Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers Caifu Chen, 4 Bai-lin Wu, 2 Tao Wei, 3 Michael Egholm, 4 William M. Strauss 1 1 Harvard Institute of Human Genetics, Harvard Medical School, Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, Massachusetts 02115, USA 2 Children’s Hospital, Harvard Medical School, Boston, Massachusetts, 02115, USA 3 Research Computing Center, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, USA 4 PE Biosytems, 850 Lincoln Center Dr., Foster City, California 94404, USA Received: 2 September 1999 / Accepted: 16 December 1999 Abstract. We have extended our earlier work to show that indi- vidual 14–20mer peptide nucleic acid probes directed against in- terspersed -satellite sequences can specifically identify chromo- somes. Peptide nucleic acid (PNA) probes were used to detect chromosomal abnormalities and repeat structure in the human ge- nome by fluorescence in situ hybridization (FISH). The hybrid- ization of a single PNA probe species directed against a highly abundant -satellite DNA repeat sequence was sufficient to abso- lutely identify a chromosome. Selection of highly repetitive or region-specific DNA repeats involved DNA database analysis. Distribution of a specific repeat sequence in human genome was estimated through two means: a computer program “whole ge- nome” approach based on ∼400 Mb (12%) human genomic se- quence. The other method involved directed search for alpha sat- ellite sequences. In total, ∼240 unique DNA repeat candidates were found. Forty-two PNA probes were designed for screening chromosome-specific probes. Ten chromosome-specific PNA probes for human Chromosomes (Chrs) 1, 2, 7, 9, 11, 17, 18, X, and Y have been identified. Interphase and metaphase results dem- onstrate that chromosome-specific PNA probes are capable of de- tecting simple aneuploidies (trisomies) in human. Another set of PNA probes showed distinct banding-like patterns and could be used as sequence-specific stains for chromosome “bar coding”. Potential application of PNA probes for investigating repeat struc- ture and function is also discussed. Introduction Peptide nucleic acids (PNAs) are nucleic acid mimics that contain a pseudo-peptide backbone, composed of charge neutral and achi- ral N-(2-aminoethyl) glycine units to which the nucleobases are attached via a methylene carbonyl linker (Egholm et al. 1992, 1993b; Nielsen et al. 1991). PNAs hybridize with high affinity to complementary DNA sequences, forming PNA-DNA complexes via Watson-Crick or Hoogsteen binding (Leijon et al. 1994). In addition to the high thermal stability of complexes, PNA-DNA binding is highly sensitive to mismatches (Egholm et al. 1993b; Jensen et al. 1997). In recent papers, it has been demonstrated that fluorochrome-labeled PNA oligomers strongly hybridize to telo- meres (Lansdorp et al. 1996; Martens et al. 1998), CAG trinucleo- tide repeats (Taneja 1998), and centromeric repeats (Chen et al. 1999). PNA-FISH can discriminate between two CENP-B centro- meric DNA sequences that differ by a single base pair in mouse and human chromosomes (Chen et al. 1999). PNA-FISH has several advantages over conventional DNA- FISH. First, the higher affinity of the PNA-DNA duplex and greater accessibility of short and charge-neutral PNA oligomers (Egholm et al. 1992, 1993a) leads to very strong FISH signals. Second, these signals are highly saturable, stable, and reproduc- ible. Third, PNA-FISH also shows a very low background, sug- gesting a potential application as a general reagent for genome- wide quantitative FISH (Chen et al. 1999; Hultdin et al. 1998; Lansdorp et al. 1996; Martens et al. 1998). Finally, these outlined advantages mean that short (<20-mer) PNA oligomers can be uti- lized in a FISH experiment. The short PNA oligomers show a superior ability over DNA oligonucleotides both in signal strength and in discriminating two DNA repeats that differ at a single base (Chen et al. 1999). Prenatal diagnosis via amniocentesis and cytogenetic evalua- tion are now the medical standard for pregnant women at increased risk (Verp and Gerbie 1981). Trisomy 21 (Down’s syndrome) remains the most frequent chromosomal anomaly, with an inci- dence of 0.12 percent or 1 in 833 live births (Adams et al. 1981; Goad et al. 1976; Griffin 1996; Huether et al. 1981; Leck 1966; Mikkelsen 1981; Zarfas and Wolf 1979). Chromosome 18 and sex chromosome aneuploidies are the next most common finding, with one XYY and one XXY in every 1000 male live births and one XXX in every 1000 female live births (Kupke and Muller 1989; Nielsen et al. 1975; Ramesh and Verma 1996; Griffin, 1996; Goad et al. 1976). In this report, using PNA-FISH with particular repetitive se- quence probes, we demonstrate the identification of chromosome- specific PNA probes. These probes have potential clinical appli- cations in prenatal diagnosis and chromosome karyotyping in hu- man. Chromosome-specific PNA probes represent a set of diagnostic reagents that may prove to be a significant improvement in the clinical setting. The rapid processing of samples, uniformity of probe preparation, and the excellent discrimination of this tech- nique represent improvements over the current technology. In this report we also demonstrate sequence-specific banding of human chromosomes by PNA-FISH. In this second pattern of hybridization, PNA signal can be found throughout the genome, exhibiting chromosome characteristic banding. In addition to the chromosome-specific probes, chromosome characteristic probes may prove to be useful in basic research for the study of structure- function analysis of the mammalian chromosome. Both classes of PNA probes may show promise in the study of small samples from large collections of normal/patient material. The study of chromosomal polymorphism in human populations may be greatly enhanced by these reagents. This study thus suggests a role for PNA-FISH in the study of chromosome evolution and conserva- tion. Correspondence to: W. Strauss; e-mail: wstrauss@hihg.med.harvard.edu Mammalian Genome 11, 384–391 (2000). © Springer-Verlag New York Inc. 2000 Incorporating Mouse Genome