Showing papers on "genomic DNA published in 2003"

Collection, Mapping, and Annotation of Over 28,000 cDNA Clones from japonica Rice

Abstract: We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Unbiased Whole-Genome Amplification Directly From Clinical Samples

Abstract: Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods.

Increased plasma DNA integrity in cancer patients

Abstract: Tumor-released DNA in blood represents a promising biomarker for cancer detection. It has been postulated that tumor necrosis causes release of DNA of varying sizes, which contrasts apoptosis in normal tissue that releases smaller and more uniform DNA fragments. To test the hypothesis that increased DNA integrity, i.e., a longer DNA strand, is a tumor-associated marker in plasma, we determined the genomic DNA integrity index in plasma DNA using real-time PCR assays. A DNA integrity index and DNA concentration in plasma were determined in 61 patients with gynecological and breast cancers and 65 female patients without neoplastic diseases. We found that the area under the receiver-operating characteristic curve for DNA integrity index was 0.911 for cancer versus nonneoplastic patients. Given 100% specificity, the highest sensitivity achieved in detecting the cancer group was 62% (95% confidence interval = 0.50-0.74) at the index cutoff of 0.59. Fifty percent of stage I cancers had a DNA integrity index above this cutoff. All 11 patients with benign adnexal masses that clinically can be confused with malignant gynecological neoplasms demonstrated DNA integrity index < 0.59. Our findings suggest that increased DNA integrity in plasma DNA is associated with cancer, and measurement of DNA integrity may provide a simple and inexpensive measure for cancer detection.

Positional cloning of a novel gene influencing asthma from Chromosome 2q14

Abstract: Asthma is a common disease in children and young adults. Four separate reports have linked asthma and related phenotypes to an ill-defined interval between 2q14 and 2q32 (refs. 1-4), and two mouse genome screens have linked bronchial hyper-responsiveness to the region homologous to 2q14 (refs. 5,6). We found and replicated association between asthma and the D2S308 microsatellite, 800 kb distal to the IL1 cluster on 2q14. We sequenced the surrounding region and constructed a comprehensive, high-density, single-nucleotide polymorphism (SNP) linkage disequilibrium (LD) map. SNP association was limited to the initial exons of a solitary gene of 3.6 kb (DPP10), which extends over 1 Mb of genomic DNA. DPP10 encodes a homolog of dipeptidyl peptidases (DPPs) that cleave terminal dipeptides from cytokines and chemokines, and it presents a potential new target for asthma therapy.

An inexpensive and high-throughput procedure to extract and purify total genomic DNA for population studies

Abstract: We describe here a procedure for the purification of high molecular weight genomic DNA that combines the economies of 'do-it-yourself', single-tube protocols with the sample throughput and DNA quality of microplate-based DNA extraction and purification kits from commercial suppliers. The procedure allows the preparation of genomic DNA of a quality suitable for polymerase chain reaction-based studies of large populations at around one-tenth of the cost of commercially available kits. Furthermore, 96 samples can be purified from crude tissue digests in around 30 min and are produced in microtitre plate format to allow efficient downstream processing of samples.

Prospecting for novel biocatalysts in a soil metagenome.

Abstract: The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an α-amylase (amyA), a 1,4-α-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

Genomic Organization and Expression Analysis of B7-H4, an Immune Inhibitory Molecule of the B7 Family

Abstract: B7-H4 is a recently identified B7 family member that negatively regulates T cell immunity by the inhibition of T cell proliferation, cytokine production, and cell cycle progression. In this study, we report that the genomic DNA of human B7-H4 is mapped on chromosome 1 comprised of six exons and five introns spanning 66 kb, of which exon 6 is used for alternative splicing to generate two different transcripts. Similar B7-H4 structure is also found in mouse genomic DNA in chromosome 3. A human B7-H4 pseudogene is identified in chromosome 20p11.1 with a single exon and two stop codons in the coding region. Immunohistochemistry analysis using B7-H4-specific mAb demonstrates that B7-H4 is not expressed on the majority of normal human tissues. In contrast, up to 85% (22 of 26) of ovarian cancer and 31% (5 of 16) of lung cancer tissues constitutively express B7-H4. Our results indicate a tight regulation of B7-H4 expression in the translational level in normal peripheral tissues and a potential role of B7-H4 in the evasion of tumor immunity.

Identification of SATB2 as the cleft palate gene on 2q32-q33

Abstract: Cytogenetic evidence, in the form of deletions and balanced translocations, points to the existence of a locus on 2q32-q33, for which haploinsufficiency results in isolated cleft palate (CPO). Here we show by high-resolution FISH mapping of two de novo CPO-associated translocations involving 2q32-q33 that one breakpoint interrupts the transcription unit of the gene encoding the DNA-binding protein SATB2 (formerly KIAA1034). The breakpoint in the other translocation is located 130 kb 3' to the SATB2 polyadenylation signal, within a conserved region of non-coding DNA. The SATB2 gene is transcribed in a telomeric to centromeric direction and lies in a gene-poor region of 2q32-q33; the nearest confirmed gene is 1.26 Mb centromeric to the SATB2 polyadenylation signal. SATB2-encoding transcripts are assembled from 11 exons that span 191 kb of genomic DNA. They encode a protein of 733 amino acids that has two CUT domains and a homeodomain and shows a remarkable degree of evolutionary conservation, with only three amino acid substitutions between mouse and human. This protein belongs to the same family as SATB1, a nuclear matrix-attachment region binding protein implicated in transcriptional control and control of chromatin remodelling. There are also sequence similarities to the Drosophila protein DVE. Whole mount in situ hybridization to mouse embryos shows site- and stage-specific expression of SATB2 in the developing palate. Despite the strong evidence supporting an important role for SATB2 in palate development, mutation analysis of 70 unrelated patients with CPO did not reveal any coding region variants.

Structure and morphogenesis of bacteriophage T4.

Abstract: Bacteriophage T4 is one of the most complex viruses. More than 40 different proteins form the mature virion, which consists of a protein shell encapsidating a 172-kbp double-stranded genomic DNA, a ‘tail,’ and fibers, attached to the distal end of the tail. The fibers and the tail carry the host cell recognition sensors and are required for attachment of the phage to the cell surface. The tail also serves as a channel for delivery of the phage DNA from the head into the host cell cytoplasm. The tail is attached to the unique ‘portal’ vertex of the head through which the phage DNA is packaged during head assembly. Similar to other phages, and also herpes viruses, the unique vertex is occupied by a dodecameric portal protein, which is involved in DNA packaging.

A method to assess genomic dna methylation using high-performance liquid chromatography-electospray ionization mass spectrometry

Abstract: The present invention provides a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The method comprises the steps of enzymatically hydrolyzing the DNA sample by sequential digestion with enzymes; separating the DNA hydrolyzates by reverse-phase high-performance liquid chromatography in isocratic mode wherein the four major DNA bases and 5-methyl-2'-deoxycytidine are resolved and eluted; identifying the 2'-deoxycytidine and 5-methyl-2'-deoxycytidine by combining diode array UV spectra analysis and mass spectra of chromatographic peaks. The isotopomes 15N3 2'-deoxycytidine and methyl-D3, ring-6-D1 5-methyl-2'-deoxycytidine are used as internal standards. Ions of m/z 126 and 130 are used to detect 5-methyl-2'-deoxycytidine and its isotopomer, and ions of m/z 112 and 115 are used to detect 2'-deoxycytidine and its stable isotopomer, respectively. The DNA methylation status is consequently calculated based on the amount of 5-methyl-2'-deoxycytidine per µg DNA with percent relative standard deviations (%RSD) for method precision of 7.1 (within-day) and 5.7 (day-to-day). The method of the present invention also allows the measurement of 5-methyl-2'-deoxycytidine expressed as a percentage of total deoxycytidine residues in genomic DNA with % RSD for method precision of 1.9 (within-day) and 1.7 (day-to-day). The LC/MS method for quantitative determination of genomic DNA methylation status is rapid, sensitive, selective and precise.

Rapid Direct Sequence Analysis of the Dystrophin Gene

Abstract: Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophy (DMD and BMD), as well as X-linked dilated cardiomyopathy. Mutational analysis is complicated by the large size of the gene, which consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. Deletions of one or more exons account for 55%–65% of cases of DMD and BMD, and a multiplex polymerase chain reaction method—currently the most widely available method of mutational analysis—detects ∼98% of deletions. Detection of point mutations and small subexonic rearrangements has remained challenging. We report the development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion. This same method, termed “SCAIP” (single condition amplification/internal primer) sequencing, is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.

Continuous segmented-flow polymerase chain reaction for high-throughput miniaturized DNA amplification.

Abstract: A continuous segmented-flow method for sequential DNA amplification is described in order to provide a basis for high-throughput genetic analysis. The approach allows an immediate distinction between amplified and nonamplified products. A mixture of sample and reagents are loaded in the form of small segments one after another in a 15-m-long narrow-bore Teflon tube, coiled such as to be repeatedly exposed to three different temperature zones. After having passed the heated zones, the samples are mixed with an intercalating dye by flow injection and sequentially detected on-line by laser-induced fluorescence. The aqueous samples travel as separate segments in a continuous flow of an immiscible, organic liquid. Perfluorodecalin was shown to be particularly suitable due to its hydrophobicity and inert properties. To reduce carryover between samples, an intermediate water plug between two consecutive samples was required. Selected regions from human genomic DNA were successfully amplified in 300-nL volumes af...

Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

Abstract: Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome.

Direct molecular haplotyping of long-range genomic DNA with M1-PCR

Abstract: Haplotypes, combinations of several phase-determined polymorphic markers, are extremely valuable for studies of disease association and chromosome evolution. Here we describe a technique called M1-PCR (M for ``multiplex'' and 1 for ``single-copy DNA molecules'') that enables direct molecular haplotyping of several polymorphic markers separated by as many as 24 kb. A genomic DNA sample first is diluted to approximately single-copy. The haplotype is directly determined by simultaneously genotyping several polymorphic markers in the same reaction with a multiplex PCR and base extension reaction. This approach does not rely on pedigree data and does not require previous amplification of the entire genomic region containing the selected markers.

Approaching real-time molecular diagnostics: Single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes

Abstract: The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and were ligated using a thermostable ligase enzyme only when the genomic DNA carried this mutation. The allele-specific primers also carried complementary stem structures with end-labels (donor/acceptor fluorescent dyes, Cy5/Cy5.5, respectively), which formed a molecular beacon following ligation. We coupled ligase detection reaction (LDR) with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the molecular beacon probes formed upon ligation. LDR-spFRET provided the necessary specificity and sensitivity to detect single-point mutations in as little as 600 copies of human genomic DNA directly without PCR at a level of 1 mutant per 1000 wild type sequences using 20 LDR thermal cycles. We also demonstrate the ability to rapidly discriminate single base differences in the K-ras gene in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA (600 copies). Real-time LDR-spFRET detection of point mutations in the K-ras gene was accomplished in PMMA microfluidic devices using sheath flows.

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Abstract: Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.

Development and validation of a T7 based linear amplification for genomic DNA

Abstract: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail. We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach. We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

Parallel gene analysis with allele-specific padlock probes and tag microarrays.

Abstract: Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.

Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries.

Abstract: A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.

Capillary electrophoretic analysis of genomic DNA methylation levels

Abstract: Changes in DNA methylation have been found in the large majority of tumors. This phenomenon includes both genome-wide hypomethylation and gene- specific hypermethylation. However, the clinical relevance of either mechanism has remained contentious. In order to determine DNA methylation levels from a large number of clinical samples, we have established a method for accurate high-throughput quantification of 5-methylcytosine in genomic DNA. Our protocol requires a small amount (<1 micro g) of DNA that is enzymatically hydrolyzed to single nucleotides. Single nucleotides are then derivatized with a fluorescent marker and separated by capillary electrophoresis. After calibration of the method, we have determined cytosine methylation levels from tumor samples of 81 patients that had been diagnosed with chronic lymphocytic leukemia (CLL). These patients showed a high variability in their methylation levels with a general trend towards hypomethylation. Because of its high accuracy and throughput our method will be useful in determining the role of genomic DNA methylation levels in tumorigenesis.

Metagenomic profiling: microarray analysis of an environmental genomic library.

Abstract: Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ∼1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.

A Novel Class of SINE Elements Derived from 5S rRNA

Abstract: Eukaryotic genomes are colonized by different retroposons, including short interspersed repetitive elements (SINEs). All currently known SINEs are derived from tRNA and 7SL RNA genes and exploit their type 2 internal pol III promoters. We report here a novel class of SINE elements, called SINE3, derived from 5S rRNA. SINE3s are transcribed from the type 1 internal pol III promoter. Approximately 10,000 copies of SINE3 elements are present in the zebrafish genome, they constitute approximately 0.4% of the genomic DNA. Some elements are as little as 1% diverged from each other, indicating that the retrotransposition of SINE3 in zebrafish is an ongoing process. The 3'-tail of SINE3 is significantly similar to that of CR1-like non-LTR retrotransposons, represented by numerous subfamilies in the zebrafish genome. Analogously to CR1-like elements, SINE3 copies are not flanked by target site duplications, and their 3' termini are composed of (ACATT)n and (ATT)n microsatellites, specific for different subfamilies of SINE3. Given the common structural features, it is highly likely that the enzymatic machinery encoded by CR1-like elements powers proliferation of SINE3.

Mixed-Genome Microarrays Reveal Multiple Serotype and Lineage-Specific Differences among Strains of Listeria monocytogenes

Abstract: Epidemiological studies and analysis of putative virulence genes have shown that Listeria monocytogenes has diverged into several phylogenetic divisions. We hypothesize that similar divergence has occurred for many genes that influence niche-specific fitness and virulence and that identifying these differences may offer new opportunities for the detection, treatment, and control of this important pathogen. To explore this issue further, we developed a microarray composed of fragmented DNA taken from 10 strains of L. monocytogenes. We then hybridized genomic DNA from 50 different strains to replicate arrays and analyzed the resulting hybridization patterns. A simple Euclidean distance metric permitted the reconstruction of previously described genetic relationships between serotypes, and only four microarray probes were needed to discriminate between the most important serotypes (1/2a, 1/2b, 1/2c, and 4). We calculated an index of linkage equilibrium from the microarray data and confirmed that L. monocytogenes has a strongly clonal population structure (IA = 3.85). Twenty-nine informative probes were retrieved from the library and sequenced. These included genes associated with repairing UV-damaged DNA, salt tolerance, biofilm formation, heavy metal transport, ferrous iron transport, and teichoic acid synthesis. Several membrane-bound lipoproteins and one internalin were identified, plus three phage sequences and six sequences with unknown function. Collectively, these data confirm that many genes have diverged between lineages of L. monocytogenes. Furthermore, these results demonstrate the value of mixed-genome microarrays as a tool for deriving biologically useful information and for identifying and screening genetic markers for clinically important microbes.