Rapid Publication

The Human Platelet Alloantigens, p1Al and pIA2 Are Associatedwith a Leucine33/Proline33 Amino Acid Polymorphism in MembraneGlycoprotein Ilia, and Are Distinguishable by DNATypingPeter J. Newman,** Rebecca S. Derbes,* and Richard H. Aster**The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233; tDepartment ofAnatomy and Cellular Biology,and §Departments of Medicine and Pathology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53233

Abstract

The human platelet alloantigens, pjAI and pjA2, comprise adiallelic antigen system located on a component of the plateletfibrinogen receptor, membrane glycoprotein (GP) MIIa. Of theknown platelet alloantigens, pIA1 which is carried by 98% ofthe caucasian population, appears to be the alloantigen thatmost often provokes neonatal alloimmune thrombocytopenicpurpura and posttransfusion purpura. The structural featuresof the GPIIIa molecule responsible for its antigenicity are asyet unknown. Using the polymerase chain reaction (PcR), weamplified the NH2-terminal region of platelet GPIIIa mRNAderived from PjAe and pIA2 homozygous individuals. Nucleotidesequence analysis of selected amplified cDNA products re-vealed a C * T polymorphism at base 196 that created aunique Nci I restriction enzyme cleavage site in the p1A2, butnot the P1Ae form of GPIIIa cDNA. Subsequent restrictionenzyme analysis of cDNAs generated by PcR from 10 p1Al/A15 P and 3 p1Al/AZ individuals showed that Nci I digestionpermitted clear discrimination between the pIA1 and pIA2 al-leles of GPIIIa. All pIA2/A2 individuals studied contain a C atbase 196, whereas PIAl homozygotes have a T at this position.This single base change results in a leucine/proline polymor-phism at amino acid 33 from the NH2-terminus, and is likely toimpart significant differences in the secondary structures ofthese two allelic forms of the GPIIIa molecule. The ability toperform DNA-typing analysis for peA phenotype may have anumber of useful clinical applications, including fetal testingand determination of the phenotype of severely thrombocyto-penic individuals.

Introduction

The human platelet membrane glycoprotein (GP)' Ilb-Illacomplex mediates platelet aggregation by acting as the func-

Address reprint requests to Dr. Newman, The Blood Center of South-eastern Wisconsin, 1701 West Wisconsin Avenue, Milwaukee, WI53233.

Receivedfor publication 24 October 1988 and in revisedform 17January 1989.

1. Abbreviations used in this paper: GP, glycoprotein; HEL, humanerythroleukemia; NATP, neonatal alloimmune thrombocytopenicpurpura; PcR, polymerase chain reaction; PTP, posttransfusion pur-pura.

tional receptor for fibrinogen on the platelet surface (1). Inaddition to this physiological role, both GPIIb and GPIIIa areknown to bear a number of clinically important alloantigenicdeterminants that are responsible for eliciting the immune re-sponse in two well-described clinical syndromes, postransfu-sion purpura (PTP) and neonatal alloimmune thrombocyto-penic purpura (NATP) (2, 3). The alloantigen system mostfrequently implicated in these disorders is P1A. There are twoserologically defined allelic forms of the pIA alloantigen, PIl"and p1A2, both of which have been localized to the GPIIIamolecule (2). The gene frequencies for these two alleles havebeen calculated to be 85% A1:15% A2 (4), based upon theobservation that p1A2 homozygous individuals represent only2% of the caucasian population. Since 98% of the populationcarries the PIA" antigen, p1A2 homozygotes are at risk of pro-ducing anti-PlAI antibodies against paternally inherited P1AIantigens present on fetal platelets, and are most likely to de-velop PTP after blood transfusion.

Molecular definition of platelet alloantigenic determinantswould contribute significantly to the understanding of themorphological features of platelet membrane glycoproteinsthat are responsible for eliciting an alloimmune response. Car-bohydrate residues have been shown not to contribute signifi-cantly to the formation of the immunogenic PIAl determinant(5), but determination of the amino acid sequence variation(s)that are presumably responsible for forming the relevant epi-topes has not yet been possible, due largely to the formidabletask of obtaining protein sequence information of both pJAland P1A2 forms of the I00-kD GPIIIa molecule. The completeamino acid sequence of GPIIIa has recently been deducedfrom the nucleotide sequences of both endothelial cell (6) andhuman erythroleukemia (HEL) cell (7, 8) GPIIIa cDNAclones, however it is likely that each of these clones representthe pIAl rather than the p1A2 allele, owing to the relative abun-dance of p1AI in the human gene pool.

Recently, we descril/ed a new approach for examiningplatelet-specific mRNAsequences from single individuals (9).Wedemonstrated that the RNAderived from the plateletspresent in 50 ml of whole blood can be converted to cDNAand then enzymatically amplified using the polymerase chainreaction (PcR) to produce microgram quantities of platelet-specific cDNA. By isolating and amplifying mRNAfrom anumber of individuals of known plA allotype, it should bepossible to examine whether phenotype-specific nucleotide se-quence variations exist in the GPIIIa gene. In this report, wepresent the first molecular description of a polymorphism as-sociated with a human platelet alloantigen, and show that thepJAl allele can be distinguished from p1A2 by differential restric-tion endonuclease digestion of amplified platelet mRNA.

1778 P. J. Newman, R. S. Derbes, and R. H. Aster

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/89/05/1778/04 $2.00Volume 83, May 1989, 1778-1781

Methods

Serological determination of Pi' phenotype. The pIA phenotype of 18different individuals was determined using a standard antigen-captureELISA assay (10, 1 1). 10 homozygous PI" individuals, 5 homozygousPIA2 individuals, and 3 heterozygotes for the pIA allotype, all of whomhad been unambiguously identified using well-characterized anti-PI"Aand anti-PIA2 antisera, were used throughout this study.

Amplification of platelet mRNA. Human platelet mRNAwas pre-pared from anticoagulated whole blood as described previously (9).Two pairs of primers were constructed; an outer pair (primers 1 and 3),and an internally nested pair (primers 2 and 4), and used to amplify aregion of the GPIIIa mRNAmolecule that encodes the amino terminal103 amino acids of mature glycoprotein IIa. Bases 1-400 of theGPIIIa mRNAmolecule are known to be encoded by at least threedifferent exons that are broken by introns comprised of > 10 kb ofsequence (personal communication from Drs. Gilbert C. White II andSusan Gidwitz, University of North Carolina-Chapel Hill and Ann B.Zimrin and Mortimer Poncz, University of Pennsylvania, Philadel-phia), permitting ready distinction between PcR products that mightbe inadvertently derived from genomic DNA, rather than mRNAse-quences. Primer 1 (5'-CGCGGGAGGCGGACGAGATGCG-3)cor-responds to the RNAstrand from bases 4-25 of the published nucleo-tide sequence (6). Primer 2 (5'-GACTCGAGACTGTGCTGG-CGCTG-3) corresponds to bases 56-71 of the RNAstrand, with anadditional 7 bases encompassing an Xho I restriction enzyme recogni-tion site incorporated onto the 5'-end to facilitate subsequent subclon-ing into plasmid vectors. The two anti-sense oligonucleotides wereprimer 3 (5'-CGCACTTGGATGGAGAAATTC-3'), which corre-sponds to nucleotides 412-392, and primer 4 (5'-CCGGATCClTG-GATGGAGAAATTC-39,which corresponds to bases 408-392 plusan additional 7 bases that contain a BamHI site. Primer 3 was used toprime first strand cDNAsynthesis, using platelet mRNAas a template,in a total volume of 50 ,d as previously described (9). All ensuing PcRreactions were performed in a programmable DNA thermal cycler(Perkin-Elmer Cetus Corp., Norwalk, CT). The first 10 rounds of PcRwere performed using primers 1 and 3 in a total volume of 100 1 usinga regimen consisting of denaturing nucleic acid strands at 94°C for 90s, annealing primers at 37°C for 2 min, and primer extending with Taqpolymerase (Perkin-Elmer Cetus Corp.) at 72°C for 3.5 min. After thefifth thermal cycle, the primer annealing temperature was increased to42°C. After the tenth cycle, the first primer pair was removed bycentrifuge-driven dialysis of the PcR reaction mixture into a nearlyidentical buffer using Centricon 30 microconcentrators (AmiconCorp., Danvers, MA). The second reaction mix was identical to thefirst, except that internally nested primers 2 and 4 were used in place ofprimers 1 and 3. After oligonucleotide exchange, the reaction volumewas again brought to 100 ,l, including 2.5 U of fresh Taq polymerase,and PcR continued for an additional 21 thermal cycles. Primer an-nealing was performed at 42°C for rounds 11-15; 47°C for rounds16-20, and 55°C for rounds 21-31. Wehave found that these condi-tions maximize specificity and yield for amplification of cDNA (un-published observations). The presence of the additional bases used toform the restriction enzyme sites at the 5' ends of primers 2 and 4 hadno detrimental effect on the quantity of specific DNAproduced duringthe PcR.

Analysis of amplified cDNAs. Selected amplified cDNAs were sub-cloned into the plasmid vector pGEM-7Zf (Promega Biotech, Mad-ison, WI), and subjected to nucleotide sequence analysis. Dideoxysequencing was performed using the modified T7 phage DNApoly-merase, Sequenase (United States Biochemicals, Cleveland, OH), ac-cording to the manufacturer's directions. Most PcR reaction productswere directly exchanged into sterile water using Centricon 30 micro-concentrators, and then digested with Nci I (purchased from eitherNew England Biolabs, Beverly, MA, or Bethesda Research Laborato-ries, Gaithersburg, MD). Restriction digests were analyzed on 1.5%agarose gels. Computer analyses of protein and nucleic acid sequenceswere performed using the program PC/GENE (Intelligenetics Inc.,

Oligo56-71

GPIIla 5'147

piAl366 bp

CrGGG

Ac/

pIA2

219

Figure 1. Diagramaticrepresentation of theNH2-terminal region ofthe GPIIIa mRNAmol-ecule. The locations ofthe two oligonucleotide

Ofig primers used for the4MM PcR are shown, as well

as the polymorphic se-quence at base 196 ofthe amplified cDNA.

Mountain View, CA) operating on an IBM PC/AT-compatible micro-computer.

Results

Nucleotide sequence analysis of GPIIIa from a plA2 homozy-gous individual. The nucleotide sequence of GPIIIa has beenindependently determined in three different laboratories(6-8). Except for a single silent polymorphism in the codon forVa1355, no interlaboratory sequence differences exist within thecoding region for the mature GPIIIa molecule. Since the PlAIphenotype is present in 98% of the population, it is likely thateach of the three previously published sequences were derivedfrom clones encoding the PlAI form of GPIIIa. To examine theamino terminal region of the PIA2 allele, we used the polymer-ase chain reaction to amplify a 366-bases region near the 5' endof platelet GPIIIa mRNA.Our amplification strategy used twosets of oligonucleotide primers, one nested internally to thefirst. The first 10 thermal cycles of the PcR amplified bases4-412 - 1,000-fold, and provided a sufficient quantity ofGPIIIa-specific cDNA to permit more stringent conditions tobe used in subsequent rounds. The remaining thermal cyclesamplified bases 56-408 using an internally nested primer pair,which are graphically depicted in the top portion of Fig. 1. Thisregion encodes the first 103 amino acids of the mature GPIIIaprotein, as well as a majority of the signal peptide. Using thisprotocol, we produced microgram amounts of the expected366 bp cDNA from a number of individuals of known pIAphenotype. After subcloning into the plasmid pGEM-7Zf, thecomplete nucleotide sequence of bases 79-408 from one p1A2homozygous individual was determined on both strands, andfound to be identical to the three previously reported se-quences for GPIIIa, except at base 196 (sequence numberingaccording to reference 6), which had a deoxycytosine (C) inplace of a deoxythymidine (T). This single base change (shownin Fig. 2) results in substitution of a Pro for Leu at amino acidresidue 33 of the mature GPIIIa molecule, and is likely toimpart significant secondary structural differences.

Restriction enzyme analysis of GPIIIa allotypes. Computeranalysis of the GPIIIa sequence from bases 56-408 revealedthat substitution of a C for a T at base 196 would create arecognition site for the restriction enzyme Nci I, which cleavesat 5'-CCGGG-3' but not 5'-CTGGG-3' sequences. To deter-mine whether the T -o C substitution found in the p1A2 indi-vidual studied above was related to plA allotype, or merelyrepresented an artifact generated in vitro during either thereverse transcriptase or Taq polymerase reactions, or an unre-lated polymorphism of GPIIIa, platelet RNA was preparedfrom a total of 18 individuals of known plA phenotype, andthen amplified using PcR to yield the same 366-bp product. As

DNA Typing for PIA Phenotype 1779

UPA

T

GG

-j~~l C.-T!~~~

riwC~AL. ...

Figure 2. DNAse-quence analysis of am-plified GPIIIa cDNAderived from a PiA2 ho-mozygous individual.The 366-bp PcR prod-uct was subcloned intothe plasmid pGEM-7Zf,and sequenced on bothstrands using primerscorresponding to theSP6 and T7 RNApoly-merase promoter se-quences. A segment ofthe autoradiograph, en-compassing bases188-203, is shown. TheDNAsequence for thepresumed PI"A allele of

GPIIIa has been previously reported, and is printed on the right innormal size lettering for reference. The single base substitution of aC for a T at base 196 is indicated with an arrow.

illustrated in Fig. 1, Nci I should not be able to cleave the366-bp cDNA from a plA' homozygous individual, whereastwo fragments of 147 and 219 bp would be generated in a p1A2homozygote. Fig. 3 shows that, as anticipated, the 366 bpcDNA amplified from plA' homozygous individuals was notcleaved by Nci I, whereas the predicted restriction fragmentswere obtained from PlA2/A2-derived cDNA. Serologically de-termined heterozygotes for the plA phenotype yielded bothuncut 366 bp cDNAand the 219- and 147-bp restriction frag-

Figure 3. Analysis ofpjA phenotype by re-

bp striction endonucleasedigestion. Bases 56-408were enzymatically am-plified from plateletRNAfrom 18 individ-uals of known plA phe-notype. The resultingPcR products, both be-

_366 fore (-) and after (+)-__ 219 digestion with the re-

* 147 striction enzyme Nci I,were analyzed on 1.5%agarose gels stainedwith ethidium bromide.

- + - + + The results from three-o _- CN 04 representative individ-

-.. .1 _ uals, one of each possi-C'J ' ble phenotype, are'< shown on this gel.

cDNAderived fromplAI homozygous individuals contain the sequence 5'-CTGGG-3',and is not cleaved by the enzyme, whereas cDNAamplified fromplA2 homozygotes yielded two fragments of the expected sizes (seeFig. 1), confirming that the base pair polymorphism at nucleotide196 segregates with pIA phenotype. Heterozygotes for the allele showthree bands, corresponding to undigested (PlA-derived) and cleaved(PlA2-derived) GPIIIa cDNA. Though not readily visible in this fig-ure, faint bands corresponding to nonspecifically amplified PcRproducts or to minute quantities of contaminating cDNAwere occa-sionally visible in the gel lanes.

ments, as expected. To date, we have successfully used thisDNAtyping procedure to predict the phenotype of 10 p1Al/A1,5 p1A2/A2 and 3 p1A1/I individuals (P = 3.8 x 10-6).

Discussion

The plAI antigen commonly stimulates the alloimmune re-sponse associated with two syndromes, neonatal alloimmunethrombocytopenia and posttransfusion purpura. The plA1 andpjA2 antigenic determinants are carried on platelet membraneGPIIIa, and have been further localized to a 17-23-kD frag-ment of this molecule (5, 12). Our ability to directly comparethe amino acid sequence of GPIIIa derived from p1A2 versusPlAI individuals was made possible by: (a) the availability ofthe complete nucleotide sequence of GPIIIa cDNA (6-8) and(b) the application of gene amplification techniques to thestudy of phenotype-specific platelet mRNAsequences (9).Based upon the published sequence of GPIIIa cDNA, we con-structed oligonucleotide primer pairs that allowed us to enzy-matically amplify a segment of the GPIIIa mRNAmoleculethat encodes the amino-terminal 103 amino acids of the ma-ture glycoprotein. Examination of the nucleotide sequence de-rived from one p1A2 homozygous individual revealed a C Tpolymorphism at base 196. This single nucleotide substitutionfortuitously results in the creation of a unique restriction en-zyme cleavage site. Utilizing the ability of the enzyme Nci I todiscriminate between these two polymorphic sequences, weanalyzed the phenotypes of 17 additional individuals, andwere able to demonstrate that the p1A2 form of GPIIIa mRNAcontains the codon CCG(Pro33) in place of the CUG(Leu33present in the plA' allele. As such, these findings represent thefirst molecular genetic characterization of a polymorphism as-sociated with a platelet-specific alloantigen system, and pro-vide compelling evidence that the plA phenotype is a geneti-cally determined, inherent property of the GPIIIa molecule,rather than a posttranslationally acquired epitope that be-comes associated with this membrane glycoprotein.

Although the plA phenotype is clearly associated with aLeu/Pro polymorphism at amino acid 33, these studies do notdirectly address the issue of which amino acids form the actualantigenic epitope. There are a number of possible ways thatthis residue might participate in the formation of the plAI/pIA2antigenic determinant. Leu/Pro33 could be physically locatedat the antigenic site, in which case this residue would consti-tute an integral part of the structure of the epitope. It is alsopossible that the presence (plA2) or absence (plAl) of Pro33alters the secondary structure of the polypeptide chain, causingthe actual antigenic determinant to be formed at another site.A third possibility, though less likely, 'is that the Leu/Pro33polymorphism is simply coinherited with an as yet unidenti-fied additional polymorphic region of GPIIIa. It should bepossible to test these models by producing phenotype-specificsynthetic peptides encompassing amino acid 33, which can beexamined for their ability to directly bind anti-PlAl or anti-PlA2antibodies, or to inhibit the binding of these alloantibodies toplatelets of the appropriate phenotype. Similarly, antipeptideantibodies can be produced and examined for their ability toreact selectively with platelets of known plAI and p1A2 pheno-type. One potential obstacle in using peptides to examine plAepitopes is that they are often incapable of mimicking complexconformationally dependent determinants. Thus, an alterna-tive strategy might involve the production of cell lines trans-

1780 P. J. Newman, R. S. Derbes, and R. H. Aster

---O-

fected with GPIIIa molecules differing only at amino acid 33.Serological evaluation of such "phenotype-specific" transfec-tants may ultimately be more useful in defining the molecularnature of these alloantigenic determinants. In any event, it isclear that more work needs to be done in order to localize theepitopes on the native GPIIIa molecule to which PlA-specificantibodies bind.

Studies are currently in progress to determine whetherother regions of the GPIIIa molecule contain polymorphismsthat correlate with pIA phenotype. Zimrin et al. (7) and Rosa etal. (8) have each noted a potential silent C '-+ A polymorphismat base 1163, which is the third base in the codon for Val355,and Burk et al. (13) have recently shown by analysis of geno-mic Southern blots that a Taq I polymorphism exists withinthe GPIIIa gene. Our inspection of the sequence surroundingbase 1163 reveals that the polymorphic sequence 5'-TCUA-3',but not 5'-TAGA-3' represents a Taq I site, and is thereforeprobably identical to the Taq I polymorphism detected byBurk et al. using Southern analysis. This Taq I site is present in

- 47% of the population (13), and is therefore unlikely tosegregate with either the PI"A or PI' allotype, which have genefrequencies of 0.85 and 0.15, respectively.

The ability to determine pIA phenotype by DNAtypingmay have several useful clinical applications. Current serolog-ically based methods involve the use of human alloantisera,which in addition to being consumable, can vary widely withrespect to titer and often contain contaminating anti-HLAantibodies. The PcR-based procedure, on the other hand, canreadily be adapted such that genomic DNA, rather than plate-let mRNA, is used as the source of the nucleotide sequence tobe amplified. Lench et al. (14) have recently reported thatsufficient DNAfor gene analysis by PcR can be isolated frombuccal epithelial cells obtained by mouthwash. One could en-vision applying this noninvasive method to the analysis ofplatelet phenotypes in a clinical setting, where the only othermaterials needed would be two primers, the restriction enzymeNci I, and access to a thermal cycler. An obvious advantageover serologically based evaluation of platelet allotype wouldbe that platelets themselves would not be needed to performthe analysis. Genomic DNAtyping for platelet phenotypemight be of greatest utility for testing severely thrombocyto-penic patients with PTP or NATP, in performing fetal testingin situations where a high-risk for developing NATP is sus-pected, and in phenotyping individuals who are reluctant todonate blood.

Finally, we anticipate that the strategy employed in thisinvestigation could readily be adapted to define polymor-phisms in other alloantigen system, such as those of erythro-cytes and granulocytes, that have thus far escaped detailedcharacterization using more classical biochemical and serolog-ical approaches.

Acknowledgments

Wethank Dr. Jack Gorski of The Blood Center of Southeastern Wis-consin for his scientific counsel, and for critically reading the manu-

script. Weare also grateful to the Platelet Antibody Laboratory of TheBlood Center of Southeastern Wisconsin for performing serologicalevaluation of platelet phenotype.

This work was supported by grants from the National Institutes ofHealth HL-38166 to Dr. Newmanand HL-1 3629 to Dr. Aster.

References

1. Phillips, D. R., I. F. Charo, L. P. Parise, and L. A. Fitzgerald.1988. The platelet membrane glycoprotein Ilb-IIIa complex. Blood.71:831-843.

2. Kunicki, T. J., and P. J. Newman. 1986. The biochemistry ofplatelet-specific antigens. In Current Studies in Hematology and BloodTransfusion. A. Hassig, editor. S. Karger, Basel. 18-32.

3. Aster, R. H. 1984. Clinical significance of platelet-specific anti-gens and antibodies. In Advances in Immunobiology and Bone Mar-row Transplantation. J. McCullough and G. Sandler, editors. Alan R.Liss, NewYork. 103-118.

4. Shulman, N. R., R. H. Aster, A. Leitner, and M. C. Hiller. 1961.Immunoreactions involving platelets. V. Post-transfusion purpura dueto a complement-fixing antigen. A proposed mechanism for thrombo-cytopenia and its relevance in "autoimmunity". J. Clin. Invest.40:1597-1620.

5. Newman, P. J., L. S. Martin, M. A. Knipp, and R. A. Kahn.1985. Studies on the nature of the human platelet alloantigen, P1AI.Localization to a 17,000-dalton polypeptide. Mol. Immunol. 22:719-729.

6. Fitzgerald, L. A., B. Steiner, S. C. Rall, Jr., S-S. Lo, and D. R.Phillips. 1987. Protein sequence of endothelial glycoprotein IlIa de-rived from a cDNAclone. Identity with platelet glycoprotein Ila andsimilarity to integrin. J. Biol. Chem. 262:3936-3939.

7. Zimrin, A. B., R. Eisman, G. Vilaire, E. Schwartz, J. S. Bennett,and M. Poncz. 1988. Structure of platelet glycoprotein IIa. A commonsubunit for two different membrane receptors. J. Clin. Invest.81:1470-1475.

8. Rosa, J-P., P. F. Bray, 0. Gayet, G. I. Johnston, R. G. Cook,K. W. Jackson, M. A. Shuman, and R. P. McEver. 1988. Cloning ofglycoprotein II1a cDNA from human erythroleukemia cells and local-ization of the gene to chromosome 17. Blood. 72:593-600.

9. Newman, P. J., J. Gorski, G. C. White II, S. Gidwitz, C. J.Cretney, and R. H. Aster. 1988. Enzymatic amplification of platelet-specific messenger RNAusing the polymerase chain reaction. J. Clin.Invest. 82:739-743.

10. Woods, V. L., E. H. Oh, D. Mason, and R. McMillan. 1984.Autoantibodies against the platelet glycoprotein Ilb/IIla complex inpatients with chronic ITP. Blood. 63:368-375.

11. Furihata, K., D. J. Nugent, A. Bissonette, R. H. Aster, and T. J.Kunicki. 1987. On the association of the platelet-specific alloantigen,Pena with glycoprotein Ila. Evidence for heterogeneity of glycoproteinIla. J. Clin. Invest. 80:1624-1630.

12. Van Der Shoot, C. E., M. Wester, A. E. G. Kr. von DemBorne,and H. G. Huisman. 1986. Characterization of platelet-specific al-loantigens by immunoblotting: localization of Zw and Bak antigens.Br. J. Haematol. 64:715-723.

13. Burk, C., C. Ingram, M. Weiner, E. F. Rappaport, E. Schwartz,and M. Poncz. 1988. A Taq I polymorphism for the human plateletglycoprotein IlIa gene (GPIIIa). Nucleic Acids Res. 16:7216.

14. Lench, N., P. Stanier, and R. Williamson. 1988. A simplenon-invasive way to obtain DNAfor gene analysis. Lancet. i:1356-1358.

DNATyping for Pt/ Phenotype 1781