA-typing
method with the following features: 1) a high de-
gree of resolution is obtained without the necessity
for numerous PCR reactions; 2) homo- or heteroz-
ygosity of an individual can easily be determined;
3) the use of the exon-2 amplification product as a
template yields clearcut typing results, the ampli-
fied product can be stored to be used for further
testing; 4) the LR-HR-PCR-SSP uses a small
amount of DNA regardless of the number of PCR
reactions; complete subtyping can be carried out
using a minimum of 100 ng of chromosomal DNA;
5 ) the method is inexpensive since no purification
of the custom-made primers is needed. It is also
not laborious since multichannel pipettes can be
used. Although 20 kt1 volumes are used in this in-
vestigation it is possible to reduce the volume to
10 p1. 6) The method can easily be extended to
other loci, i.e., DRB3-6. DQ and/or DP, possibly

47
Savelkoul et al.
by a multiplex PCR-1 assay. 7) The method can be
adapted for automatic reading. By adding ethidi-
um homodimer to the reaction mixture the PCR
product can be identified by fluorescent reading
(19). Typing time will thus be further shortened
since agarose gel electrophoresis is omitted. 8) Last
but not least the method is suitable for routine
subtyping in cadaveric renal transplantation. Even
though typing for the DRBl alleles is extremely
complex, high-resolution DRB.1 typing with this
method is obtained in 4.5 h.
Acknowledgment
The authors wish to thank Sabine Timmermans-
Stevens for excellent technical assistance.
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Address:
Pniil H. M. Savelkoril
Tissue Typing Department
University Hospital Maastricht
PO.Box 5800
6202 AZ Maastricht
The Netherlands