Tissue Antigens 1993: 42: 146-149 Prmtrd in Denmark . All rights reserved

CoDyrighr Munksgoard 1993 ~

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

Brief Communication

Direct detection of PCR products for HLA class I1 typing

D. Chia, P. Terasaki, H. Chan, R. Tonai, P-A. Siauw. Direct detection of PCR products for HLA class I1 typing. Tissue Antigens 1993: 42: 146-149. 0 Munksgaard. 1993

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Abstract: Direct detection of the PCR, or DD-PCR is proposed as an efficient method for performing PCR assays. Following the PCR reaction, ethidium homodimer dye is added to the reaction mixture and read by fluorescence. The dye step circumvents the necessity of running reactions on agarose gel electrophoresis, which is the current standard. This simple modification should find wide application for assays utilizing the PCR reaction. Here we show the ready detection of HLA class I1 polymorph-

1 ism.

The PCR reaction (1) has revolutionized possi- bilities for detecting DNA polymorphisms. Here we describe another simple technique for detecting the PCR product; addition of ethidium homodimer dye (2) which simplifies tasks such as typing for the HLA class I1 polymorphisms for it is only necessary to perform the PCR reaction with sets of primers to have the answers as detection or non- detection of the product. Adding dye directly to the reaction mixture replaces the standard method of agarose gel electrophoresis to detect the product as a band, a method that should be usable for other applications of the PCR method.

Today, HLA Class I1 typing is usually performed by oligonucleotide probes as introduced by Saiki (3), and Tiercy (4) and used in the Eleventh Inter- national Histocompatibility Workshop. However, we have advocated the use of simpler sequence- specific primers (5), for which further evidence of efficacy has been provided by Bein (6) and Olerup (7). Although the most cumbersome step in this method is the agarose gel electrophoresis, replacing this step by merely adding the dye and photo metri- cal detection renders the SSP method much simpler than the ‘standard’ oligonucleotide probe methods for HLA typing.

DNA was isolated from 1.5 ml of blood after lysis of red cells by ammonium chloride. Leuko-

David Cbia, Paul Tansaki, Henry Chaa, Richard Tamai and ’ Pee-An S l a w UCLA nssua Typing Laboratory, Deparbnent of Surgery. UCLA School of Mediine, Los Angels. Camornu USA.

Key words SSP - PCR - DDPCR - HLA typing

Received 22 January, revised, accepted tor puMk

- DAB

I cation 25 May 1993

cytes were extracted with 2.5% SDS and 1.9 M guanidine hydrochloride at 65°C for 30 min and precipitated with 100% ethanol. The precipitated DNA was dissolved in water and the DNA concen- tration determined by comparing it to dilutions of a standard solution of salmon sperm DNA (Phar- macia, Piscataway, N.J.) after addition of 8 pl of ethidium homodimer- 1 (Molecular Probe, Eugene, OR) at a concentration of 6 pg/ml to 2 pl of DNA. Fluorescence was measured with excitation at 535 nm, 575 nm dichromic mirror, and 635 nm emis- sion filters in the Lambda Scan reader (One Lambda, Canoga Park, CA) at 90% power.

PCR reaction was performed in 96-well standard microtiter trays. Primer pairs used in the study are shown in Table 1. We added 5 p1 of the primer mixture composed of 4.5-7 pg/ml of each primer, 500 pM dNTPs, and PCR buffer (20 mM Tns- HCl, pH 8.3 100 mM KCI, 4 mM MgC12, 0.002% (w/v) gelatin) to each well and overlaid 50 p1 of mineral oil to prevent evaporation. Then 5 pl of DNA-Taq polymerase mixture (9 pg/ml DNA and 5 units/100 pl Taq) was added to each well. PCR reactions were carried out in an MW-2 Thermal Cycler (Techne Inc., Princeton, NJ). Primer pair for the positive control is listed in Table 1, and DNA without primers was used as the negative control. The program for the thermal Cycler was:

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Brief communication

Table 1. The primer paw nudeotide sequences and its specifiuhes

96°C fro 2 min, 5 cycles of 96°C for 20 sec, 65°C for 20 sec. 72°C for 30 sec, 28 cycles of 96°C for 20 sec, 63 "C for 20 sec, 72°C for 30 sec, and finally, 72°C for 2 min. Fluorescence measurement of the product was performed after completion of the PCR reaction by aliquoting 2 p1 of the product together with 4 pl of the 6 pg/ml ethidium homodi- mer dye into a 72-well Terasaki micro tray. Read- ings were made as described above. The fluor- escence reading of each well would be subtracted from the mean value of all the wells for that par- ticular DNA, and if the value was equal or less than zero would be considered as zero. The fluorescence results for each primer were then normalized against the positive control (PC).

A comparison of fluorescence measurements with agarose gel electrophoresis is shown in Fig. 1. The positive bands shown with electrophoresis correlated well with the direct fluorescence read- ings. As noted, the strength of the bands generally correlated with the fluorescence readings.

HLA class I1 typing by fluorescence measure- ment was performed for 10 different individuals, as shown in Table 2. The bold italic letters were detected as bands by gel electrophoresis. Note that

all the bands were detected by high fluorescent values. Concordance of readings with the bands was 100% if readings below 700 were taken as negative. When these results were compared with the standard method of SSP-PCR (8) (Table 2), the same DRB genes were identified. The normalized fluorescence results between positive and negative primers were at least two times and most of them three times for the 10 DNA studied.

It is apparent from these preliminary studies that the PCR reaction products can be detected by a much simpler method than that which is used cur- rently, namely fluorimetric measurement of the product. Our initial concern was that the ethidium dye would detect both the primer and product, so we experimented with membranes which would separate them. This step, however, proved to be unnecessary. Grosse-Wilde has proposed the use of exonuclease I and alkaline phosphatase to remove excess dNTPs and primers from the double-stran- ded DNA produced by PCR (9). We found that the key to rendering these steps unnecessary was to measure the concentration of DNA first used in the test. If this was not excessive, then direct ad- dition of the dye without a separation step was

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Table 2. fluorescence results of 10 human DNA samples by PCRSSP

possible. The distinction between positive and negative reactions can be improved by designing better primer pairs, while the concentration of primer and magnesium can also affect the PCR reaction. Primers will be able to initiate PCR reac- tion with alleles that are complementary but can also do so with alleles with a difference in the nucleotide sequence of about 10 base pair from the 3’ end.

The direct detection by fluorimetric measure- ment of the PCR (DD-PCR) test should be usable in essentially all applications of the PCR test. The method is particularly suitable for applications which require the performance of many tests simul- taneously, as in HLA typing. The critical feature is that performance of the PCR reactions and read- ing the tests can be done in the same wells. In the present studies, the reactions were performed in a standard microtiter tray with a thermal cycler designed for the trays and the products were trans- ferred for reading. However, in the future, with a flat plate thermal cycler modification, the PCR reaction can be performed on a much smaller scale of 1 microliter volumes in the Terasaki tray and detected directly in these trays. Primers can be

preloaded into micro trays and the entire test per- formed in the trays with automated devices, much as serological reactions are performed today. This method would be the micro modification of the DD-PCR or MDD-PCR.

The main drawback of the SSP method of HLA typing is therefore solved by the present modifi- cation. The need to perform excessive numbers of PCR reactions is solved by the use of the micro tray and simple fluonmetric measurement which permits the performance of a large number of reac- tions with ease.

References

1 . Saiki R, Scharf S. Faloona F. et al. Enzymatic amplification of beta-globulin genomic sequences and restriction analysis for diagnosis of sickle cell anemia. Science 1985: 230: 1 3 5 0 4 .

2. Markovits J, Roques BP, Le Peco J-B. Ethidium dimer: A new reagent for the fluonmetric determination of nucleic acids. Anal Biochem 1979: 94 2 5 9 6 4 .

3. Saiki RK. Walsh PS, Levenson CH, et al. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Nod Acad Sci USA 1989: 86: 62304 .

4. Tiercy JM, Jeannet M, Mach B. A new approach for the

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F i w r e s c e n c p measurement 2 ’. aqarose qei stained w i t h E. B.

analysis of HLA class I I po1ymorphism:HLA oligotyping. Blood Review 1990: 4: 9- 15.

5 . Tonai RJ. Geer L, Wang Y. Park MS, Terasaki PI. Oligo- nucleotide typing of HLA DR by allele-specific PCR ampli- fication. 16th Annual meeting of m e American Society far Histocompatibility and Immunogenetics. LA, California, USA 1990: Abstract.

6. Bein G, Glaser R. Kirhner H. Rapid HLA-DRBI genotyp ing by nested PCR amplification. Tissue antigens 1992: 39: 68-73.

7. Olerup 0, Zetterquist H. HLA-DR typing by PCR amplifi- cation with sequence-specific primers (PCR-SSP) in two hour: An alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric

8. Park MS, Tonai R. Phenotype frequencies of the Class II(DR, DQ) DNA alleles by the patterns of Sequence- Specific Primer Mixtures (SSPM) in four different popula- tions and the probable haplotypes between DRBl allele and DQBl allele. Clinical Transplants 1992: 39: 475-99.

9. Ferencik S. Grosse-Wilde H. A simple photometry detec- tion method for HLA-DRBI specific PCR-SSP product. Eur J Immunol (in press).

. transplantation. Tissue Antigens 1992: 39: 225-35.

Address: David Cliici, Ph.D. 950 Veteran Avenue Los Angeles

i 2;s i : z California 90024 I. U.S.A.

Figure 1. A comparison of PCR results between gel visualization and fluorescence measurement for DNA I (raw data). 2%) agar- ose gel electrophoresis was run at 110 volts for 1.5 h in TBE buffer (45 mM Tris-Borate pH 8.3, 12.5 mM Disodium EDTA) with 0.5 lg/ml ethidium bromide. The primers used are indi- cated in Table I . &here primer pair i i l started from the bottom of the figure and ended with PC a t the top.

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