Design factors that influence PCR amplification success of cross-species primers among 1147 mammalian primer pairs

BACKGROUND: Cross-species primers have been used with moderate success to address a variety of questions concerning genome structure, evolution, and gene function. However, the factors affecting their success have never been adequately addressed, particularly with respect to producing a consistent method to achieve high throughput. Using 1,147 mammalian cross-species primer pairs (1089 not previously reported), we tested several factors to determine their influence on the probability that a given target will amplify in a given species under a single amplification condition. These factors included: number of mismatches between the two species (the index species) used to identify conserved regions to which the primers were designed, GC-content of the gene and amplified region, CpG dinucleotides in the primer region, degree of encoded protein conservation, length of the primers, and the degree of evolutionary distance between the target species and the two index species. RESULTS: The amplification success rate for the cross-species primers was significantly influenced by the number of mismatches between the two index species (6–8% decrease per mismatch in a primer pair), the GC-content within the amplified region (for the dog, GC ≥ 50%, 56.9% amplified; GC<50%, 74.2% amplified), the degree of protein conservation (R(2 )= 0.14) and the relatedness of the target species to the index species. For the dog, 598 products of 930 primer pairs (64.3%) (excluding primers in which dog was an index species) were sequenced and shown to be the expected product, with an additional three percent producing the incorrect sequence. When hamster DNA was used with the single amplification condition in a microtiter plate-based format, 510 of 1087 primer pairs (46.9%) produced amplified products. The primer pairs are spaced at an average distance of 2.3 Mb in the human genome and may be used to produce up to several hundred thousand bp of species-specific sequence. CONCLUSION: The most important factors influencing the proportion of successful amplifications are the number of index species mismatches, GC-richness of the target amplimer, and the relatedness of the target species to the index species, at least under the single PCR condition used. The 1147 cross-species primer pairs can be used in a high throughput manner to generate data for studies on the genetics and genomics of non-sequenced mammalian genomes

Organization of the Mouse and Human Carbonic Anhydrase II Genes a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75491/1/j.1749-6632.1984.tb12355.x.pd

Gene-specific universal mammalian sequence-tagged sites: Application to the canine genome

We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail: CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB , and RB1 . We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as “anchored reference loci” for the development of mammalian genetic maps [O'Brien, S. J., et al., Nat. Genet. 3 :103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets “universal mammalian sequence-tagged sites” because they should be useful for many mammalian genome projects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44161/1/10528_2004_Article_BF00553904.pd

Comparison of the 5' regions of human and mouse carbonic anhydrase II genes and identification of possible regulatory elements

The nucleotide sequence of the 5' region of the human carbonic anhydrase II gene has been determined. This sequence begins 643 base pairs upstream from the ATG start site and continues through exon 1, intron 1, exon 2 and the adjoining 125 nucleotides of intron 2. The human sequence is compared with homologous regions of the mouse (YBR strain) carbonic anhydrase II gene by aligning the two sequences for optimal homology. In addition to a TATA box and a putative CCAAT box (CCACC in human and CCACT in mouse), three conserved tandem-repeat elements in mouse and two in human (consensus: cCNGTCACCTCCgC) are located 15 and 22 base pairs upstream, respectively, from the CCAAT boxes in the human and mouse sequences. This repeat element is similar to a tandem repeat sequence located at about the same position in mammalian [beta]-globin genes, and may represent regulatory elements common to both the carbonic anhydrase and [beta]-globin genes. The regions surrounding exon 1 are extremely G + C-rich in both human and mouse genes. In addition, severel CCGCCC or GGGCGG sequences which may be important for transcriptional efficiency are found in the 5' flanking regions of the human and mouse genes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25466/1/0000004.pd

The deduced amino acid sequence of human carbonic anhydrase-related protein (CARP) is 98% identical to the mouse homologue

A recently reported mRNA, encoding `carbonic anhydrase-related polypeptide' (CARP) from the Purkinje cells of mouse cerebellum, was shown to have a 30-40% deduced amino acid sequence identity with the carbonic anhydrases (CA) of mammals. In order to compare the mouse and human CARP sequences, we used the polymerase chain reaction (PCR) to amplify human CARP sequences from several cDNA libraries (salivary gland, testis and placenta). The sequence has an 89.3% sequence identity with mouse CARP at the nucleotide level and 97.9% at the amino acid level. This extremely high evolutionary conservation suggests an important function for the CARP gene product.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30832/1/0000494.pd

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products

Two, electrophoretically distinct, forms of carbonic anhydrase I (CA Ia and CA Ib) are found at high polymorphic frequencies in red cells of natural populations of pigtail macaques, Macaca nemestrina , from southeast Asia. By use of the polymerase chain reaction, exons of the CA I gene were amplified from homozygous ( a/a, b/b ) and heterozygous ( a/b ) animals. Direct sequencing of the amplified DNA from four animals revealed differences between the a and the b electrophoretic alleles ranging from three to six nucleotides, and from one to three differences within each allele. These results indicate a greater genetic variability at the CA I locus in this macaque species than previously realized.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44158/1/10528_2004_Article_BF00553755.pd

Characterization of the genes encoding carbonic anhydrase I of chimpanzee and gorilla: comparative analysis of 5' flanking erythroid-specific promoter sequences

The genes encoding carbonic anhydrase I (CA I) have been characterized for chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla). In addition, 44 nucleotides (nt) at the 5' end of the noncoding first exon (exon la), which is unique to the erythroid CA I mRNA, together with 188 nt of the adjacent 5' flanking regions, were sequenced for the corresponding positions of the CA I of orangutan, pigtail macaque, and squirrel monkey. When these 5' flanking regions are compared, along with those published for human and mouse CA I, they were found to contain several conserved sequences that may bind factors involved in the erythroid-specific expression of CA I. Comparisons of the human, chimpanzee, and gorilla coding and noncoding CA I sequences do not significantly deviate from a pattern of trichotomy for the evolutionary origins of these three hominoid species.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30572/1/0000207.pd

Carbonic anhydrase II is induced in HL-60 cells by 1,25-dihydroxyvitamin D3: A model for osteoclast gene regulation

Carbonic anhydrase II (CA II) generates the H+ required for osteoclast-mediated bone resorption in humans. We have developed the human promyelocytic cell line HL-60 as a model system with which to study the osteoclast-specific expression of the CA II gene. Treatment of the cell line with 1,25-dihydroxyvitamin D3 resulted in a dramatic de novo induction of CA II at both the protein and mRNA levels. CA II mRNA was also induced to a lesser extent by 12-O-tetradecanoyl phorbol 13-acetate. Treatment with dimethyl sulfoxide did not increase CA II mRNA. These findings indicate that the HL-60 cell line will be a useful model system to study the osteoclast-specific expression of the CA II gene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27889/1/0000303.pd

Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry as a Rapid Screening Method to Detect Mutations Causing Tay–Sachs Disease

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay–Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster. © 1997 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35066/1/981_ftp.pd

Molecular Characterization of Three Canine Models of Human Rare Bone Diseases: Caffey, van den Ende-Gupta, and Raine Syndromes

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