1356–1358 Nucleic Acids Research, 1998, Vol. 26, No. 5
1998 Oxford University Press
Sensitive detection of p53 gene mutations by a ‘mutant enriched’ PCR-SSCP technique Moira Behn and Marcus Schuermann* Zentrum für Innere Medizin, Abteilung Hämatologie/Onkologie, Philipps-Universität Marburg, Baldingerstrasse, D-35033 Marburg, Germany Received September 24, 1997; Revised and Accepted December 30, 1997
ABSTRACT For the rapid and sensitive detection of p53 ‘hot spot’ mutations, we combined polymerase chain reaction based single-strand conformational polymorphism (PCR-SSCP) analysis with sequence specific-clamping by peptide nucleic acids (PNAs) in a one-step reaction tube protocol. For this purpose, we designed two PNA molecules comprising aa 246–250 of exon 7 and aa 270–275 of exon 8, respectively, to suppress the amplification of wild-type p53 allelic variants during PCR amplification. Using this method in a survey of 20 brush cytology samples from lung cancer patients, we were able to detect five p53 point mutations occurring in codons 248, 249 and 273 which could not be retrieved by conventional PCR-SSCP. Thus, allelic suppression by PNA molecules opens a way to largely improve the sensitivity of existing PCR-SSCP protocols (∼10–50-fold) and could be useful in the detection of ‘hot spot’ oncogene lesions in histological samples containing only a small number of cancer cells. Loss of heterozygosity at the p53 locus has been extensively documented in many solid cancers. Part of the allelic variants found are due to point mutations which in the p53 gene frequently affects codons at positions 248, 249 and 273, so-called ‘hot spot’ mutations. In the past, the existing PCR-SSCP technique as established by Orita et al. (1), allowed the detection of variant alleles only if the number of mutant alleles in material examined exceeded a certain percentage (usually >2–5%). In several tissues and body fluids however, the number of cancer cells may be much smaller than this, which is beyond the current detection limit. To overcome this problem, several combinations have been perfomed in the past, such as tumour cell enrichment by cell sorting (2), microdissection of tumour tissue (3) or excision of DNA fragments from SSCP gels and subsequent reamplification (4). Given the apparent wide spread use of PCR-SSCP in routine analysis, these protocols have the disadvantage of additional and time-consuming experimental steps. In this study, we assessed the ability of selective mutant allele-specific enrichment using peptide nucleic acids (PNAs; 5, for review see 6, 7). Due to the different chemical nature, PNAs cannot serve as primer molecules during PCR amplification.
However, PNAs can form PNA–DNA hybrids with much higher thermal stability than corresponding DNA–DNA hybrids which permits their application in PCR reactions if higher annealing temperatures are favoured. In addition, PNA–DNA hybrids are more destabilized by single base pair mismatches. We reasoned that this feature specifically, would make PNAs useful in the discrimination of single point mutations since cross-hybridisation to wild-type allelic fragments is less probable at the given Tm value (8). Figure 1 gives a schematic view of the protocol applied whereby the following PNA oligonucleotides were chosen (obtained from TIB Molbiol, Berlin): PNA248-9 (H2N-GGGCCTCCGGTTCAT-N2H) complementary to codon 246–250 and PNA273 (H2N-ACAAACACGCACCTC-N2H) complementary to codon 271–275 of the p53 gene (not shown in Fig. 1). DNA–oligonucleotide primers consisted of the following primer pair combinations: p53, exon 7; p53p5 (5′-CCTCATCTTGGGCCTGTGTTATCTCCTAGGTTGGCT-3′) and p53p6 (5′-CCAGTGTGCAGGGTGGCAAGTGGCTCCTGACCTGGA-3′) yielding a fragment of 169 nt. For the analysis of codon 273 mutations in exon 8, primer pair p53p7 (5′-CTCTTGCTTCTCTTTTCCTATCCTGAGTAGTGGTAA-3′) and p53p8 (5′-CCTCCACCGCTTCTTGTCCTGCTTGCTTACCTCGCT-3′) were taken to obtain a fragment of 196 nt. DNA preparation from brush cytology material was performed as described in (9). The PCR clamping reaction itself was performed in a total volume of 25 µl, consisting of reaction buffer (25 mM TAPS, pH 9.3, 50 mM KCl, 1.5 mM MgCl2 and 1 mM DTT), 0.2 mM of each dNTP, 50 ng of DNA, 12.5 pmol of each primer, 75 pmol of PNA and 1 U of ELONGase (Gibco BRL). Following a hot start at 94C for 5 min, PCR was then performed over 24 cycles for 60 s at 94C, 60 s at 73C in case of PNA248-9 (or alternatively 68C in case of PNA273, 60 s at 54C and 120 s at 72C.) The additional step at 73 or 68C, respectively, was chosen in order to allow the preferential annealing of the PNA to DNA. Alternatively, conventional PCR-SSCP was performed in the absence of PNAs omitting the additional annealing step. As can be seen in Figure 2, the analysis of cytology material for mutations in p53 exon 7 by conventional SSCP-PCR revealed a uniform pattern of allele separation indicating that no mutation in exon 7 could be detected. This changed, however, if PNA248-9, a PNA complementary to the codon 246–250 region, was added to the PCR reaction. This time strands of different mobility became apparent in lanes 2, 3, 5 and 16 (Fig. 2, bottom). The
*To whom correspondence should be addressed. Tel: +49 6421 286990; Fax +49 6421 282700; Email:
[email protected]
1357 Nucleic Acids Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.15 Nucleic
1357
Figure 1. Schematic diagram of ‘mutant enriched’ PCR-SSCP using sequence-specific PNA molecules spanning the p53 codon 248 and 249 ‘hot spot’ region (codons 246–250). Amplification of alleles mutated in the respective positions is not blocked and therefore occurs preferentially.
Figure 2. ‘Mutant enriched’ SSCP analysis by the use of PNA-mediated PCR clamping. Example of enhanced sensitivity in PCR-SSCP using a PNA oligonucleotide largely blocking the amplification of wild-type p53 alleles. A clear polymorphism becomes visible in lanes 3, 5 and 16 which is not seen in the absence of added PNA. Following ‘mutant enriched’ SSCP-PCR (described in the text) 3 µl of [α-33P]dCTP labelled PCR amplification product was added to 12 µl of stop solution [95% formamide, 10 mM NaOH and tracking dyes, heated to 95C for 3 min and loaded onto a MDE-gel (Hydrolink)] according to the manufacturer’s specification. The gel was run at 4C and 4 W for 14 h and the labelled fragments were visualised by autoradiography. Confirmatory sequence analysis was performed on excised and reamplified DNA contained within normally and aberrantly migrating single strand fragments using an ABI PRISM 377 sequencing automater (Applied Biosystems), according to the manufacturer’s ‘cycle sequencing’ protocol.
1358 Nucleic Acids Research, 1998, Vol. 26, No. 5 and the general frequency of ‘hot spot’ mutations in many tumours would make it useful to screen for these oncogene mutations in exfoliated material from body fluids. Despite the current costs of PNA oligonucleotide synthesis, these molecules can be used in a concentration which allows several thousand reactions to be performed from one batch. If routinely applied, the extra costs for the synthesis of PNA oligonucleotides therefore, would not contribute excessively to the overall costs of conventional PCR-SSCP. Table 1. Analysis of brush cytology specimen from 20 patients with lung cancer by conventional and ‘mutant enriched’ PCR-SSCP Number of mutations
‘conventional’ PCR-SSCP
‘mutant enriched’ PCR-SSCP
codon 248
02
(CGG→CAG) (CGG→TGG)
codon 249
02
(AGG→AGC) (AGG→AGC)
codon 273
01
(CGT→CAT)
REFERENCES Figure 3. Serial dilution experiment using DNA containing a homozygous codon 248 mutation into DNA of a wild-type status. The concentration (%) of mutant alleles was as follows: 100 (1), 50 (2), 5 (3), 1 (4), 0.5 (5), 0.1 (6) and 0% (7). PCR-SSCP conditions were the same as described in Figure 2. With conventional PCR-SSCP (top), detection is possible up to a limit of 5% mutant DNA, suppression of wild-type alleles by an additional PNA allows detection up to a limit of 0.5% mutant alleles in the original DNA.
different nature of the PCR products in these lanes was confirmed by sequence analysis after gel excision and reamplification (30 cycles). This way, we found two mutations to have occurred in codon 248, two in codon 249 and one in codon 273. All mutations gave rise to amino acid substitutions (Table 1). To test the effect of the PNAs on the suppression of normal alleles more quantitatively, we performed serial dilution experiments using DNA of two different p53 genotypes. Figure 3 shows that suppression by PNA248-9 leads to the safe detection of a codon 248 mutation in a 200-fold excess of normal DNA. The ‘mutant enriched’ PCR-SSCP method we describe, thus, allows the rapid, efficient and sensitive detection of oncogene ‘hot spot’ mutations in material containing only a minority of tumour cells. Similarly, this method would be useful in the detection of K-ras mutations since these are confined to three codons (at positions 12, 13 and 61) two of which could be detected by using only a single PNA. Although the majority of p53 lesions would not be detected this way, the gain in sensitivity
1 Orita,M., Suzuki,T. and Hayashi,K. (1989) Genomics, 5, 874–879. 2 Maesawa,C., Tamura,G., Suzuki,Y., Ishida,K., Saito,K. and Satodate,R. (1992) Jpn. J. Cancer Res., 83, 1253–1256. 3 Kawamata,N., Park,D., Wilczynski,S., Yokota,J. and Koeffler,H.P. (1996) Prostate, 29, 191–193. 4 Sherratt,E.J., Thomas,A.W., Gagg,J.W. and Alcolado,J.C. (1996) Biotechniques, 20, 430–432. 5 Orum,H., Nielsen,P.E., Egholm,M., Berg,R.H., Buchardt,O. and Stanley,C. (1993) Nucleic Acids Res., 21, 5332–5336. 6 Nielsen,P.E. (1995) Annu. Rev. Biophys. Biomol. Struct., 24, 167–183. 7 Agrawal,S. and Iyer,R.P. (1995) Curr. Opin. Biotechnol., 6, 12–19. 8 Buchardt,O., Egholm,M., Berg,R.H. and Nielsen,P.E. (1993) Trends Biotechnol., 11, 384–386. 9 Behn,M., Qun,S., Pankow,W., Havemann,K. and Schuermann,M (1998) Clincal Cancer Res., in press.
RELATED PAPERS RECENTLY PUBLISHED IN NUCLEIC ACIDS RESEARCH Head,S.R., Rogers,Y.H., Parikh,K., Lan,G., Anderson,S., Goelet,P. and Boyce-Jacino,M.T. (1997) Nested genetic bit analysis (N-GBA) for mutation detection in the p53 tumor suppressor gene. Nucleic Acids Res. 24, 5065–5071. Roberts,E., Deeble,V.J., Woods,C.G. and Taylor,G.R. (1997) Potassium permanganate and tetraethylammonium chloride are a safe and effective substitute for osmium tetroxide in solid-phase fluorescent chemical cleavage of mismatch. Nucleic Acids Res. 24, 3377–3378. Bi,W. and Stambrook,P.J. (1997) CCR: a rapid and simple approach for mutation detection. Nucleic Acids Res. 24, 2949–2951.
