A successful strategy for comparative mapping with human ESTs: 65 new regional assignments in the pig

Large-scale sequencing of cDNAs from numerous tissues is currently being performed within the framework of the Human Genome Project. These expressed sequence tags (ESTs) are then mapped on a radiation hybrid panel to produce a high-resolution map of human genes. In this report, we estimate the efficiency of mapping these ESTs in the pig. A total of 344 human ESTs from Généthon were selected for amplification in other species by Zoo-PCR: 186 of these could be reproducibly amplified by use of pig DNA and the corresponding human primer pairs. One-hundred seven of these were tested on a porcine–rodent somatic cell hybrid panel, permitting regional localizations of 65 ESTs with agarose or single-strand conformation polymorphism analysis gels. The corresponding pig PCR products were sequenced: 60 ESTs matched significantly with the expected human sequences. Fifty-one of these localizations in the pig are in agreement with the comparative mapping data between humans and pigs based on heterologous chromosome painting. Seven ESTs that were localized in an unexpected region may indicate new chromosomal correspondences. This work significantly increases the number of genes mapped on the pig genome and demonstrates that this approach can be successfully applied to improve the gene density of mammalian genomic maps in chromosomal regions of interest, such as those in which QTL (Quantative Trait Loci) have been identified. Received: 31 July 1998 / Accepted: 14 October 1998     

Mapping of 22 expressed sequence tags isolated from a porcine small intestine cDNA library

Complementary DNA sequences were selected from a resource of tentatively identified clones from a porcine small intestine cDNA library. Forty PCR primer pairs were designed to amplify 101–309 base pairs of the 3′ untranslated region of the genes. The PCR conditions were optimized by altering both formamide and magnesium concentrations on samples of pig, mouse, and hamster DNA. Twenty primer pairs that, under stringent conditions, were pig-specific and amplified the expected fragments were chosen for regional assignment in a pig/rodent hybrid cell panel. Furthermore, 22 primer pairs were chosen to amplify DNA from the parental animals of the PiGMaP shared reference families in order to detect possible polymorphisms. Primer pairs that generated polymorphisms were used for genetic mapping. A total of 22 porcine expressed sequence tags (ESTs) were cytogenetically or genetically mapped by this approach. Twelve of the mapped ESTs could be added to the human–porcine comparative map. Received: 8 December 1996 / Accepted: 31 January 1997     

Amplification and detection of polymorphic sequence-tagged sites in<Emphasis Type="Italic">Lathyrus sativus</Emphasis>

A simple procedure was developed to convertLathyrus sativus defence-related expressed sequence tags (ESTs) into mappable genetic markers by using PCR. Twenty-nine STS primer pairs were generated on the basis of sequence information from anL. sativus cDNA library. These primers were used to screen for polymorphisms between 2L. sativus accessions, ATC 80878 and ATC 80407, resistant and susceptible, respectively, toMycosphaerella pinodes infection. All 29 primer pairs amplified PCR products in both accessions, 11 of which amplified multiple RAPD-like products. The remaining 18 primer pairs amplified single monomorphic products. Following cloning, sequencing, and database searches, 17 of 18 PCR products were confirmed to have amplified the targeted genome region. Ten of these 17 STS primer pairs revealed polymorphisms between ATC 80878 and ATC 80407 when PCR products were digested with a range of restriction endonucleases. These results suggest that the STS-based PCR analysis will be useful for generating informative molecular markers inL. sativus for future genome mapping experiments.     

Optimization of RAPD‐PCR conditions in cattle

Abstract

Random Amplified Polymorphic DNA polymerase chain reaction (RAPD‐PCR) is a fast and easy way of identifying DNA polymorphisms generated from several regions of the genome. This could expedite the process of identifying informative polymorphic markers that may be linked to important genes controlling economic traits. In cattle, failure to obtain consistent amplification patterns in RAPD‐PCR has been a cause for concern. This has been attributed to the fact that decamer primers that are used in RAPD‐PCR reactions are likely to amplify regions of DNA where the primer‐template base pairing has some degree of mismatch and that these mismatches fail to repeat from reaction to reaction. This paper describes the use of tricine buffer along with changes in reaction components and thermal cycling conditions that has yielded consistent and reproducible RAPD‐PCR amplifications using single primers and double primer combinations on bovine DNA.     

Mapping of 1400 expressed sequence tags in the bovine genome using a somatic cell hybrid panel

A bovine/hamster hybrid cell panel consisting of 30 independent hybrids was developed to locate genes. Polymerase chain reaction analysis of 279 microsatellites on the cattle linkage map in this panel revealed the presence of all chromosomes in either entire or fragmented form. Among primer pairs prepared from bovine 3'-expressed sequence tags (ESTs), 1400 ESTs were assigned to specific chromosomes, of which 1303 were newly assigned in this study, and mapped 854 (61%) to 1 of 192 chromosomal segments using this panel. The regional mapping of new genes to cattle chromosomes can be rapidly achieved using this panel.     

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.     

Traced orthologous amplified sequence tags (TOASTs) and mammalian comparative maps

Progress on mammalian comparative maps could be significantly accelerated by developing reagents defining orthologous landmarks in the genome of many mammalian species. Using the large databases of gene sequences, we designed 225 orthologous gene-specific primer pairs corresponding to 146 functional genes. Of these 225 primer pairs, 155 (68.9%), 182 (80.9%), 126 (56.0%), and 82 (36.4%) produced a single PCR product when tested against human, pig, dog, and hamster genomic DNA, respectively. In addition to the general rules of primer designing, particular factors must be taken into consideration when choosing gene-specific universal primers—for instance, preference for single-exon regions or highly conserved segments among species, avoidance of GC-rich regions. Sequencing all the canine PCR products traced by these primers demonstrated that of 123 traced canine fragments with readable and reliable sequences, 121 (98.4%) were found to match the GenBank orthologous sequences used for designing the primers, after a BLAST search. Comparative characterization of PCR fragments among human, pig, dog, and hamster revealed that the length of a single exon was much conserved among species, with few exceptions. As the fragments were traced with amplification by orthologous gene-specific primers, we suggest they be termed Traced Orthologous Amplified Sequence Tags (TOASTs). Received: 22 December 1997 / Accepted: 16 March 1998     

The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species

The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.     

Mining of expressed sequence tag libraries of cacao for microsatellite markers using five computational tools

Expressed sequence tags (ESTs) provide researchers with a quick and inexpensive route for discovering new genes, data on gene expression and regulation, and also provide genic markers that help in constructing genome maps. Cacao is an important perennial crop of humid tropics. Cacao EST sequences, as available in the public domain, were downloaded and made into contigs. Microsatellites were located in these ESTs and contigs using five softwares (MISA, TRA, TROLL, SSRIT and SSR primer). MISA gave maximum coverage of SSRs in cacao ESTs and contigs, although TRA was able to detect higher order (>5-mer) repeats. The frequency of SSRs was one per 26.9 kb in the known set of ESTs. One-third of the repeats in EST-contigs were found to be trimeric. A few rare repeats like 21-mer repeat were also located. A/T repeats were most abundant among the mononucleotide repeats and the AG/GA/TC/CT type was the most frequent among dimerics. Flanking primers were designed using Primer3 program and verified experimentally for PCR amplification. The results of the study are made available freely online database (). Seven primer pairs amplified genomic DNA isolated from leaves were used to screen a representative set of 12 accessions of cacao.     

EasyExonPrimer

EasyExonPrimer is a web-based software that automates the design of PCR primers to amplify exon sequences from genomic DNA. EasyExonPrimer is written in Perl and uses Primer3 to design PCR primers based on the genome builds and annotation databases available at the University of California, Santa Cruz (UCSC) Genome Browser database (http://genome.ucsc.edu/). It masks repeats and known single nucleotide polymorphism (SNP) sites in the genome and designs standardised primers using optimised conditions. Users can input genes by RefSeq mRNA ID, gene name or keyword. The primer design is optimised for large-scale resequencing of exons. For exons larger than 1 kb, the user has the option of breaking the exon sequence down into overlapping smaller fragments. All primer pairs are then verified using the In-Silico PCR software to test for uniqueness in the genome. We have designed >1000 pairs of primers for 90 genes; 95% of the primer pairs successfully amplified exon sequences under standard PCR conditions without requiring further optimisation. AVAILABILITY: EasyExonPrimer is available from http://129.43.22.27/~primer/. The source code is also available upon request. CONTACT: Xiaolin Wu (forestwu@mail.nih.gov).     

EST derived PCR-based markers for functional gene homologues in cotton.

We investigated the utility of the Gossypium arboreum EST sequences in the GenBank database for developing PCR-based markers targeting known-function genes in cultivated tetraploid cottons, G. hirsutum and G. barbadense. Four hundred sixty-five randomly selected ESTs from this library were subjected to BLASTn search against all GenBank databases, of which putative function was assigned to 93 ESTs based on high nucleotide homology to previously studied genes. PCR primers were synthesized for 89 of the known-function ESTs. A total of 57 primer pairs amplified G. arboreum genomic DNA, but only 39 amplified in G. hirsutum and G. barbadense, suggesting that sequence divergence may be a factor causing non-amplification for some sites. DNA sequence analysis showed that most primer pairs were targeting the expected homologous loci. While the amplified products that were of larger size than the corresponding EST sequences contain introns, the primer pairs with a smaller amplicon than predicted from the flanking EST sequences did not amplify the expected orthologous gene sequences. Among the 39 primer pairs that amplified tetraploid cotton DNA, 3 detected amplicon size polymorphisms and 10 detected polymorphisms after digestion with one of six restriction enzymes. Ten of the polymorphic loci were subsequently mapped to an anchor RFLP map. Digestion of PCR-amplified sequences offers one means by which cotton genes can be mapped to their chromosomal locations more quickly and economically than by RFLP analysis.     

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.     

Exhaustive computational identification of pathogen sequences far-distant from background genomes: Identification and experimental verification of human-blind dengue PCR primers

We recently developed novel algorithms for exhaustive identification of all nucleotide subsequences present in a pathogen genome which differ by at least a chosen number of mismatches from the sequences of host/background organisms. This type of exhaustive computational analysis will be useful in reducing false positives and cross-reactivity in PCR and hybridization assays. We present the first experimental test of the method by showing that the subsequences identified when used as 18-mer PCR primers can detect the presence of dengue virus (DENV) even in the presence of a large excess of complex human genomic DNA. From our computations, 715 serotype-specific primer pairs were identified for three different DENV serotypes in which each primer sequence lies at least two mismatches from the nearest human sequence. DNA clones of representative strains of DENV-1, DENV-2, and DENV-4 viruses were subjected to real-time PCR testing using eight primer pairs each. Efficiencies were uniformly very high (mean+/-S.D.=99.6+/-3%), and amplification of human DNA was never observed within 35 cycles, even at a 5.5-fold molar excess of human DNA. Exhaustive primer/probe screening can potentially produce more selective and sensitive diagnostic assays for pathogens, especially in the presence of complex backgrounds.     

Long DOP-PCR of rare archival anthropological samples

The application of molecular DNA technologies to anthropological questions has meant that rare or archival samples of human remains, including blood, hair, and bone, can now be used as a source of material for genetic analysis. Often, these samples are irreplaceable, and/or yield very small quantities of DNA, so methods for preamplifying as much of the whole genome as possible would greatly enhance their usefulness. DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction) is an amplification method that uses a degenerate primer and very low initial annealing temperatures to amplify the whole genome. We adapted a published DOP-PCR protocol to long PCR enzyme and amplification conditions. The effectiveness of these modifications was tested by PCR amplification of DOP-PCR products at a mixture of genomic targets including 66 different microsatellites, 11 Alu insertion polymorphisms, and variable-length segments of the human lipoprotein lipase gene (LPL). The selected microsatellite markers were chosen to represent every chromosome, with expected product sizes ranging from 150 base pairs to 8,000 base pairs in length, while the 22 Alu insertion polymorphisms were selected to reveal biases in the recovery of alleles of different sizes. To determine nucleotide sequence variation, 2 kilobases (kb) of the LPL gene in 30 Mongolian individuals were sequenced. All gene-specific targets from DOP-PCR product template were amplified. No unexpected polymorphisms in the sequence results attributable to the DOP-PCR step were found, and 93% to 95% of Alu genotypes that have been amplified from total genomic DNA were replicated. The incorrect typings were all due to the preferential amplification of the shorter of two possible alleles in individuals heterozygous for an Alu insertion and were all correctly typed on subsequent reamplification of the gene-specific PCR products. This method of whole-genome amplification promises to be an efficient way to maximize the genetic use of rare anthropological samples.     

Mapping of ESTs derived from pre-implantation stage cattle embryos to allocate 16 new additions to the ordered comparative map of cattle and human

Twenty expressed sequence tags (ESTs) derived from cDNA libraries of different developmental stages of embryos were mapped using a whole genome bovine hamster radiation hybrid panel. These include 14 markers representing genes, most of which have not so far been mapped in cattle, with another three being novel in both cattle and human. The markers were placed on specific chromosomes with high LOD scores and except two all localizations fit the current human and cattle comparative map. The assignment of these genes further enriches the cattle genome map and also contributes to the international effort of generating comparative maps.     

亚麻EST-SSR信息分析与标记开发

与基因组SSR相比,以EST为基础的EST-SSR分子标记具有自身的优点。本研究从11240条亚麻（Linum sitatissmum L.）EST序列中检索出877条含有SSR的序列,其出现频率为7.8%。其中以三核苷酸重复出现的频率最高,占总SSR序列的60.1%;其次是二核苷酸重复,占21.9%;四、五和六核苷酸重复占18%。根据这些含SSR的EST序列共设计了73对SSR引物,在8份亚麻材料间通过PCR扩增检测,有63对引物扩增出清晰条带,引物可用率86.3%;有17对引物在8份亚麻材料间显现出多态性,占可扩增引物的26.3%。     

A set of 99 cattle microsatellites: characterization,synteny mapping,and polymorphism

Cattle microsatellite clones (136) were isolated from cosmid (10) and plasmid (126) libraries and sequenced. The dinucleotide repeats were studied in each of these sequences and compared with dinucleotide repeats found in other vertebrate species where information was available. The distribution in cattle was similar to that described for other mammals, such as rat, mouse, pig, or human. A major difference resides in the number of sequences present in the bovine genome, which seemed at best one-third as large as in other species. Oligonucleotide primers (117 pairs) were synthesized, and a PCR product of expected size was obtained for 88 microsatellite sequences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a panel of 36 hamster/bovine somatic cell hybrids. Of our bovine microsatellites, eighty-six could be assigned to synteny groups of chromosomes. In addition, 10 other microsatellites—HEL 5, 6, 9, 11, 12, 13 (Kaukinen and Varvio 1993), HEL 4, 7, 14, 15—as well as the microsatellite found in the -casein gene (Fries et al. 1990) were mapped on the hybrids. Microsatellite polymorphism was checked on at leat 30 unrelated animals of different breeds. Almost all the autosomal and X Chr microsatellites displayed polymorphism, with the number of alleles varying between two and 44. We assume that these microsatellites could be very helpful in the construction of a primary public linkage map of the bovine genome, with an aim of finding markers for Economic Trait Loci (ETL) in cattle.