Patricia Bray-Ward

Mutation detection and single-molecule counting using isothermal rolling-circle amplification

Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the – strand of DNA, a complex pattern of DNA strand displacement ensues that generates 109 or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

Comprehensive human genome amplification using multiple displacement amplification

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

Improvements in Cytogenetic Slide Preparation: Controlled Chromosome Spreading, Chemical Aging and Gradual Denaturing

BACKGROUND Metaphase spreading is an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M-FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization. Materials and Methods Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation, aging, and hybridization was developed. RESULTS This protocol yields good chromosome spreads from even the most difficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing benchwork. Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing. CONCLUSION The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results.

Cloning, characterization, and chromosomal mapping of a human electroneutral Na(+)-driven Cl-HCO3 exchanger.

The electroneutral Na+-driven Cl-HCO3 exchanger is a key mechanism for regulating intracellular pH (pH i ) in neurons, glia, and other cells. Here we report the cloning, tissue distribution, chromosomal location, and functional characterization of the cDNA of such a transporter (NDCBE1) from human brain (GenBankTM accession number AF069512). NDCBE1, which encodes 1044 amino acids, is 34% identical to the mammalian anion exchanger (AE2); ∼50% to the electrogenic Na/HCO3 cotransporter (NBCe1) from salamander, rat, and humans; ∼73% to mammalian electroneutral Na/HCO3 cotransporters (NBCn1); 71% to mouse NCBE; and 47% to a Na+-driven anion exchanger (NDAE1) fromDrosophila. Northern blot analysis of NDCBE1 shows a robust ∼12-kilobase signal in all major regions of human brain and in testis, and weaker signals in kidney and ovary. This human gene (SLC4A8) maps to chromosome 12q13. When expressed inXenopus oocytes and running in the forward direction, NDCBE1 is electroneutral and mediates increases in both pH i and [Na+] i (monitored with microelectrodes) that require HCO 3 − and are blocked by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The pH i increase also requires extracellular Na+. The Na+:HCO 3 − stoichiometry is 1:2. Forward-running NDCBE1 mediates a36Cl efflux that requires extracellular Na+ and HCO 3 − and is blocked by DIDS. Running in reverse, NDCBE1 requires extracellular Cl−. Thus, NDCBE1 encodes a human, electroneutral Na+-driven Cl-HCO3 exchanger.

Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Simultaneous detection of microsatellite repeats and SNPs in the macrophage migration inhibitory factor (MIF) gene by thin-film biosensor chips and application to rural field studies

Microsatellite repeat and single nucleotide polymorphisms (SNPs) are abundant sources of genetic variation, but existing methodologies cannot simultaneously detect these variants in a facile or inexpensive way. We describe herein a thin-film biosensor chip based on an allele-discriminating oligonucleotide array that enables genotyping for both microsatellite repeats and SNPs in a single analysis. We validated this methodology for the functionally polymorphic −794 CATT5–8 repeat and −173 G/C SNP present in the promoter of the human gene for macrophage migration inhibitory factor (MIF). In a comparison of 30 samples collected at a rural hospital in Zambia, we observed a 100% concordance for both the CATT repeat and G/C SNP between the biosensor methodology and the conventional capillary electrophoresis. The biosensor chips are low in cost and once printed, they are robust and require no instrumentation for analysis. When combined with multiple displacement amplification, this methodology can be utilized in primitive settings for the genotyping of nanogram quantities of DNA present in blood, dried and stored on filter paper samples. We applied this methodology to a field study of MIF genotype in children with malaria, and provide first evidence for a potential association between MIF alleles and malaria infection. We also present data supporting significant population stratification of the low- versus high-expression forms of MIF that may bear on the role of this gene in infectious diseases.

Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling

The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources . Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides , for use in common nucleic acid labeling reactions, such as PCR and nick translation. The modified nucleotides were synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptenes such as biotin or digoxigenin, which require fluorescently labeled proteins for detection. This method allows custom preparation of most common fluorescent nucleotides and rapid testing of new ones, while reducing the cost of procedures such as multiplex fluorescent in situ hybridization (M-FISH) by 100-200 fold.

Human Sperm Maintain Their Shape Following Decondensation and Denaturation for Fluorescent In Situ Hybridization: Shape Analysis and Objective Morphometry

Abstract The relationship between abnormal sperm morphology and chromosomal aberrations has been of interest. Thus far, however, studies have focused on frequencies of sperm with either abnormal morphology or aneuploidies in semen samples, not on detection of individual spermatozoa exhibiting both abnormal morphology and aneuploidy. To assess the feasibility of simultaneous evaluation of both attributes in an individual sperm cell, we investigated whether sperm shape is preserved after decondensation and denaturation, procedures that are required for fluorescent in situ hybridization (FISH). On 21 slides, 395 sperm were fixed, photographed, and then digitized by the computer-assisted Metamorph morphometry program for individual evaluation before decondensation. To establish whether sperm of various shapes would behave in similar manners, the cells were also classified, according to their head shapes, into symmetrical (n = 115), asymmetrical (n = 115), irregular (n = 115), and amorphous (n = 50) categories. Following decondensation and subsequent denaturation, sperm that had been photographed initially were relocalized and digitized for morphometry. Head area, perimeter, long axis, short axis, shape factor, and tail length were evaluated in each of the 395 sperm in both the native and decondensed states. After the decondensation and denaturation protocol of the FISH procedure, the sperm exhibited a proportional increase in dimensions as compared to their original sizes. Their initial shapes were preserved with high fidelity whether the sperm were in the symmetrical, asymmetrical, irregular, or amorphous categories. Hybridization with the chromosome probes had no further effect on sperm shape or size. We provide images to demonstrate how these findings facilitate studies about the relationship between sperm shape and chromosomal content or aberrations in individual spermatozoa.

Characterization of the human myeloid leukemia-derived cell line GF-D8 by multiplex fluorescence in situ hybridization, subtelomeric probes, and comparative genomic hybridization.

The human myeloid leukemia cell line GF‐D8 was established from the peripheral blood blasts of a patient with acute myeloid leukemia FAB subtype M1 (AML‐M1). The karyotype, which has not changed significantly over several years of culture, was described initially as 44,XY,‐5,del(7q),inv(7q),add(8q),add(11q),del(12p),‐15,‐17,+mar. With the advent of multicolor fluorescence in situ hybridization (FISH) techniques, the prospect of accurately characterizing this complex karyotype became feasible. In the present study, we applied 24‐color whole‐chromosome painting and analyzed the results using a filter‐based detection system and proprietary software for multiplex FISH (M‐FISH). This resulted in the refinement of the karyotype and the identification of hitherto unsuspected chromosome rearrangements. M‐FISH identified the origin of the add(8q) and add(11q) as well as the small marker chromosome. Both the del(7q) and del(12p) were redefined as unbalanced translocations and an apparently normal chromosome 11 was shown to be t(11;17). Importantly, the del(12p) was shown to be a der(12)t(7;12). Single‐color whole‐chromosome painting studies confirmed these findings, but also identified a cryptic t(Y;12) not seen in the original M‐FISH analysis. We then carried out a FISH screening assay using a complete set of chromosome‐specific subtelomeric probes. This allowed the identification of p and q subtelomeric regions involved in the translocations and indicated amplification of the 8q subtelomeric region. Comparative genomic hybridization (CGH) revealed a highly unbalanced karyotype, as deletions accompanied the majority of translocations, and identified the regions of amplification as 8q22.3‐qter and 11q21‐qter. Finally, conventional FISH with centromeric and unique sequence probes was necessary to elucidate all of the rearrangements. Genes Chromosomes Cancer 24:213–221, 1999.

Small marker chromosome identification in metaphase and interphase using centromeric multiplex fish (CM-FISH).

Multicolor karyotyping procedures, such as multiplex fluorescence in situ hybridization (M-FISH), spectral karyotyping, or color-changing karyotyping, can be used to detect chromosomal rearrangements and marker chromosomes in prenatal diagnosis, peripheral blood cultures, leukemia, and solid tumors, especially in cases where G-banding is not sufficient. A regular M-FISH analysis requires relatively large amounts of labeled DNA (microgram quantities), is not informative in interphase nuclei, hybridization can take up to 2 to 3 days, and unlabeled human chromosome-painting probes are not available commercially. Unique probes (plasmids, PAC), specific for centromeric or subtelomeric chromosomal regions, can replace the painting probes in M-FISH to address specific issues, such as the identification of marker chromosomes and aneuploidies. A set of plasmid probes carrying repetitive sequences specific for the α-satellite region of all human chromosomes were combined in a metaphase assay and an interphase assay, allowing identification of aneuploidies in one hybridization step, on a single cytogenetic slide. The fluorophore-dUTP and the labeled antibodies required to label and detect the DNA probes can be prepared in any laboratory. All DNA probes can be easily isolated and labeled using common molecular cytogenetic procedures. Because of the repetitive nature of the probes, hybridization time is short, usually less than 1 hour, and the analysis can be performed with nonspecialized image-processing software.