Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries

We present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Our primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, we adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called "contigs." These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.     

Investigation of pseudoautosomal and bordering regions in avian Z and W chromosomes with the use of large insert genomic BAC clones

To study pseudoautosomal and bordering regions in the avian Z and W chromosomes, we used seven BAC clones from genomic libraries as DNA probes of fragments of different gametologs of the ATP5A1 gene located close to the proximal border of the pseudoautosomal region (PAR) of sex chromosomes of domestic chicken and Japanese quail. Localization of BAC clones TAM31-b100C09, TAM31-b99N01, TAM31-b27P16, and TAM31-b95L18 in the short arm of Z chromosomes of domestic chicken and Japanese quail (region Zp23-p22) and localization of the BAC clones CHORI-261-CH46G16, CHORI-261-CH33F10, and CHORI-261-CH64F22 on W chromosomes of these species and in the short arm of Z chromosomes (region Zp23-p22) were determined by fluorescence in situ hybridization with the use of W-specific probes. The difference in the localization of the BAC clones on the Z and W chromosomes is probably explained by divergence of the nucleotide sequences of different sex chromosomes located beyond the pseudoautosomal region.     

Five-color-based high-information-content fingerprinting of bacterial artificial chromosome clones using type IIS restriction endonucleases

We have developed a high-information-content fingerprinting (HICF) system for bacterial artificial chromosome (BAC) clones using a Type IIS restriction endonuclease, HgaI, paired with a Type II restriction endonuclease, RsaI. In the method described, unknown five-base overhangs generated with HgaI are partially or fully sequenced by modified fluorescent dideoxy terminators. Using an in-lane size standard labeled with a fifth dye, fragments are characterized by both the size and the sequence of its terminal one to five bases. The enhanced information content associated with this approach significantly increases the accuracy and efficiency of detecting shared fragments among BAC clones. We have compared data obtained from this method to predicted HICF patterns of 10 fully sequenced BACs. We have further applied HICF to 555 BAC clones to assemble contigs spanning 16p11.2 to 16p13.1 of human chromosome 16.     

Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells

We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication. This element provides nuclear retention in cells expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography of expanding colonies. A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days. Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.     

One step cloning of defined DNA fragments from large genomic clones

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.     

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.     

In silico simulation of fingerprinting techniques based on double endonuclease digestion of genomic DNA

We have developed an online generic tool for simulation of fingerprinting techniques based on the double endonuclease digestion of DNA. This tool allows modelling and modifications of already existing techniques, as well as new theoretical approaches not yet tried in the lab. It allows the use of any combination of recognition patterns and discrimination of end types yielded by restriction with non palindromic recognition sizes. Re-creation of experimental conditions in silico saves time and reduces laboratory costs. This tool allows simulation of Amplified Fragment Length Polymorphism (AFLP-PCR), Subtracted Restriction Fingerprinting (SRF), and additional novel fingerprinting techniques. Simulation may be performed against custom sequences uploaded to the server, or against all sequenced bacterial genomes. Different endonuclease types may be selected from a list, or a recognition sequence may be introduced in the form. After double digestion of DNA, four fragment types are yielded, and the program allows their customised selection. Selective nucleotides may be used in the experiment. Scripts for specific simulation of AFLP-PCR and SRF techniques are available, and both include a suggestion tool for the selection of endonucleases. This is the first program available for the simulation of SRF fingerprinting. Availability: This free online tool is available at http://www.in-silico.com/DDF/.     

Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones

By determining spatial-temporal expression patterns, reporter constructs provide significant insights into gene function. Although additionally providing information on subcellular distribution, translational reporters, where the reporter is fused to the gene coding sequence, are used less frequently than simpler constructs containing only putative promoter sequences. Because these latter constructs may not contain all necessary regulatory elements, resulting expression patterns must be interpreted cautiously. To ensure inclusion of all such elements and provide details of subcellular localization, construction of translational reporters would, preferably, utilize genomic clones, containing the complete locus plus flanking regions and permit seamless insertion of the reporter anywhere within the gene. We have developed such a method based upon λ Red-mediated recombineering coupled to a robust two-step counter-selection protocol. We have inserted either gfp or cfp precisely at the C-termini of three Caenorhabditis elegans target genes, each located within different fosmid clones, and examined previously with conventional reporter approaches. Resulting transgenic lines revealed reporter expression consistent with previously published data for the tagged genes and also provided additional information including subcellular distributions. This simple and straightforward method generates reporters highly likely to recapitulate endogenous gene expression and thus represents an important addition to the functional genomics toolbox.     

Genomic mapping by fingerprinting random clones: a mathematical analysis

Results from physical mapping projects have recently been reported for the genomes of Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans, and similar projects are currently being planned for other organisms. In such projects, the physical map is assembled by first "fingerprinting" a large number of clones chosen at random from a recombinant library and then inferring overlaps between clones with sufficiently similar fingerprints. Although the basic approach is the same, there are many possible choices for the fingerprint used to characterize the clones and the rules for declaring overlap. In this paper, we derive simple formulas showing how the progress of a physical mapping project is affected by the nature of the fingerprinting scheme. Using these formulas, we discuss the analytic considerations involved in selecting an appropriate fingerprinting scheme for a particular project.     

REF Select: expert system software for selecting restriction endonucleases for restriction endonuclease fingerprinting

REF Select, expert system software, has been developed to assist in the selection of optimal restriction endonucleases for restriction endonuclease fingerprinting (REF), a method for rapid and sensitive mutation screening of long DNA segments (1-2 kb). The REF method typically involves six separate digestions with up to two restriction endnonucleases used in each digestion. If done manually, performing a comprehensive review of the large number of possible sets of restriction endonucleases that could be used (over 10(19) in the example presented here) and making an optimal choice is not feasible. Furthermore, the typical nonoptimal manual selection takes approximately 8 h by someone experienced with REF. REF Select enables a comprehensive review of the possible sets and a consistent, objective and fast selection of an optimal set by using a two-step strategy: the selection of sets that meet specific constraints, which is followed by a ranking of those sets by an optimality score. Based on our experience with REF, we chose default selection and ranking parameters to help the user get started quickly. These parameters form a knowledge base that can be customized and then saved by the user. In conclusion, REF Select facilitates the general application of REF by serving as an expert system for the selection of optimal restriction endonucleases. We demonstrated REF Select using an example segment from the human p53 gene.     

Application of the restriction landmark genomic scanning (RLGS) method to rice cultivars as a new fingerprinting technique

The restriction landmark genomic scanning (RLGS) method was applied to rice, using two Japanese cultivars, Nipponbare and Koshihikari, and a Chinese landrace, LiuZhou'Bao'Ya'Zao'. More than 3000 landmarks were detected as spots on the individual autoradiograms of each cultivar. Nipponbare and LiuZhou' Bao'Ya'Zao' showed apparently different RLGS profiles, from which the genetic similarity (GS) between them was estimated as 0.344. Although the two Japanese cultivars, Nipponbare and Koshihikari showed quite similar RLGS profiles, they were easily distinguished on the basis of the presence or absence of specific spots; the GS value between them was calculated as 0.980. The RLGS method is shown to be a powerful fingerprinting technique, especially for the classification and identification of cultivars in rice and probably in other crops as well.     

Nonisotope variant of genomic fingerprinting based on the phage M13 DNA in kinship studies in man

DNA "fingerprinting" with the 32P-labeled or biotinylated M13 phage DNA was used in man parentage studies. It was shown that the non-isotopic method, having sensitivity, similar to that of isotopic one, gave higher level of band resolution. The method is safe and requires 3-5 h to visualize the bands.     

The genomic fingerprinting of the causative agents of sapronoses

The genome polymorphism of the causative agents of sapronoses (Vibrio cholerae, Legionella and Leptospira) has been studied. The use of the method of genome fingerprinting [correction of dactyloscopy] has been shown to permit the differentiation of closely related strains of such causative agents. The epidemically significant strains of the causative agents of sapronoses, isolated in different geographical regions, have been found to be genotypically related, i.e., they are probably of clonal origin. Avirulent and nontoxigenic strains are genotypically heterogeneous and differ both from one another and from epidemically significant strains. Using V. cholerae as an example, the hypothesis of the appearance of potentially dangerous variants at the epidemic period in the absence of their release at the period between epidemics is considered.     

Retrotransposon characterisation and fingerprinting of apple clones by S-SAP markers

Retrotransposons have been found to comprise the most common class of transposable elements in eukaryotes and to occur in high copy number in plant genomes. Several of these elements have been sequenced and were found to display a high degree of heterogeneity and insertional polymorphism, both within and between species. The dispersion, ubiquity and prevalence of retrotransposons in plant genomes provide an excellent basis for the development of marker systems and, hence, may be good molecular candidates in distinguishing among apple clones, when they represent bud mutations of the original variety, considering that the random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) used thus far in fingerprinting analyses have failed to meet discrimination expectations. The technique called sequence-specific amplified polymorphism (S-SAP), which makes it possible to identify dominant markers for the detection of variation in the DNA flanking the retrotransposon insertion site, was used in the present study to distinguish several clones of the cultivars ‘Gala’ and ‘Braeburn’ in apple fingerprinting. Moreover, our results suggest that the bud mutations, which have generated new patented varieties of ‘Gala’ and ‘Braeburn’, appear to derive from retrotransposon insertion.     

Generation of large insert yeast artificial chromosome libraries

The development of YAC cloning technology has directly enhanced the relationship among genetic, physical, and functional mapping of genomes. Because of their large size, YACs have enabled the rapid construction of physical maps by ordered clone mapping and contig building, and they complement other molecular approaches for mapping complex genomes. Large insert libraries are constructted by size fractionating large DNA embedded in agarose and protecting DNA from degradation with polyamines.