Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.     

Integration of animal linkage and BAC contig maps using overgo hybridization

The alignment of genome linkage maps, defined primarily by segregation of sequence-tagged site (STS) markers, with BAC contig physical maps and full genome sequences requires high throughput mechanisms to identify BAC clones that contain specific STS. A powerful technique for this purpose is multi-dimensional hybridization of "overgo" probes. The probes are chosen from available STS sequence data by selecting unique probe sequences that have a common melting temperature. We have hybridized sets of 216 overgo probes in subset pools of 36 overgos at a time to filter-spotted chicken BAC clone arrays. A four-dimensional pooling strategy, including one degree of redundancy, has been employed. This requires 24 hybridizations to completely assign BACs for all 216 probes. Results to date are consistent with about a 10% failure rate in overgo probe design and a 15-20% false negative detection rate within a group of 216 markers. Three complete rounds of overgo hybridization, each to sets of about 39,000 BACs (either BAMHI or ECORI partial digest inserts) generated a total of 1853 BAC alignments for 517 mapped chicken genome STS markers. These data are publicly available, and they have been used in the assembly of a first generation BAC contig map of the chicken genome.     

Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.

The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.     

A two-dimensional YAC pooling strategy for library screening via STS and Alu-PCR methods.

We have developed a simple two-dimensional YAC pooling strategy to facilitate YAC library screening via STS and Alu-PCR approaches. The method has been implemented using the human total genomic YAC library of Olson and coworkers, and its validity tested by isolation of many chromosomes 19- and 21-specific YACs. The Alu-PCR approach is notable in that it is hybridization-based, such that PCR primer pairs do not need to be repeatedly synthesized and tested for each screening step.     

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.     

The generation of DNA probes to chromosome 11q23 by Alu PCR on small numbers of flow-sorted 22q- derivative chromosomes

A strategy for the isolation of DNA probes from small numbers of flow-sorted human chromosomes has been developed. A lymphoblastoid cell line carrying the 22q- derivative chromosome product of the constitutional t(11;22) translocation was used as the source of chromosomes. Synthetic oligonucleotide primers, based on the consensus Alu sequence, were used to amplify inter-Alu sequence from 500 flow-sorted 22q- derivative chromosomes. The amplified sequences were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range of 400 to 1000 bp. Approximately 70% of these clones hybridized to human DNA as single-copy probes. To identify clones derived from chromosome 11, the library was screened with a heterogeneous probe prepared by Alu-PCR amplification from the DNA of a somatic cell hybrid containing one homology of chromosome 11. All the positive clones found were mapped to within the q23-q25 region of chromosome 11 known to be translocated onto the 22q- derivative chromosome. Further mapping studies showed that most of these probes (7/8) lay between the breakpoints for the t(4;11) translocation of acute lymphocytic leukemia and the t(11;22) of Ewing sarcoma. Thus, the use of Alu-PCR on the small derivative chromosome 22q- has provided a greatly enriched source of probes to region 11q23, a part of the genome that is currently of great interest. This approach will be particularly appropriate to small numbers of chromosomes when high specificity rather than total representation is required.     

Isolation of new probes from Xq12-->q13: an example of the screening of reference libraries with Alu-PCR products from radiation hybrids.

In order to isolate new probes from the juxtacentromeric region of the long arm of the human X chromosome, we used Alu-mediated polymerase chain reaction (Alu-PCR) products as probes to directly screen a chromosome X-specific gridded cosmid library. These Alu-PCR products were synthesized from radiation hybrids containing the loci DXS159, PGK1, and PGK1P1. This approach allowed us to select 18 cosmids capable of hybridizing with at least two Alu-PCR products. Four cosmids hybridized to more than three Alu-PCR products. Three of these four cosmids were contiguous, and the fourth was independent. Two cosmids that hybridized with two Alu-PCR products were further characterized. Physical mapping indicated that all of these clones are located in the expected region on Xq, confirming the validity of our approach.     

Mapping Genomic Library Clones Using Oligonucleotide Arrays

We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adapters. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density oligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection.     

Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.

To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs). In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5. The probe produces a unique fingerprint profile characteristic of each YAC clone. The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated. The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments. We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.     

Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

Background  

Comparison of data produced on different microarray platforms often shows surprising discordance. It is not clear whether this discrepancy is caused by noisy data or by improper probe matching between platforms. We investigated whether the significant level of inconsistency between results produced by alternative gene expression microarray platforms could be reduced by stringent sequence matching of microarray probes. We mapped the short oligo probes of the Affymetrix platform onto cDNA clones of the Stanford microarray platform. Affymetrix probes were reassigned to redefined probe sets if they mapped to the same cDNA clone sequence, regardless of the original manufacturer-defined grouping. The NCI-60 gene expression profiles produced by Affymetrix HuFL platform were recalculated using these redefined probe sets and compared to previously published cDNA measurements of the same panel of RNA samples.     

Multilocus DNA fingerprint analysis of cellbanks: Stability studies and culture identification in human B-lymphoblastoid and mammalian cell lines

The technique of multilocus DNA fingerprinting has great potential for the authentication of animal cell cultures and in identification of cross-contamination. The Alec Jeffreys probes 33.6 and 33.15 were used as multilocus probes to demonstrate the consistent DNA fingerprint profiles in human peripheral blood and its derivative Epstein-Barr virus (EBV) transformed B-lymphoblastoid cultures maintained by repeated subculture for six months. However, fingerprint analysis of EBV transformed cultures generated from small numbers of cells showed that the majority (seven of eight cultures) had anomalous profiles. Some of these altered profiles shared common features not seen in the peripheral blood pattern. Analysis of seven murine hybridoma clones from a single fusion experiment revealed only two clones which could not be distinguished using probe 33.15. Further studies of master and distribution cell banks for eleven cell lines demonstrated consistent fingerprint profiles in all cases except one (U937). However, this cell line showed only minor differences in the master and distribution bank profiles. These data indicate that, while changes in fingerprint profile may be identified in exceptional instances, the multilocus fingerprinting method using probes 33.6 and 33.15 is a powerful and reliable tool in the quality control of animal cell cultures.     

Clustering binary fingerprint vectors with missing values for DNA array data analysis.

Oligonucleotide fingerprinting is a powerful DNA array-based method to characterize cDNA and ribosomal RNA gene (rDNA) libraries and has many applications including gene expression profiling and DNA clone classification. We are especially interested in the latter application. A key step in the method is the cluster analysis of fingerprint data obtained from DNA array hybridization experiments. Most of the existing approaches to clustering use (normalized) real intensity values and thus do not treat positive and negative hybridization signals equally (positive signals are much more emphasized). In this paper, we consider a discrete approach. Fingerprint data are first normalized and binarized using control DNA clones. Because there may exist unresolved (or missing) values in this binarization process, we formulate the clustering of (binary) oligonucleotide fingerprints as a combinatorial optimization problem that attempts to identify clusters and resolve the missing values in the fingerprints simultaneously. We study the computational complexity of this clustering problem and a natural parameterized version and present an efficient greedy algorithm based on MINIMUM CLIQUE PARTITION on graphs. The algorithm takes advantage of some unique properties of the graphs considered here, which allow us to efficiently find the maximum cliques as well as some special maximal cliques. Our preliminary experimental results on simulated and real data demonstrate that the algorithm runs faster and performs better than some popular hierarchical and graph-based clustering methods. The results on real data from DNA clone classification also suggest that this discrete approach is more accurate than clustering methods based on real intensity values in terms of separating clones that have different characteristics with respect to the given oligonucleotide probes.     

On the maximal cliques in c-max-tolerance graphs and their application in clustering molecular sequences

Given a set S of n locally aligned sequences, it is a needed prerequisite to partition it into groups of very similar sequences to facilitate subsequent computations, such as the generation of a phylogenetic tree. This article introduces a new method of clustering which partitions S into subsets such that the overlap of each pair of sequences within a subset is at least a given percentage c of the lengths of the two sequences. We show that this problem can be reduced to finding all maximal cliques in a special kind of max-tolerance graph which we call a c-max-tolerance graph. Previously we have shown that finding all maximal cliques in general max-tolerance graphs can be done efficiently in O(n ³ + out). Here, using a new kind of sweep-line algorithm, we show that the restriction to c-max-tolerance graphs yields a better runtime of O(n ² log n + out). Furthermore, we present another algorithm which is much easier to implement, and though theoretically slower than the first one, is still running in polynomial time. We then experimentally analyze the number and structure of all maximal cliques in a c-max-tolerance graph, depending on the chosen c-value. We apply our simple algorithm to artificial and biological data and we show that this implementation is much faster than the well-known application Cliquer. By introducing a new heuristic that uses the set of all maximal cliques to partition S, we finally show that the computed partition gives a reasonable clustering for biological data sets.     

Rational design of landmark probes for quantitative DNA fiber mapping (QDFM)

Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs. We recently developed a procedure termed ‘quantitative DNA fiber mapping’ (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs. QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to ~2.3 kb/µm. In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping. Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs. As demonstrated in representative hybridizations, vector-specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences.     

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.     

Fine-mapping of an Arabidopsis cell death mutation locus

An Arabidopsis cell death mutation locus was mapped to chromosome 2 between lGS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.     

The gene for human erythrocyte protein 4.2 maps to chromosome 15q15.

Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5' and the 3' coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). Our results demonstrate that the locus of the P4.2 gene is located within 15q15.     

Evaluating oligonucleotide properties for DNA microarray probe design

Most current microarray oligonucleotide probe design strategies are based on probe design factors (PDFs), which include probe hybridization free energy (PHFE), probe minimum folding energy (PMFE), dimer score, hairpin score, homology score and complexity score. The impact of these PDFs on probe performance was evaluated using four sets of microarray comparative genome hybridization (aCGH) data, which included two array manufacturing methods and the genomes of two species. Since most of the hybridizing DNA is equimolar in CGH data, such data are ideal for testing the general hybridization properties of almost all candidate oligonucleotides. In all our data sets, PDFs related to probe secondary structure (PMFE, hairpin score and dimer score) are the most significant factors linearly correlated with probe hybridization intensities. PHFE, homology and complexity score are correlating significantly with probe specificities, but in a non-linear fashion. We developed a new PDF, pseudo probe binding energy (PPBE), by iteratively fitting dinucleotide positional weights and dinucleotide stacking energies until the average residue sum of squares for the model was minimized. PPBE showed a better correlation with probe sensitivity and a better specificity than all other PDFs, although training data are required to construct a PPBE model prior to designing new oligonucleotide probes. The physical properties that are measured by PPBE are as yet unknown but include a platform-dependent component. A practical way to use these PDFs for probe design is to set cutoff thresholds to filter out bad quality probes. Programs and correlation parameters from this study are freely available to facilitate the design of DNA microarray oligonucleotide probes.     

Fine-mapping of an Arabidopsis cell death mutation locus

An Arabidopsis cell death mutation locus was mapped to chromosome 2 between /GS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.