Sequence-specific priming and exonuclease-released fluorescence assay for rapid and reliable HLA-A...

Copyriglrr 0 Muitk.r.qci<ircl 1996

T I S S U E A N T I G E N S ISSN 0001-2815

Sequence-specific priming and exonuclease-released fluorescence detection of HLA-DQB1 alleles

S . J. Faas, R. Menon, E. R. Braun, W. A. Ruden, M. Trucco. Sequence-specific priming and exonuclease-released fluorescence detection of HLA-DQB 1 alleles. Tissue Antigens 1996: 48: 97-1 12.0 Munksgaard, 1996

Molecular typing of HLA DQB 1 alleles, employing sequence-specific primers (SSP) for PCR amplification, was used to test a novel method that eliminates the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We have evaluated the performance of this fluorescence-based oligonucleotide probe assay to assign the most common DQB 1 alleles on DNA from 14 homozygous cell lines and in a blind study of 50 diabetic patient samples that had been previously typed at the DQB 1 locus using SSOP and conventional SSP-based approaches. We used a panel of 14 DQB 1 SSP primer pairs, internal control primers, and a combination of 4 fluorescent oligonucleotide probes to detect 14 alleles or groups of alleles and controls. We can reliably detect single-base allelic differ. ences, observe 100% concordance with the results obtained using both of the standard methods, and are able to further subtype several alleles that are not easily distinguished using SSOP (e.g. DQB I *0401/0402 and DQB I *0302/ 0303). Sequence-specific priming and exonuclease-released fluorescence (SSPERF) detection is technically simple and can be performed in less than 2 hours, including DNA extraction, PCR amplification, data analysis and allele identification. This method is particularly useful for the analysis of large num. bers of samples, for which high throughput is critical and for which gel-based approaches are difficult to perform. This technique may also be useful for small-scale class I and class I1 molecular typing in clinically oriented labora-

High-resolution HLA class I1 typing at the molecular level has become a routine laboratory assay, providing considerable information in such diverse areas as transplantation biology (1 -3), population genetics (4- 6 ) and disease susceptibility (7-9). These advances in class I1 molecular typing have been made possible by the development and application of a variety of tech- niques including restriction fragment length polymorphism (RFLP) analysis (10-14), polymerase chain reaction (PCR) DNA amplification (15.16). PCR-RFLP ( 17-20), sequence-specific oligonucleotide probe (SSOP) hybridization (2 1-23). PCR ani- plification using sequence-specific primers (PCR- SSP) (24-26). “reverse” dot blot hybridization (27- 29). and finally. direct sequencing (30).

S. J. Faas, R. Menon, E. R. Braun, W. A. Rudert and M. Trucco Division of Immunogenetics, Department of Pediatrics, Ufliversity of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA

Key words: fluorescence detection -sequence- specific primer- HLA-DQBI -molecular typing - 5’ exonuclease Received 15 November 1995, revised, accepted for publication 5 February 1996

Serology, SSOP and SSP are the principal techni- cal approaches used for the detection of class I and class I1 alleles. Each, approach has its own advan- tages and.disadvantages. Current serologic methods, while highly specific, demand the use of nonrenew- able pools of antisera that must be continually tested and validated, and which may not adequately detect alleles associated with different racial or ethnic groups. For example, some of the phenotypic polymorphisms within the DQB I locus are not revealed by serological typing (3 1, 32).

The SSOP-based assay is currently widely used for the detection of HLA-DRB polymorphisms. While this assay is more feasible for testing large numbers of samples, it has the disadvantage of tak-

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ing approximately four days from DNA extraction to allele designation. High-resolution analysis of the DQB 1 alleles is more challenging than DRB 1 analysis using SSOP. Laboratories employing SSOP analysis of DQB 1 alleles often report difficulties in assigning some alleles due to cross reactivities among the probes used. SSOP analysis is also lim- ited in identifying certain heterozygous combinations that yield identical probe patterns with other heterozygous combinations.

The third type of approach described by a number of groups (24-26) uses sequence-specific primers (SSP) and PCR in a gel-based assay, in which the presence and length of the PCR product is the final readout. For example, Olerup and colleagues have described a set of SSP primers that defines 13 of the most common alleles at the DQBl locus (24). SSP has also been employed to identify alleles at the DRB 1 (25) and HLA class I loci (33-36). The specificity of PCR priming is augmented by inhibiting nonspecific amplification through the introduction of a nucleotide mismatch near the 3’ end of the primer (37). Although this strategy demands that a panel of PCR reactions be performed on each sample tested, an SSP-based approach can offer higher resolution than SSOP alone. The number of speci- ficities identified by this approach can continue to expand with the implementation of new or slightly modified primers, or by performing a “two-step” assay that incorporates a second round of PCR with additional primer sets.

The SSP approach offers several positive features for molecular typing: first, a DNA template can be used, thus obviating the requirement and expense of using viable cells as the source of mRNA for typing (a prerequisite for certain typing strategies (30)). Second, the amplified fragments are short enough that they can be validated or confirmed by sequencing during the course of assay development. Third, from DNA extraction to allele identification, reliable and easily interpretable results can be generated in a few hours (24, 25). This short timeframe makes the SSP molecular alternative especially ap- pealing for clinical HLA laboratories involved with cadaveric transplantation.

Although the SSP strategy is particularly well- suited for laboratories that analyze small numbers of samples, a gel-based strategy is less feasible for laboratories performing large-scale molecular typing. We wanted to design a molecular typing strategy that would combine the high-throughput advantage of SSOP with the speed, high resolution and relative ease of SSP analysis. To this end, we have used SSP together with a modification of a recently described method that permits the amplification and direct detection of specitic target DNA (38) with no require-

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ment for post-amplification hybridization or gel analysis steps. This assay takes advantage of the 5’ to 3’ exonuclease activity of the Taq DNA polymerase normally used in PCR DNA amplification (39). An oligonucleotide probe, labelled at the 5’ end with a “reporter” fluorescent dye and at the 3’ end with a “quencher” fluorescent dye, is added to each PCR reaction containing allele-specific primers. Internal control primers and a second doubly-labelled fluorescent probe containing a spectrally distinct 5‘ reporter dye are also present in each PCR reaction. The annealing of either probe to its comple- mentary PCR template strand during the course of amplification generates a substrate suitable for exonuclease attack. Cleavage of the hybridized probe generates smaller fragments that physically release the reporter from the quenching residue, enabling its detection by an increase in sample fluorescence (Fig. 1). Thus the accumulation of specifically-amplified DNA product is detected only under conditions in which the fluorogenic probe hybridizes to

A

B

C

D Figure I. Schematic of sequence-specific priming and exonuclease-released fluorescence detection. A) Hybridization of a doubly-labelled fluorogenic probe to target DNA sequences during PCR amplification. The positions of the reporter dye (FAM or HEX) and the quencher dye (TAMRA) are indicated by the symbols 0 and 0, respectively. The 3’ phosphate is indicated by the k. The increased length of the probes (relative to that of the primers) favors the hybridization of the probe prior to the annealing of the primers. B) Primer annealing to the template DNA. C) Extension of the primer by Tat1 polymerase. D) Encounter of the double-stranded templnte Formed by hybrid- izution of the probe initiates clenvnge of the probe at its 5’ end by T q polymerase. Clenvnge of the probe physically separates the reporter and quencher dye iiiolecules, which ithrogates the quencher effect nnd results i n ;in iiicre;ise in rcportcr Huores- cence.

SSPERF detection of HLA/DQBl alleles Table 1. Homozygous cell lines, primers and fluorescent probes used for the SSPERF assay

HLA-DQB1 Other alleles Cell line allele Primer set5 Amplified Probe

EHM ‘0501 (1) 8-5’014-3’01 Q B O ~ SP0010 ‘0502 (2) 8-5‘024-3’02 Q B O ~

FUR0 ‘0601 (4) 8-5’034-3’04 Q B O ~

wT47 ‘0604 (7) B-5‘06tB-3’06 Q B O ~ H0301 ‘0605 Y (8) 8-5’134-3’06 P B O ~ cox ’0201 (9) 8-5’074-3’07 111302 BM16 ‘0301 (10) B - S O ~ + B - ~ ’ O ~ *a304 Q B O ~

YT ‘0401 (13) 8-5’104-3’11 0805 MADURA ‘0402 (14) 6-5’1 1 tB-3‘11 Q B O ~

EK ‘0503 (3) 8-5’024-3‘03 0805 GN009 ‘0504 8-5’124-3’02 * 0805

PGF ‘0602 (5) 8-5’04tB-3‘05 0605 CB6B *0603 (6) 8-5’05tB-3’05 “0607 R 0605

WT5 1 ‘0302 (1 1) 8-5‘08tB-3‘08 t ‘0201 0803 OKB ‘0303 (1 2) 8-5‘08tB-3’10 t 0803

ORB intron All reactions all ORB C5tC3

the amplified DNA and is enzymatically cleaved by the Taq polymerase. The fluorescence signal is read directly from the PCR reaction mixture using a fluorescence spe’ctrometer.

Material and methods Cells and cell lines

A panel of homozygous cell lines from the 11 th In- ternational Workshop covering 15 different DQB 1 alleles was used as the source of DNA to establish the method (Table 1). DNA was prepared from cell lines and from peripheral blood lymphocytes obtained from 50 unrelated patients with insulin-dependent diabetes mellitus (IDDM) using a modified salting-out protocol (40).

Sequence specific oligonucleotide probe (SSOP) based typing

Dot blotting and SSOP hybridization of generic DQBl PCR reaction products were performed as previously described (23) with some modifications. Briefly, DNAs from homozygous cell lines and patient samples were subjected to PCR using primers that amplify all HLA-DQB 1 alleles. The amplification products were dotted onto positively-charged nylon membranes (Boehringer Mannheim, India- napolis, IN) and hybridized using a panel of fluores- ceinated oligonucleotide probes. Hybridization was carried out for 2 hours at 42°C in buffer containing 50mM Tris-HC1 (pH 8.0), 3.OM tetramethylammo- nium chloride (TMAC), 2mM EDTA and 0.1% SDS. Following hybridization and washing, the annealing of specific probes was revealed by incubation of the membranes with an anti-fluorescein- horseradish peroxidase (HRP) conjugate, followed by detection of the bound peroxidase using an ECL detection system (Amersham, Arlington Heights, IL) .

Sequence-specific primers (SSP) for DQBI typing

The SSP assay was tested and validated using 14 sequence-specific pairs of primers for HLA-DQB 1 allele DNA amplification, selected according to Ole- rup et al. (24). In each PCR reaction, a segment of the third DRBl intron was co-amplified as a positive control (24). Sense and anti-sense DQBl and control primers are listed in Table 1. All primers were generated in our laboratory by standard methods using an ABI 394 automated DNA synthesizer (Applied Biosystems, Foster City. CAI.

Cell lines are homozygous for the indicated OQBl allele with the exception of GN009, which is a ‘0504/’0201 heterozygote. 5 The combination of primers used for detection of each 0081 allele and for

detection of the ORB intron are taken directly from reference 24. Note that some primer pairs amplify more than one allele.

4 This primer pair was included in the initial testing to establish the method, but was not included in routine assays because of the rarity of the ‘0504 allele. The remainder of the primer pairs (14 total) constituted the panel described in the text.

# In addition to ‘0603, the primer pair 8-5‘05tB-3’05 amplifies 0081 ‘0607 (45) rather than ‘0608 as was originally described (24).

Y The H0301 cell line has been recently designated as ‘0609 (46). The amplification of a true ‘0605 allele using this primer pair has not yet been confirmed in our laboratory.

t The sequence of primer 8-3’08 was modified from that originally described (24) to include two additional bases at the 5’ end, as well as an inosine sub- stitution as follows: 5’ggctgttccagtactcgicgg-3’. The sequence of primer B- 3’10 was similarly modified as follows: Sggctgttccagtactiggcgt-3’. These changes improved the efficiency and specificity of the primers.

Fluorescent DQBI-specific and DRBI control probes

Three fluorescent probes (QB02, QB03 and QB05) were generated that differentially hybridize with DNA sequences internal to the sequence-specific primers used for DQBI-SSP. A fourth fluorescent probe, specific for a segment of the DRBl third intron, was also generated to reveal amplification of the internal-control sequences in each PCR reaction. Each DQB 1 probe was synthesized with the reporter dye 6-carboxyfluorescein (6-FAM, Applied Biosys- tems, Foster City, CA) at the 5’ end and at the 3’ end, an amino-modifier C6 dT linker (L) arm nucleotide (Glen Research, Sterling, VA) followed by a 3’ phosphate (Phosphalink, Applied Biosysterns, Foster City, CA) to prevent extension of the hybridized probe during PCR amplification. (These reagents were diluted with acetonitrile and coupled to the ex- tending oligonucleotides under conditions specified by the rnanufxturers.) A different reporter dye. 4.7, 2’. 4’. 7’. 5’-hexachloro-6-carboxyHuol-escei1~ (HEX, Applied Biosystenis. Foster City, CA) was siniilnrly

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Faas et al.

attached to the 5' end of the control probe. Following deprotection, the singly-labelled oligonucleotides were ethanol precipitated, resuspended in 0.25 M NaHCO,/Na,CO, (pH 9.0), and incubated overnight at 37°C with the quencher dye 6-carboxy-tetrameth- ylrhodamine (TAMRA NHS ester, Applied Biosys- tems, Foster City, CA). Unreacted dye was removed by passage of the reaction mixture over a PD-10 Sephadex column (Pharmacia Biotech Inc., Piscat- away, NJ). FAM-TAMRA and HEX-TAMRA doubly-labelled probes were purified by reverse-phase HPLC using a DeltaPak C18 column (Waters, Bed- ford, MA) and an acetonitrile gradient that ranged from 10-80% acetonitrile in 0.1M triethyl ammo- nium acetate (pH 7.0). The detailed conditions of HPLC purification will be published elsewhere (Braun, E. et al., manuscript in preparation). Individ- ual fractions were collected, dried down, ethanol precipitated, resuspended in sterile water and evaluated both for DNA concentration (by measuring the absorbance at 260nm) and for low ( 4 ) reporter-to- quencher ratios by measuring emission intensities at 518, 556 and 580nm (the approximate maximum emission intensities for FAM, HEX and TAMRA, respectively) with the excitation wavelength set at 488nm. The sequences and melting temperatures of the fluorescent probes are as follows:

QB02:5'FAM-ccgagaagagatcgcacgcttcgaca- L(TAMRA)PO,-3' (8OOC)

QB03: 5'FAM-cgagaggagtacgcacgcttcgacagc- L(TAMRA)PO,-3' (8 1°C)

7 ,

QB05: 5'F~-ataaccgagag&gtacgtgcgcttcgac-

DRCON: S'HEX-tcccacatcctattttcatttgctcca- L(TAMRA)PO4-3' (80°C)

L(TAMRA)PO,-3' (75°C).

Probe QB02 reacts with DQB1*0201 and *0202; probe QB03 reacts with DQB1*0301, *0302, *0303 and *0304; and probe QB05 was designed to reveal hybridizations with alleles DQB 1*0501, "0502, "0503, *0504, "0601, *0602, *0603, *0604, *0605 *0401 and *0402. The location of each probe relative to the DQBl primers is shown in Fig. 2. The control probe (DRCON) hybridizes with a consen- sus sequence contained in the third intron of all DRBl alleles. All probes were designed with melting temperatures higher than those of the primers to ensure binding of the probes prior to primer extension during PCR.

ATP-dependent nuclease assay

Doubly-labelled probes were incubated for 4 hours at 37°C in the presence or absence of 13 units of ATP-dependent DNAse (41) (United States Bio- chemical, Cleveland, OH) at a final concentration of OSpm, in a volume of lOOmL of buffer containing 20mM (NH4),SO4/ 5mM MgCL,/ 0.5m.M ATP and 80mM Tris-HCl ph 8.9. Following incubation, 4pL of the digested and undigested probes were mixed and diluted to 40pL in PCR buffer to yield the following groups: 1) F M A M R A probe (digested) plus HEX/TAMRA probe (undigested); 2) FAM/ TAMRA probe (undigested) plus HEXfTAMRA probe (digested); 3) FAM/"AMRA probe (digested) plus HEX/TAMRA probe (digested). The probe mixtures were transferred into wells of a white 96- well microtiter plate (Perkin Elmer, Norwalk, CT) and scanned on a fluorescence spectrometer equipped with a plate reader (TaqMan LSSOB, Per- kin ElmedApplied Biosystems, Foster City, CA). Fluorescence was measured at 518, 556 and 580nm

100

SSPEFW detection of HLA/DQBl alleles

sophistication is certainly not required, we used i t to initially test the method and to provide an alternate means of confirming data obtained using a fluorescence spectrometer. Intact probes could be easily resolved from cleaved probes, both in the gel image and by histogram display of the migrated probe. Once the feasibility of the assay had been demon- strated, we began to use the following methodology to analyze the data from subsequent experiments: 40ml of each PCR amplification mixture were transferred from the thermocycler tubes into wells of white 96-well microtiter plates (Perkin Elmer, Nor- walk, CT) and scanned on a fluorescence spectrometer equipped with a plate reader (TaqMan LSSOB, Perkin ElrnedApplied Biosystems, Foster City, CA). In each experiment, 40pL of the ATP-dependent DNAse digested and undigested probe mixtures were also transferred to certain wells of the microtiter plate for comparison of fluorescence val-

as described above. Ratios of fluorescence intensity for FAM/TAMRA and HEXRAMRA were calculated as described below.

PCR reactions

In preliminary experiments, “generic” amplifications were performed using 1 lth International Histo- compatibility Workshop DQBampA and DQBampB primers (42), together with a single DQB 1-specific fluorogenic probe. PCR mixtures consisted of 500ng genomic DNA, lx PCR buffer (50mM KCV 2.0mM MgCL,/ lOmM Tris-C1 pH 8.3), 200mM of each dNTP, 0.25yM DQBl generic primers, 50nM fluorescent probe and 1.25 Ulreaction Taq polymerase. All subsequent experiments were performed using SSP primers: each PCR reaction mixture contained two DQBI-specific primers, one DQB 1-specific fluorescent probe, two DRB 1 -specific control primers and one fluorescent DRB 1-positive control probe. The PCR mixtures consisted of 500ng genomic DNA, l x PCR buffer (50mM KCV2.0mM MgCL,/ lOmM Tris-C1 pH 8.3), 200mM of each dNTP, 0.25yM DQBl primers, 0.075pM DRB control primers, 50nM each fluorescent probe and 1.25 U/ reaction Taq polymerase. Negative controls consisted of lx PCR buffer containing both allele-specific and internal control fluorescent probes and dNTPs. In a single 96-well format, a total of 6 samples, including all controls, can be typed using the 14 DQBl primer pairs. PCR amplifications were performed in a GeneAmp PCR System 9600 thermocycler (Perkin Elmer, Norwalk CT) using both two-step and three-step cycling parameters with es- sentially similar results. Two-step reactions were performed as follows: 35 cycles of 95°C for 20 secl 65°C for 1 min, followed by a final extension of 72°C for 5 min. Three-step reactions consisted of 30 cycles of 96°C for 20sec/ 65°C for 30 sec and 72°C for 30 sec. Positive and negative amplifications were visualized by ethidium bromide staining following electrophoresis on a 1.5% agarose gel.

Fluorescence measurements

In preliminary experiments, a 0.3 pL aliquot of each PCR reaction was evaluated for evidence of probe cleavage by electrophoresis on 6% native polyacrylamide gels in lx TBE (pH 8.3) and analysis of the size of the fluorescent probe and/or its cleavage products on a 373 DNA Sequencer using the GENESCAN analysis program (Applied Biosys- tems, Foster City, CA). Electrophoresis was performed for 90 minutes at constant power of 35 watts, and the gel visiinlized using the 53 1, 545. 560 and 580 nrn filter set. While this degree of analytical

2 5 0 300 350 2 5 0 300 350 , I

I I I

Figure.3. Nonspecific cleavage of the QB03 probe tinder conditions of “generic” amplification of DNA from homozygous cell lines. PCR reactions were performed using the generic DQBampA and DQBampB primers described in the 1 1 th Inter- national Histocompatibility Workshop (42) together with the QB03 fluorogenic probe. Amplified products were evaluated for probe cleavage following electrophoresis on a 6% native polyacrylamide gel and detection using GENESCAN software. The homozygous cell lines and primer pairs used for detection of the indicated alleles are listed in Table 1 . The negative control consisted of all of the elements of the PCR reaction except template DNA. The scale at the top of the panel indicates the position in the gel scanned by the laser (expressed in scan numbers). Reac- tions exhibiting probe cleavage are highlighted in black. The intact probe migrates as a single peak detected iit approximately scan #335, while cleavage products migrate kister in the gel nnd uppear iis smaller peaks between scan #260 and #320. The high ratio of intact to cleaved probe reHrcts the fact that the probe is added to each PCR reaction in luge excess.

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250 300 350 400 250 300 350 400

0602 I

250 300 3 5 0 400 1

n

0402 A Figure 4. Specific cleavage of the appropriate Ruorogenic probe under conditions of SSP-PCR amplification of DNA from a heterozygous (DQB 1 *0201/*0302) sample. PCR reactions were performed using the allele-specific primer pairs and probe combinations described in Table 1. Amplified products were evaluated as described in Materials and Methods and in the legend to Figure 3. The negative control consisted of all of the elements of the PCR reaction except template DNA. The scale at the top of the panel indicates the relative position of the peaks in the gel expressed as scan numbers. Reactions exhibiting probe cleavage are highlighted in black. The intact probe migrates as a single peak detected at approximately scan #325, while cleavage products migrate faster in the gel and appear as smaller peaks between scan #260 and #320. The high ratio of intact to cleaved probe reHects the fact that the probe is added to each PCR reaction in large excess.

ues. Fluorescence was measured at three wavelengths as described above. At each wavelength, the emission intensity of a PCR buffer blank was sub- tracted from the intensities measured for each sample and control well. For each reaction mix, two ratios were calculated: the first represents the fluorescence intensity at 518 divided by the fluorescence intensity at 580 (i.e., FAMRAMRA), while the second represents the fluorescence intensity at 556 divided by the fluorescence intensity at 580 (i.e., HEXlTAMRA). These ratios are represented as RQ- FAM and RQ,,,. The fluorescence emission generated by TAMRA is unaffected by the presence or absence of amplification, and serves to normalize for well-to-well variation in probe concentration, pipetting errors or microtiter well inconsistencies. Fi- nally, a value for ARQ is generated according to the following example for FAM fluorescence: RQF.4, minus RQFAM,N-,-,, where RQF,qC,(NT, is the FAM/ TAMRA fluorescence ratio associated with the no- template controls.

Results Evaluation of reporter dye quenching

In order to determine the extent of quenching of the reporter dye (i.e. FAM or HEX) by the TAMRA dye, we compared the reporter fluorescence of intact probes with that of probes that were maximally degraded by the activity of ATP- dependent DNAse. This enzyme hydrolyzes nucle- otides from both the 5’ and 3’ ends of linear double-stranded and single-stranded DNA, and requires ATP for its activity (41). The efficiency of quenching can be estimated by comparing the emission intensities of the reporter dye in the intact and digested probes. The quenching efficien- cies of the probes tested range from approximately 69% to 94% (data not shown). In addition, the fluorescence associated with the undigested and digested probe mixtures predicts the range of potential fluorescence of positive samples (discussed in detail below).

,

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SSPERF detection of HLA/DQBl alleles

Experimental Design 5 6 7 8 9 10 1 2 3 4 11 12

DRCON DRCON DRCON DRCON PCR

BUFFER * DRCOA

QB05 -l3R€m

QB05 DRCOP

QB05 DRCOb

QB05 DRCON

QB05 DRCON

QB02 DRCON

5.13.3.06

5.073.07

A

B

C

D

E

F

. c

H

P:

QB05 DRCON 5.01,3.01

QB05 DRCON

5.013.01

QB05 DRCOh 5.01,3.01

QB05 DRCON

QB05 DRCOP

5.01,3.01

5.02,3.02

QB05 DRCOA

4805 DRCOK

QB05 DRCON

QB05 DRCON

5.01,3.01

502,3.02

5.02,3.03

5.033.04

QB05 DRCOA

QB05 DRCOIi

QB05 DRCON

5.01,3.01

5.02,3.02

5.02,3.03

4805 DRCOb

QB02 DRCOA 5.073.07

5.13.3.06 DRCON 5.131.06

DRCON 5.07.3.07

~

QB05 DRCOh 5.0‘2,3.02

QB05 DRCON 5.02,3.02

QB05 DRCON 5.023.02

QB05 DRCON 5.023.03

QB05 DRCON 5.023.03

QB03 DRCON 5.093.09

QB03 DRCON 5.09,3.09

QB03 DRCON 5.09.3.09

QB05 DRCON

QB05 DRCON

QB05 DRCON

5.022.03

5.03,3.04

5.04.3.05

QB05 DRCON

QB05 DRCON

5.02,3.03

5.03,3.M

~~

QB05 DRCON

QB05 DRCON

QB05 DRCON

5.03,3.04

3.04,3.03

5.05,3.05

QB05 DRCON 506.3.06

QB03 DRCON 5.08,3.08

4803 DRCON 5.083.08

4803 DRCON

4803 DRCON

QB05 IRCON

5.08,3.08

5.08,3.10

5.10,3.11

QB05 IRCON 5.11.3.11

QB05 DRCON

QB05 DRCON

5.03,3.01

5.04,3.05

QB05 DRCON

QB05 DRCON

QB05 DRCON

5.03,3.04

5.01,3.05

5.052.05

QB05 DRCON 5.04.3.05

4805 DRCON 5.043.05

QB03 DRCON

QB05 DRCON

5.08.3.10

5.10,3.11

DRCON 5.11.3.11

4803 DRCON

4805 3RCON

5.08,3 10

5.10.3.11

QB05 3RCON 5.113.11

QB05 DRCON 5.053.05

QB05 DRCON 5.052.05

QB05 DRCON 5.06,3.06

QBO5 DRCON 5.05,3.05

QB05 DRCON 5.063.06

QB05 DRCON 5.05,3.05

QB05 DRCON 5.06,3.06

QB05 DRCON 5.063.06

6

QB05 DRCON 5.06,3.06

5 1 2 3 4 4 5 1 2 3 Figure 5. Experimental layout of a typical assay in which 6 sample DNAs are analyzed for DQB I in il single 96-well tray. ROW A contains various probe controls as indicated. DRCON refers to the DR intron control probe. QB02, QB03 and QB05 refer to the DQB 1 specific probes. Wells A2-A4 contain mixtures of equal amounts of intact or ATP-dependent DNAse-digested (e.g.,Q€W probes at a final concentration of 50nM. Wells A5-Al2 lack template DNA but contain allele-specific and control primers and the two indicated fluorogenic probes in Ix PCR buffer. (Note that the unusual order of these control probes is simply to enable the multichan- nel pipetting of a group of horizontal probe master mixes that correspond, in order, to the 7 vertical master mixes containing the same probes). The primer pairs used to amplify each allele are indicated. The patient number is indicated at the bottom of each column (e.g DNA from Patient # I is distributed in Column 1, wells B-H and in Column 7, wells B-H).

WT51 (*0302), DKB (*0303), PGF (“0602) or EHM (*0501) cells; the negative control lacking template DNA did not exhibit probe cleavage (Fig. 3). Thus cleavage of this fluorogenic probe was observed despite differences of up to two base mismatches between probe and template DNA. SSP amplifications also resulted in nonspecific cleavage of a particular probe under conditions of one or two-base mismatches, but only if the “correct” primer pair was present in the reaction. Results from a heterozygous sample (*0201/*0302) illustrate this point (Fig. 4). Sample # 23001 0 I was subjected to analysis using the fourteen DQB I primer pairs listed in Table I together with the appropriate Huorescent probe (i.e.

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Fluorescent probe specificity

Initial experiments evaluated the exonuclease cleavage of several fluorescent probes during both generic- and SSP-PCR reactions using DNAs from DQB 1 homozygous cell lines. In these experiments, the fluorescence signal in each reaction was visualized by electrophoresis of an aliquot of each PCR reaction on polyacrylamide gels and analysis of the cleavage products using GENESCAN software. Un- der conditions of generic amplification using DQBampA and DQBampB primers (421, the QB03 probe was cleaved regardless of whether the reaction contained template DNA from B M 16 p030 I ) .

Faas et al.

9

86.009

64.126

47.027

50.436

347.519

51.826

84.558

84.960

FAM 10 11

26.563 32.231

46.699 57.824

169.938 43.837

60.848 64.018

92.204 327.134

56.942 54.991

89.006 82.657

93.976 89.677

A

B

C

D

E

F

G

H

P:

A

B

C

D

E

F

G

H

P:

115.m

85 046

67655

78.758 78.591 81.537 82.790 82.682 202.683

83.323 84.531 85.061 302.259 337.683 81.629

90420 87383 92537 87922 69869 200 142

1 2

1 2

28.226 33.216

59.580 57.681

68.146 66.373

71.335 69.065

31.319

59.927

50.288

55.560

96.767

59.298

64.947

69.904

68.081

69.513

3 4

172.637

103.201

67.900

70.636

74.510

71.646

-0.071

1 2

112.750

3 4

54.747

62.168

65.427

70.213

77.201

72.288

80.911

5 6 1

92.927

56.484

63.379

67.633

68.934

70.657

80.010

HEX

62.486

68.827

60.162

79.352

115.605

48.849 59.329 61.827

111.474 67.287 108.579

60.698 61.864 61.593

75.964 78.654 72.411

76.963 87.829 81.769

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5 for exDerimenta1 Figure 6. Raw fluorescence values from six patient samples. Forty microliters from each PCR reaction (see Figun layout) were transferred to a 96-well plate and the fluorescence measured at three wavelengths as described in Materials A d Meth- ods. Patient numbers are indicated below each plate. The fluorescence values shown for FAM (A), HEX (B) and TAMRA (C) are direct readings from the LSJOB fluorescence spectrometer prior to normalization. The shaded wells highlight an increase in FAM fluorescence that indicates the amplification and specific detection of the test allele. Note the minor increase in the FAM fluorescence value associated with nonspecific amplification of the *0303 allele by the B-5‘04/J3-3‘05 (e.g. the *0602-specific) primer pair (indicated in well F1, by hatched lines).

either QB02, QB03 or QB05) in each reaction. Deg- radation of probes QB02 and QB03 occurred only in the presence of primer pairs B-5’07+B-3’07 and B- 5’08+B-3’08, respectively (Fig. 4). These results indicated that a positive fluorescence signal was dependent, as expected, upon both the specificity of the probe (which could anneal under conditions of up to two base mismatches), and the specificity of the primers within each reaction. The final combined “functional” specificity of both probes and primers is therefore increased. This analysis also confirmed that an SSP-based amplification strategy would require relatively few probes compared with a strategy using generic primers and a large panel of more specific doubly-labelled fluorescent probes.

Heterozygote analysis

We evaluated the ability of the SSPERF assay to detect HLA-DQB 1 heterozygosity in DNAs prepared

from peripheral blood lymphocytes from 50 diabetic patients that had previously been typed by the SSOP method. These samples were subjected to SSP amplification with the fourteen DQBl primer pairs, control primers and fluorescent probes. Six patient samples plus all controls occupied a single 96-well plate; the experimental outline is shown in Fig. 5 . The raw data representing FAM, HEX and TAMRA fluorescence values for a single 96-well plate are given in Fig. 6, and a confirmatory gel for these same samples is shown in Fig. 7. Both fluorescence and gel analyses confirmed the following typing of these six individuals: Patient #I: DQB1* 0303/ *0402; Patient #2: DQB I* 0501/*0402; Patient #3: DQB1* 0302 homozygote; Patient #4: DQBI* 0501/*0201; Patient #5: DQB 1* 0603P0302; Pa- tient #6: DQB l* 0603/*0201. Cross-reactive bands were occasionally observed, but did not generate positive fluorescence signals because of the specificity of the relevant probes in these reactions. For

105

Faas et al.

1

patient 1

1

7 1

patient 2

7 1

7 1 7

patient 3

7 1 7

c

patient 4 patient 5 patient 6

Figwe 7. Agarose gel electrophoresis of the samples analyzed in Fig. 6. An aliquot of the a'mplified products from each PCR reaction was resolved by gel electrophoresis. For each patient. iimplifications generated by primer pairs 1-7 (listed in Table I and Fig. 5 ) are shown in the top seven wells and those generated by primer piirs 8-14 are shown in the bottom seven wells. Bands in each well representing amplifications of the DRB I intron internal conrol are indicated by the arrow. Bands representing allele-specitic oniplitica- tions are included in the areil in each gel indicated by the bracket. Weak cross-reociive kinds arc evident in samples from Pntient # I (well 5) . Patient #4 (well I I ) and Patient #6 (well I I ) and are discussed in the text.

106


generate an enormous increase in FAM (DQB I-specific) fluorescence values that was easily separable from the fluorescence associated with amplifications of the DRB 1 control alone.

example, a band amplified by the "0602 primer pair was evident in the gel representing the typing for Patient # 1 (Fig. 7. patient #1, lane 5 ) , but did not hybridize with the QB05 probe used to detect authen- tic "0602 alleles; thus no significant cleavage of the probe occurred (Fig. 6, well Fl). Similarly, although the *0201/*0302 primer pair can amplify either of these alleles, the QB03 probe in this reaction mix efficiently hybridized to amplified *0302 sequences (see Fig. 7, Patients #3 and #5, lane 11 and Fig. 6 wells E9 and E l l ) but was not cleaved in the presence of an amplified "0201 allele (see Fig. 7, patients #4 and #6, lane 11 and Fig. 6, wells E l0 and E12). The DRBl control band was easily visualized in each reaction. The intensity of this band was often characteristically weaker than that of the specific band in positive specific reactions, due to PCR conditions that favor DQB 1 -specific amplifications by using lower concentrations of DRBl primers relative to specific primers.

Figure 8 displays representative alleles detected by each of the three DQBI fluorescent probes; the data represent the typing results obtained for all 50 diabetic patient samples. An example of an allele that was not detected in this population (DQBl"0401) is included. The group of points that represents negative controls (i.e. probes alone) are tightly clustered around the origin of each graph. The negative amplifications for each indicated allele from all fifty samples are represented by another tight cluster of points that is shifted to the right in each graph due to the increased HEX fluorescence generated by the DRB 1 positive control amplification in these samples. The ellipse around each cluster of data points reflects 5 standard deviations from the mean fluorescence generated by these DRB 1 amplifications. The minimal observed increase in ARQHEX fluorescence observed in these reactions versus the mean fluorescence of the negative controls also varied slightly and ranged from 4.7-8.5 standard deviations. The points depicting samples that were positive for the indicated allele appear along a diagonal whose slope approximates the increase in FAM fluorescence as measured at both the FAM and HEX emission maxima. These points fall within a range, indicated by the dotted lines that represent the fluorescence associated with increasing FAMRAMRA cleavage in combination with either minimal or maximal cleavage of the HEX/ TAMRA probe, as determined by ATP-dependent nuclease digestion. The difference in ARQFAC, Ruo- rescence between the weakest positive signal und the negative amplifications also varied from allele to allele and ranged from 8.6 to 74 standard deviations. Thus even a "weak positive" anipliticntion resulted i n sufficient cleavage of the hybridized probe to

Discussion Probe design

In the study described herein, we have used previously described primer pairs (24) in combination with doubly-labelled fluorescent probes to amplify and directly detect the presence of 14 different DQB 1 alleles in DNA prepared from both homozygous cell lines and from heterozygous IDDM patient samples. The SSPERF method for the detection of HLA-DQBl alleles is highly specific, sensi- tive and rapid. This approach offers all of the advan- tages of a conventional SSP-PCR assay without the requirement for post-PCR gel analysis. In addition, the ability to construct probes with different reporter dyes permits the simultaneous amplification and detection of an internal control target sequence within the same PCR tube.

The most critical element in the SSPERF assay is the synthesis of appropriate fluorogenic probes. Such probes must exhibit 1) low background fluorescence (i.e. efficient quenching of the reporter dye fluorescence by the quenching dye); 2) efficient cleavage by Taq polymerase between the nucle- otides carrying the reporter and quencher dyes; 3) high specificity for the target DNA sequence; 4) compatibility with the temperature, MgCI, and cycling conditions optimal for SSP-amplifications; and 5) an inability to form hairpins, dimers or other significant secondary structures with the other primers or probes within the reaction.

The factors affecting reporter dye quenching are not completely predictable. In our experience to date, the position of the quencher dye does not appear to influence the efficiency of quenching to a great degree; however, others have reported that a G residue next to the reporter dye can impart a quenching effect that is independent of probe cleavage (43). The choice of reporter and quencher dyes also potentially affects quenching, although we have not systematically compared probes synthesized with different combinations of fluorescent dyes. The degree of TAMRA labelling and the purity of the probe may affect probe performance because the presence of significant levels of singly-labelled oligonucleotides results in a poorly quenched probe and a high background reporter fluorescence. An ideal intact fluorogenic probe should exhibit approximately 90% quenching, although in practice. depending upon the probe sequence. this level of quenching is not always achievable. Every probe

107

Faas et al.

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Figure 8. Fluorescence data for several DQBl alleles from 50 patient samples. Forty microliters from each PCR reaction (see Figure 5 for experimental layout) were transferred to wells of a 96-well plate. In each graph all 50 sample are represented. Each data point represents the ARQ,, and ARQ,, normalized fluorescence values for a single patient. The scales on both abscissa and ordinate are identical for all graphs. The test allele as well as the DQB 1-specific probe used for its detection are indicated. Normalized baseline fluorescence of the intact probes (i.e., probes alone) are represented by the cluster of points at the origin of each graph. Fluorescence associated with the negative amplification of the indicated test allele in the presence of amplification of the DRB 1 intron internal control from the 50 samples is represented by the encircled cluster of points. The size of the ellipse around these control amplifications represents 5 standard deviations from the mean fluorescence of these reactions. Increased fluorescence, associated with amplifications specific for the indicated test allele, is represented by individual data points in a diagonal orientation (for example, 8 patients were positive for the DQB 1*0501 allele, 4 were positive for the DQBl*Q502, etc.). These points fall within a range (e.g., delineated by the dotted lines flanking the data for test allele *0501), corresponding to the values (empty circles) of the combinations of intact and digested probes (e.g,, wells A2, A3, and A4 of Fig.5). The frequency of each DQB 1 allele detected by the SSPERF assay from the diabetic patient samples was as follows: allele *0201: heterozygous 16. homozygous 3; *0301: 8, l: *0302: 22.2; *0303: 3,O: *0402: 5. 0: *0501: 7, I ; *0502: 4, 0; *0602: 6, I ; *0603: 4. 0: *0604: 7, I . Alleles '0401, *0503, *0601 and *0605 were not present in this population. These frequencies are consistent with the known HLA DQB I alleles associated with IDDM.

108


mented and widely used method of increasing typing resolution, or clarifying “ambiguous alleles” flagged by SSOP. Adding fluorogenic probes to these reactions not only eliminates the requirement for a gel-based readout, but, in some cases, also adds a second degree of specificity. For example, the primer pair B-5’08-B-3’08 originally described by Olerup et al. (24) amplifies both DQB1*0201 and “0302 alleles. The detection of both of these alleles in a conventional SSP assay demands two additional amplifications using B-5‘07-B-3’07 primers (for the detection of the *0201 allele) and B- 5’08-B-3’09 primers (for the detection of the *0302/ *0303 alleles). In our assay system, however, the QB03 probe discriminates between *0302 and *020 1 alleles amplified by our modified primer pair B-5’08-B-3’08, and only the *0302 allele is detected (Fig. 6 and 7). Similarly, the QB02 probe hybridizes only with *0201 target sequences and is used in combination with the B-5’07-B-3’07 primer pair to detect this allele. Thus the resolution of these two alleles is accomplished with only two amplification reactions, each using a different probe, rather than with three reactions as in conventional SSP. Elimi- nating this third reaction allowed us to include an additional primer pair for the detection of the *0605 allele (Table l), while limiting the total number of reactions to 14. This array permits us to analyze 6 DNA samples (including controls) on a single 96- well plate.

Since the SSPERF assay detects the presence of specific amplification, but does not reveal any information regarding the size of the amplimer, it is important to validate the specificity of the SSP primers used before relying solely on the fluorescence readout. Obviously, primer pairs that give rise to nonspecific amplification of other HLA alleles, which can still anneal to the included probe, will yield false-positive results. However, the specificity of “problem” primers can often be enhanced by modifications such as those we describe for the B-3’08 and B-3’10 primers: the primer efficiency and specificity of these primers was improved upon by mak- ing the inosine substitutions detailed in the footnote to Table 1. Under our conditions, the modified B- 3’10 primer, in combination with the B-5’08 primer, amplifies the DQB1*0303 allele in the absence of cross-reactive amplification of the DQB 1 *0302 allele. These primer modifications thus permit better discrimination between *0302 and *0303 alleles despite the use of a single probe (QB03) in both reactions. Taken together, these examples illustrate how the combined specificity of primers and probes increase the total functional specificity of an individual reaction beyond that of either component alone. A similar approach may be used to reduce the cross-

that we have used is not quenched to this extent, but still worked well in this assay.

Factors that affect the efficiency of probe cleavage include the placement of the quenching dye relative to the reporter dye and the extent of hybridization of the probe to target DNA. While some investigators have placed the quencher dye internally (44), others suggest that placing the quencher dye at the 3’ end both maximizes the potential to cleave the probe between the two dyes, and minimizes any likelihood that the dye will interfere with hybridization (43). All of the probes that we describe in these experiments contain the reporter dye at the 5’ end and the quencher dye at the 3’end of the oligonucleotide. Since most of the specificity of this assay comes from the choice of primers, probe design must conform to the con- straints set by the primers themselves. As with conventional primer-probe combinations, adjustments in the’length or in the choice of the particular sequence of a given probe are often sufficient to resolve additional problems associated with Specificity, cycling parameters, secondary structure or incompatibility with other primers and probes.

Considering all of the criteria for appropriate probe design, we chose to implement an approach that would require as few fluorogenic probes as possible. Thus, we used only three fluorescent probes, designed to absorb up to two-base mismatches, to detect all of the most common HLA-DQB 1 alleles. Analysis of other loci by this method may require even fewer probes: our experiments using the SSPERF strategy to detect 34 HLA-DRB 1 alleles indicates that for this locus, only one DR-specific probe is required (Rud- ert, Menon, Braun, Faas and Trucco, manuscript in preparation). Although the preparation and purification of fluorescent probes is more time-consuming than the simple synthesis and labelling of conventional fluorescent or radioactive probes for SSOP- based typing, such probes, once validated in the assay system, can be generated in large-scale (1 - 1Op.M) syntheses that will provide sufficient reagents for many thousands of reactions. For example, a single 1 pM oligonucleotide synthesis would generate enough purified probe for approximately 200,000 reactions, taking into account the anticipated losses that are inevitable at each step in the synthesis and purification scheme. Once generated, the doubly-labelled probes can be aliquotted, together with the relevant SSP primer pairs, into appropriate master mixes and stored indefinitely at -20°C in PCR strip tubes.

Specificity

The use of SSP primers to generate allele- or group- specific amplification products is a well-docu-

109

Faas et a].

amplification of “0303 by *0602-specific primer pairs (see for example Fig. 7, Patient #l), although this cross-reactivity does not interfere with the assay, as discussed above.

We have observed complete concordance between the results we obtained using our SSPERF method and those using the conventional SSP method. In addition, even poorly amplified specific products, that are weak or are not easily visible in a gel, are readily detectable by our assay. As expected, this assay proved to be more reliable than SSOP for the analysis of the 50 diabetic patient samples. In some cases, we have been able to subtype certain alleles not resolved by SSOP (for example, our assay, as well as conventional SSP, differen- tiates “0401 from “0402 alleles). Also, in two cases we were able to assign *0302 homozygosity to samples that had previously been typed as potential *0302/*0302/3 heterozygotes by SSOP (for example, see the data for Patient #3 in Figs. 6 and 7). In two additional samples, we were able to reliably identify a heterozygous *0301/ *0302 typing where previous SSOP typing results had been ambiguous (i.e., unable to clearly differentiate between *0302 and “0303 alleles). In one other case we were able to discount the possible presence of a *0301 allele and confirm homozygosity of the *020 1 allele. Sim- ilarly, in an additional sample, we could reject the previous typing results of a *0302/:k0603 heterozygote in favor of a clear *0302 homozygote. For the routine DQB I typing of diabetic patients, this level of discrimination is invaluable, since, for example, it permits the discrimination between alleles that are important for the determination of IDDM susceptibility or resistance (e.g. alleles expressing non-Asp versus Asp amino acids at position 57 of the DQP chain) (9, 22,23).

The fluorescence data summarized in Fig. 8 illustrate several points that require discussion. Al- though the two reporter dyes, FAM and HEX, are spectrally distinct at 518 and 556nm, there is still some spectral overlap between them. (This is physically unavoidable since one prefers to excite both dyes at the same wavelength in this assay). Thus the signal generated by an increase in FAM fluorescence is detected primarily at the 518 wavelength, but is also observed to a lesser degree at the 556 (HEX) wavelength. These increases in apparent HEX fluorescence, due to FAM, can readily be observed in the raw data (see Fig. 6B) and translate graphically into the diagonal line that represents samples that are positive for the test allele (see Fig. 8). Although it would be possible to mathematically separate the fluorescent components into those inde- pendently contributed by the FAM and the HEX dyes. this degree of sophisticntion W;IS l~nnecessnry

110

for our application. Since the HEX signal was infor- mative only for the case of the negative amplifications of the test alleles, it was only necessary to ac- curately determine the HEX signal in the absence of a strong FAM signal.

The differences observed between high and low positives are likely due to sample-related excipient effects, such as trace salts or other impurities, that may affect probe hybridization and/or cleavage, as well as DNA amplification. However, it should be stressed that even the weakest positive sample generated a fluorescence signal that was almost 9 standard deviations higher than the average negative amplification. Of course, the threshold for what can be considered a positive sample depends upon the number of samples analyzed and the confidence limits one wishes to assign to the typing. In this assay, we permitted a sample to be called “positive” if its fluorescence value were at least 3 standard deviations above that demarcated by the ellipse around the control amplifications negative for the test allele. (Note that the size of these ellipses also represents a variation in fluorescence equivalent to 5 standard deviations from the mean, although in most cases we observe differences of less than 2 standard deviations from the mean among these negative samples).

The data representing two of the test alleles shown in Fig. 8 indicate DRBl control amplifications that exhibit slightly higher FAM fluorescence than those associated with the other test alleles (see Fig. 8, DQB1*0501 and “0302). This increased FAM fluorescence did not interfere with our ability to discriminate between positive and negative allele-specific amplifications. We suspect that this effect may be due to a low level of cleavage of the QB05 and QB03 probes in these negative reactions. Analysis of the potential interactions between these probes and each of the other oligonucleotides present in these particular reactions has revealed a weak dimerization between probe QB05 and primer B-5’01, and between probe QB03 and primer B- 3’08. The stability of this dimerization may be sufficient to initiate a low level of Taq polymerase-medi- ated cleavage of the probe in the absence of target DNA amplification. However, dimerization does not appear to significantly inhibit target DNA amplification or reduce the level of fluorescence associated with samples positive for the test allele.

The SSPERF assay should be directly applicable to the molecular typing of alleles at other HLA loci as well. We have already made considerable progress in establishing the iisefiilness of this technique for DRB I molecular typing (Rudert. Menon, Braun, Faas and Trucco. manuscript in preparation), and have begun testing the method for HLA class I


ments describe allelic series. Proc Natl Acad Sci U S A

Cascino I, Rosenshire S, Turco E. Marrari M, Duquesnoy RJ, Trucco M. Relationship between DQ alpha and DQ beta RFLP and serologically defined class II HLA antigens. J Immunogenet 1986: 13: 387-400. Carsson B, Wallin J, Bohme J, Moller E. HLA-DR-DQ haplotypes defined by restriction fragment length analysis: correlation to serology. Hum Immunol 1987: 20: 95-1 13. Trucco M, Ball E. RFLP analysis of DQ-beta chain gene: workshop report. In: Histocompatibility testing 1987. Vol.

Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. In: Diego RW, ed. Methods in enzymology. San Diego, CA: Academic Press, 1987: 335-50. S a k i RK. Gelfand OH, Stoffel S et al. Primer-directed en- zymatic amplification of DNA with a thermostable DNA polymerase. Science 1989: 239: 487-9 1. Trucco G. Fritsch R, Giorda R, Trucco M. Rapid detection of IDDM susceptibility, using amino acid 57 of the HLA- DQ beta chain ils a marker. Diabetes 1989: 38: 1617-22. Nomura N, Ota M, Tsuji K, Inoko H. HLA-DQB 1 genotyping by a modified PCR-RFLP method combined with group-specific primers. Tissue Antigens 199 1: 38: 53-9. Ota M, Seki T, Nomura N et al. Modified PCR-RFLP method for HLA-DPB I and -DQAI genotyping. Tissire An- tigens 199 1 : 38: 60-7 1. Tong JY, Hsia S, PYris GL et al. Molecular compatibility and renal graft survival: the HLA DQB I genotyping. Transplantation 1993: 55: 390-5. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified beta-globin and HLA- DQ alpha DNA with allele-specific oligonucleotide probes. Nature 1986: 324: 163-6. Todd JA. Acha-Orbea H, Bell JI et al. A molecular basis for MHC class 11-associated autoimmunity. Science 1988: 240:

Morel PA, Dorman JS, Todd JA, McDevitt HO, Tnicco M. Aspartic acid at position 57 of the HLA-DQ beta chain pro- tects against type I diabetes: a family study. Proc Ncitl Accrrl Sci U S A 1988: 85: 8111-16. Olerup 0, Aldener A, Fogdell A. HLA-DQB 1 and -DQA I typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours. Tissue Antigens 1993: 41: 119- 34. Olerup 0, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992: 39: 225-35. Bunce M, Taylor CJ, Welsh KI. Rapid HLA-DQB typing by eight polymerase chain reaction amplifications with sequence-specific primers (PCR-SSP). Hum Immunol 1993:

Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequence- specific oligonucleotide probes. Proc Narl Acad Sci U S A

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1. 1989 860-7.

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111

typing. We anticipate that the typing of selected alleles or groups of alleles for DRB l , DQB l , HLA-A, HLA-B and HLA-C will be possible using 2 full 96- well trays for a single individual. If the trays are set up in advance, such an assay could be performed in less than 2 1/2 hours including DNA extraction (15 min), 2-step PCR (1 1/4 hours), fluorescence measurements (20min/tray), data analysis and allele designation (10 min).

Acknowledgments

We thank Angela Alexander and Theresa Castelli for providing the SSOP typing results and IDDM patient sample DNAs, Dr. Gayle Rosner for helpful discussions, Read Fritsch for preparation of the fig- ures and Patrick Hnidka for preparation of the manuscript. This work was supported in part by grant #N00014-94-1-1199 from the Office of Naval Research.

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Sequence-specific priming and exonuclease-released fluorescence assay for rapid and reliable HLA-A...

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