Multilocus markers for mouse genome analysis: PCR amplification based on single primers of arbitrary nucleotide sequence

Polymerase chain reaction (PCR) based on single primers of arbitrary nucleotide sequence provides a powerful marker system for genome analysis because each primer amplifies multiple products, and cloning, sequencing, and hybridization are not required. We have evaluated this typing system for the mouse by identifying optimal PCR conditions; characterizing effects of GC content, primer length, and multiplexed primers; demonstrating considerable variation among a panel of inbred strains; and establishing linkage for several products. Mg²⁺, primer, template, and annealing conditions were identified that optimized the number and resolution of amplified products. Primers with 40% GC content failed to amplify products readily, primers with 50% GC content resulted in reasonable amplification, and primers with 60% GC content gave the largest number of well-resolved products. Longer primers did not necessarily amplify more products than shorter primers of the same proportional GC content. Multiplexed primers yielded more products than either primer alone and usually revealed novel variants. A strain survey showed that most strains could be readily distinguished with a modest number of primers. Finally, linkage for seven products was established on five chromosomes. These characteristics establish single primer PCR as a powerful method for mouse genome analysis.     

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.

Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.     

Strain-specific fingerprints of Rhizobium galegae generated by PCR with arbitrary and repetitive primers

Strain-specific genomic patterns of Rhizobium galegae were generated by PCR using both arbitrary and repetitive (BOX, ERIC and REP) primers. The identification of the strains was achieved also by RFLP analysis. However, the PCR genomic fingerprinting has significant advantages: it is not only simpler and faster, but it is also much more discriminative because it deals with the full bacterial genome and not only with parts of it as is the case with RFLP. In addition, both kinds of PCR fingerprinting (using arbitrary or repetitive primers) generated highly specific and reproducible patterns when parallel reactions with total bacterial DNA, extracted from independent liquid cultures were performed. The latter shows that AP- and rep-PCR are convenient for controlling the production and application of Rhizobium inoculants.     

Using universal degenerate primers for restriction digestion mapping by PCR.

In this study, a polymerase chain reaction (PCR) is developed to determine the restriction map without using restriction endonucleases. A 937 bp fragment of pUC 19 which contained one cut site for EcoRI and two recognition sites for PvuII was used as a model. The PCR was carried out using designed degenerate primers and the products were analyzed on 1.5% agarose gel. The number of cut sites, length of fragments and the arrangement of the fragments from 3' or 5' end of desired sequence were determined.     

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region ofrrn operon ofFrankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS 1 region of the nuclearrrn operon ofAlnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.     

Genomic fingerprints produced by PCR with consensus tRNA gene primers.

The polymerase chain reaction using only a single 'consensus' tRNA gene primer, or a pair of primers facing outward from tRNA genes, amplifies a set of DNA fragments in bacterial, plant and animal genomic DNAs. Presumably, these PCR fingerprints are mainly derived from the regions between closely linked tRNA genes. The pattern of the PCR products is determined by which genomes and which primer(s) are used. Genomic fingerprints are largely conserved within a species and, in bacteria, most products in the fingerprint are conserved between closely related species. Thus, PCR with tRNA gene consensus primers helps to identify species and genera.     

Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping.

Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between strains of almost any organism. We applied the technique to the mouse (Mus musculus). The characteristic differences in the AP-PCR genomic fingerprints between strains will be of value in strain identification and verification. Using one primer, we genetically mapped four polymorphisms in a set of C57BL/6J x DBA/2J recombinant inbreds. One of these polymorphisms is a length variant. The method will allow rapid genetic mapping of DNA polymorphisms without Southern blotting.     

Genotype relationships inMicroseris elegans (Asteraceae,Lactuceae) revealed by DNA amplification from arbitrary primers (RAPDs)

The genetic relationships among 10 inbred lines representing 10 populations of the autogamous annualMicroseris elegans from throughout California has been determined using random amplified polymorphic DNAs (RAPDs). Seventeen arbitrary 10 base pair primers produced 134 amplification products; 81 of these were shared by two or more strains. The 3 genotypes from Northern California are closely related as are 3 genotypes from Middle Californian populations which are not nearest neighbors. DNA fingerprinting with the oligonucleotide (GATA)₄ gave compatible results, but the comparison was limited to samples run on one gel. Isoenzyme patterns are compatible with the DNA results, but limited by the very low number of informative polymorphisms. The clustered relationship among genotypes within a species and their geographic distribution suggests very restricted genetic recombination and an origin of new populations from randomly dispersed achenes within the range of the species.     

Photomorphogenesis in<Emphasis Type="Italic"> Phycomyces</Emphasis>: differential display of gene expression by PCR with arbitrary primers

The zygomycete fungus Phycomyces blakesleeanus develops two types of fruiting bodies of very different size, macrophores and microphores. Blue light stimulates macrophorogenesis and inhibits microphorogenesis. I have adapted a method based on the polymerase chain reaction with arbitrary primers to investigate the role of differential gene expression during photophorogenesis in Phycomyces. Several cDNAs for genes induced in vegetative mycelium have been observed, but only one gene induced by blue light has been detected. I have demonstrated the feasibility of this approach by the isolation of a cDNA segment for the heat-shock protein HSP100 that is induced by blue light at the onset of sporangiophore development. The heat-shock protein HSP100 is an ATP-binding protein that has the ability to disassemble protein complexes. In plants, the gene for HSP100 is induced by light. The cDNA segment for HSP100 obtained from Phycomyces is 686 bp long and the predicted amino acid sequence contains one of the ATP-binding sites.     

Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified with an ENA residue

When we placed an ENA residue into primers at the 3' end, or the n-1, n-2, or n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3' end, only primers containing the ENA residue at the n-2 position were read by Taq DNA polymerase for amplification. The use of the ENA primers avoided the generation of undesired short products, which are thought to be derived from primer-dimers. A greater discrimination of the SNP site by these primers containing the ENA residue was observed compared with that of the corresponding unmodified DNA primers that are often used for allele-specific polymerase chain reaction (AS-PCR). This improvement is probably due to the difficulty of incorporating a nucleotide into the mismatched ENA primer by Taq DNA polymerase in the modified primer-template duplex. These results demonstrate that ENA primer-based AS-PCR would enable a rapid and reliable technique for SNP genotyping.     

Rapid identification of Enterococcus italicus by PCR with primers targeted to 16S rRNA gene

AIMS: To develop a species-specific PCR assay with primers targeted to 16S rRNA gene for the identification of Enterococcus italicus, a new species of Enterococcus, involved in the production of Italian cheeses. METHODS AND RESULTS: The type strain of E. italicus (DSM 15952(T) - 16S rRNA gene accession no. AJ582753) and other strains of the species were subjected to a rapid identification by PCR using primer pairs located within the 16S rRNA gene. A species-specific PCR product of approximately 323 bp was obtained after amplification of all E. italicus strains tested. The specificity of the primers was validated with representatives of the most closely related genera and species and a number of other bacterial species. In addition, the technique enabled the recognition of E. italicus from cheeses. CONCLUSIONS: The protocol was highly efficient and sensitive, enabling the identification of E. italicus from cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific PCR offers a reliable and rapid alternative to conventional phenotypic methods for the identification of E. italicus within the heterogeneous genus Enterococcus.     

Designing and optimizing comparative anchor primers for comparative gene mapping and phylogenetic inference

Here we describe protocols for designing, optimizing and implementing conserved anchor primers for use in genome mapping or phylogenetic applications, with particular emphasis on homologous gene sequences among mammals. The increasing number of whole genome sequences in public databases makes this approach applicable across a wide range of taxa. Genome sequences from representatives of two or more divergent subclades within a taxonomic group of interest are used to identify candidate local alignments (i.e., exons, exons spanning introns or conserved 5'- or 3'-untranslated regions) that contain sequences with appropriate variability for the chosen downstream application. PCR primers are designed to maximize amplification success across a broad range of taxa, and are optimized under a touchdown thermocycling protocol. Based on the initial optimization results, primers are selected for application in a diverse sampling of species, or for mapping the genome of a target species of interest. We discuss factors that have to be considered for experimental design of broad-scope phylogenetic studies. With this protocol, primers can be designed, optimized and implemented within as little as 1-2 weeks.     

Significant differences between mouse and human trophinins are revealed by their expression patterns and targeted disruption of mouse trophinin gene.

Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21-22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.