(-D-l 1991 Oxford University Press

2794 Nucleic Acids Research, Vol. 19, No. 10

Studies on clonality by POR analysis of the PGK-1 gene H.van Kamp, R.Jansen, R.Willemze, W.E.Fibbe and J.E.Landegent Laboratory of Experimental Hematology, University Medical Center, Leiden, The Netherlands Submitted February 20, 1991 Analysis of the methylation pattern of the polymorphic Xchromosome linked genes PGK-1 and HPRT (1) have been widely used to assess the clonal nature of human tumour cells in female patients heterozygous for one of these markers. The applicability of this procedure would be greatly facilitated if it could be analysed by PCR (2). The PGK-1 gene seemed to be a good candidate since the methylation sites (Hpafl sites) and the polymorphic BstXl site are within 1 kb of DNA. About 33 % of females are expected to be heterozygous at this site (1). For PCR amplification, the known nucleotide sequence from exon 1 (3) had to be extended into the region containing the polymorphism. A 'Sau 3A partial' genomic library in the X-phage EMBL3 was screened with a 333 bp probe that was obtained by PCR amplification using the Al sense and Bl antisense primers (Fig. 1). One of the X clones isolated, (X 7.4), was subjected to restriction mapping and Southern hybridization to determine the appropriate DNA fragment for subcloning into pUC 13. The sequence of a subclone containing a 0.8 kb BamHI segment (pP3), was established by the dideoxy chain-termination method using the universal 'forward and reverse' sequencing primers. Because the DNA analysed did not reveal a BstXI site, 2 additional primers were synthesized, and sequence analysis was also performed on A2-B2 PCR-generated fragments from a male who did exhibit the BstXI site, and from a heterozygous female. Clonality was analysed using the Al primer and a new primer B3, located 3' of the BstXI site. The amplified fragment thus encompasses two HpaII sites, previously shown to be differentially methylated, as well as the BstXI polymorphism. Since PCR does not maintain methylation patterns, genomic DNA has to be digested with Hpal and amplified in addition to an aliquot (0.1 Mg) of undigested DNA. Both amplicons are subsequently digested with BstXI and analysed on gel. The procedure was tested on DNA derived from peripheral blood leucocytes of 2 female patients who were known to exhibit a clonal and a polyclonal hematopoiesis respectively, as determined by Southern hybridization with the 333 bp PGK-l probe. As can be seen in Pig. 2, the patterns observed after PCR analysis are concordant with the Southern data. In case of a monoclonal pattern (A), one band is eliminated or significantly reduced in intensity after HpaH digestion due to inactivation of either the paternal or the maternal X-chromosome, whereas in the polyclonal case (B), the intensity of the 2 alleles is unaffected by HpaII digestion and subsequent amplification. In conclusion, we have shown that PCR of the PGK-1 gene can be successfully applied for studies on clonality, allowing analysis of a limited number of cells.

REFERENCES 1. Vogelstein,B., Fearon,E.R., Hamilton,S.R., Preisinger,A.C., Willard,H.F., Michelson,A.M., Riggs,A.D. and Orkin,S.H. (1987) Cancer Res. 47, 4806-4813. 2. Saiki,R.K., Scharf,S., Faloona,F., Mullis,K.B., Horn,G.T., Erlich,H.A. and Arnheim,N. (1985) Science 230, 1350-1354. 3. Michelson,A.M., Blake,C.C., Evans,S.T. and Orkin,S.H. (1985) Proc. Natl. Acad. Sci. USA 82, 6965 -6969.

ACKNOWLEDGEMENT This work was supported by grants from the Dutch Cancer Society (IKW90-07) and from the J.A. Cohen Institute for Radiopathology and Radiation Protection.

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Figure 1. Restriction map, showing the X7.4 clone, the 0.8 kb subclone pP3, and the region containing exon 1. The position of the primers Al (TGT TCC GCA TTC TGC AAG CC), A2 (GTC ATT TTA CTT TCC CUT CC), Bi (GGA AAA TGC GGC TAG AAA CC), B2 (ACA TGC GCA GAG TAA GAG AC) and B3 (TAT CCT TTT GTG CAG GAA CC) is also indicated.

4"'o ft Figure 2. Clonal analysis by PCR of the PGK-l gene (upper part) and Southern hybridization (lower part). DNA was derived from peripheral blood leucocytes of 2 female patients exhibiting a clonal (A) and polyclonal (B) hematopoiesis. For Southern hybridization, 12 gg DNA was digested with PstI and BstXI and divided into 2 aliquots, of which one was not further digested (lane 1) and the other was digested with HpaII (lane 2). Hybridization was performed with the 333 bp Al-Bl probe, yielding bands of 1.05 and 0.90 kb. PCR analysis was performed as described in the text. Lane 1 shows the BstXI polymorphism (619 and 557 bp), lane 2 the result of amplification after Hpafl digestion.

Studies on clonality by PCR analysis of the PGK-1 gene.

(-D-l 1991 Oxford University Press 2794 Nucleic Acids Research, Vol. 19, No. 10 Studies on clonality by POR analysis of the PGK-1 gene H.van Kamp, R...
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