A simple and efficient method for constructing high resolution physical maps.

This paper describes a simple and efficient walking method for constructing high resolution physical maps and discusses its applications to genome analysis. The method is an integration of three strategies: (1) use of a highly redundant library of 3Kb-long subclones; (2) construction of a multidimensional pool from the library; (3) direct application of a PCR (polymerase chain reaction)-based screening technique to the pooled library, with two PCR primers, one from the end of the subcloning vector and the other from the leading edge of the walk. This technique allows not only detection of each overlapping subclone but simultaneous determination of its orientation and the size of its overlap. The end of the subclone with the smallest overlap is sequenced and a primer is designed for the next step in the walk. Iteration of the screening procedure with minimum overlapping subclones results in completion of the high resolution map. Using this method, a 3Kb-resolution map was constructed from an 80Kb region of the bithorax complex of Drosophila melanogaster. The method is general enough to be applicable to DNA from other species, and simple enough to be automated.     

Highly sensitive feature detection for high resolution LC/MS

Background  

Liquid chromatography coupled to mass spectrometry (LC/MS) is an important analytical technology for e.g. metabolomics experiments. Determining the boundaries, centres and intensities of the two-dimensional signals in the LC/MS raw data is called feature detection. For the subsequent analysis of complex samples such as plant extracts, which may contain hundreds of compounds, corresponding to thousands of features – a reliable feature detection is mandatory.     

A simple method for high temporal resolution calcium imaging with dual excitation dyes.

Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cells. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a potential drawback of ratiometry is that it requires equally fast switching of the excitation light between two wavelengths. To circumvent continuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined with the acquisition of a single ratiometric pair of fluorescence images at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change during the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs acquired at regular intervals during the recording period, that furthermore includes a correction for the changing dye concentration in the calculation of [Ca2+].     

A sensitive peptide mapping method. Identification of three amino acid substitutions within two anti-azobenzenearsonate monoclonal antibody light chains

Peptide mixtures, precolumn-derivatized with dimethylaminoazobenzene isothiocyanate, have been separated by reversed-phase high-performance liquid chromatography to generate a dimethylaminoazobenzene thiocarbamoyl peptide map. The eluted peptide derivatives are detected in the visible region with a sensitivity of 2-5 pmol and can be collected for direct structural analysis. This technique was applied to compare the sequence homology of two immunoglobulin light chains which were derived from two anti-azobenzenearsonate monclonal antibodies, namely 10K44-7A1 and 10K26-12A1. The complete variable region sequences of 10K44-7A1 and 10K26-12A1 light chains were established based on the sequence analysis of tryptic peptides, intact light chains and reference sequences obtained previously [Siegelman M. and Capra, J.D. (1981) Proc. Natl Acad. Sci. USA, 78, 7679-7683]. Altogether, three amino acid substitutions have been detected within complementary determining regions 1 and 2, and framework region 3, all requiring only a single base change at the DNA level. This new technique provides detection limits and the feasibility of analysing peptides which are not obtainable with conventional techniques.     

A novel method for the genome-wide high resolution analysis of DNA damage

DNA damage occurs via endogenous and exogenous genotoxic agents and compromises a genome's integrity. Knowing where damage occurs within a genome is crucial to understanding the repair mechanisms which protect this integrity. This paper describes a new development based on microarray technology which uses ultraviolet light induced DNA damage as a paradigm to determine the position and frequency of DNA damage and its subsequent repair throughout the entire yeast genome.     

A new method of DNA denaturation mapping.

Described is a new method for DNA denaturation mapping utilizing glyoxal (ethanedial) to stabilize the denatured regions. The extent of glyoxal reaction can be easily and sensitively measured using an assay based on the intercalation of ethidium into duplex DNA. Thus denturation maps can be produced in a controlled way under a wide variety of conditions.     

The human SOX18 gene: cDNA cloning and high resolution mapping

SOX genes comprise a family of genes that are related to the mammalian sex determining gene SRY and these genes play key roles during animal development. We report here cloning and characterisation of the human SOX18 gene. SOX18 gene is expressed in foetal brain as well as in a wide range of foetal and adult tissues indicating its function is not restricted to early development. Mapping analysis has revealed that SOX18 gene is located on human chromosome 20q13.3, 27.29 cR distal from the marker D20S173.     

Rapid high resolution single nucleotide polymorphism-comparative genome hybridization mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ~200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.     

A sensitive automated method for adenosine triphosphatase kinetics.

An automated method for measuring adenosine triphosphatase (ATPase) activity is described. A modified version of a Technicon Autoanalyzer utilizing a sensitive colourimetric technique for determining inorganic phosphate concentrations (1 nmol/ml) allows investigations on enzymes of low specific activities. Dialysis may be used for measuring tissue homogenate activities or bypassed by examining purified enzyme preparations. When combined with a gradient apparatus, the proposed technique is particularly well suited for the study of enzyme kinetics in relation to cation or anion concentrations.     

A sensitive high performance liquid chromatographic method for determining small amounts of glycosylproteins

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.     

A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.     

A simple method for high resolution banding of chromosomes in amniotic fluid cells

Summary A simple technique which results in good quality early mitotic stages of amniotic fluid (AF) cells is presented. Two days after trypsinization AF cell cultures are incubated for 4 h in culture medium containing 20 U/ml liquemin. During the last hour 5 g/ml ethidium bromide (EB) is added and 15 min before harvest 0.04 g/ml colcemid is applied as usual. G-banded and Q-banded chromosomes corresponding to at least 550–850 bands per haploid genome can be obtained in sufficient numbers.     

Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping

The success of high resolution genetic mapping of disease predisposition and quantitative trait loci in humans and experimental animals depends on the positions of key crossover events around the gene of interest. In mammals, the majority of recombination occurs at highly delimited 1-2 kb long sites known as recombination hotspots, whose locations and activities are distributed unevenly along the chromosomes and are tightly regulated in a sex specific manner. The factors determining the location of hotspots started to emerge with the finding of PRDM9 as a major hotspot regulator in mammals, however, additional factors modulating hotspot activity and sex specificity are yet to be defined. To address this limitation, we have collected and mapped the locations of 4829 crossover events occurring on mouse chromosome 11 in 5858 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. This chromosome was chosen for its medium size and high gene density and provided a comparison with our previous analysis of recombination on the longest mouse chromosome 1. Crossovers were mapped to an average resolution of 127 kb, and thirteen hotspots were mapped to <8 kb. Most crossovers occurred in a small number of the most active hotspots. Females had higher recombination rate than males as a consequence of differences in crossover interference and regional variation of sex specific rates along the chromosome. Comparison with chromosome 1 showed that recombination events tend to be positioned in similar fashion along the centromere-telomere axis but independently of the local gene density. It appears that mammalian recombination is regulated on at least three levels, chromosome-wide, regional, and at individual hotspots, and these regulation levels are influenced by sex and genetic background but not by gene content.     

Noninvasive assessment of local myocardium repolarization changes using high resolution surface ECG mapping

A method using body surface potential maps for assessment of myocardium lesions with changed repolarization is presented and suitable mapping system is introduced. Differences between normal and altered QRST integral maps together with torso volume conductor model were used to determine the equivalent dipole representing the lesion. Performance of the method was studied on simulated data. Changed repolarization was modeled by shortening of myocyte action potentials in regions typical for stenosis of the main coronary arteries. The equivalent dipole estimated the positions of small lesions with a mean error of 9+/-4 mm (17+/-14 mm for larger transmural lesions). The subepicardial or subendocardial character of the lesions was reflected in the dipole orientation. Tests of the method on patients after myocardial infarction that underwent coronary intervention on a single coronary vessel showed that in 7 of 8 successfully treated patients the dipole position matched well with the treated vessel. A small dipole moment in another patient indicated unsuccessful treatment. The method was implemented in a new 128-channel mapping system. Its active electrodes, battery powered measuring unit and optical computer interface help to minimize noise in ECG and guarantee patient's safety. The results suggest that the method and mapping system offer useful tools for noninvasive identification of local repolarization changes in the myocardium.     

A simple method for DNA restriction site mapping.

When a DNA molecule, enzymatically labelled with 32p at one end, is partially digested with a restriction enzyme labelled tdna fragments are obtained which form an overlapping series of molecules, all with a common labelled terminus. ta restriction map can then be constructed from an analysis of the size distribution of these molecules. This technique has been used for the restriction site mapping of cloned histone DNA (h22) where as many as 35 cleavage sites may be accurately determined in a single experiment.