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1.
Biotechniques ; 33(3): 532-4, 536, 538-9, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12238763

ABSTRACT

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.


Subject(s)
DNA, Bacterial/genetics , Plasmids/genetics , Sepharose , Sequence Analysis, DNA/instrumentation , Sequence Analysis, DNA/methods , Cell Fractionation/methods , DNA, Bacterial/isolation & purification , Electrophoresis, Agar Gel/instrumentation , Electrophoresis, Agar Gel/methods , Plasmids/isolation & purification , Quality Control , Reproducibility of Results , Sensitivity and Specificity
3.
Genetica ; 111(1-3): 43-58, 2001.
Article in English | MEDLINE | ID: mdl-11841186

ABSTRACT

Sex chromosomes in fish provide an intriguing view of how sex-determination mechanisms evolve in vertebrates. Many fish species with single-factor sex-determination systems do not have cytogenetically-distinguishable sex chromosomes, suggesting that few sex-specific sequences or chromosomal rearrangements are present and that sex-chromosome evolution is thus at an early stage. We describe experiments examining the linkage arrangement of a Y-chromosomal GH pseudogene (GH-Y) sequence in four species of salmon (chum, Oncorhynchus keta; pink, O. gorbuscha; coho, O. kisutch; chinook, O. tshawytscha). Phylogenetic analysis indicates that GH-Y arose early in Oncorhynchus evolution, after this genus had diverged from Salmo and Salvelinus. However, GH-Y has not been detected in some Oncorhynchus species (O. nerka, O. mykiss and O. clarki), consistent with this locus being deleted in some lineages. GH-Y is tightly linked genetically to the sex-determination locus on the Y chromosome and, in chinook salmon, to another Y-linked DNA marker OtY1. GH-Y is derived from an ancestral GH2 gene, but this latter functional GH locus is autosomal or pseudoautosomal. YY chinook salmon are viable and fertile, indicating the Y chromosome is not deficient of vital genetic functions present on the X chromosome, consistent with sex chromosomes that are in an early stage of divergence.


Subject(s)
Chromosome Mapping , Genetic Markers , Salmon/genetics , Y Chromosome , Animals , Base Sequence , Blotting, Southern , Female , Male , Sequence Homology, Nucleic Acid , Sex Determination Processes , Species Specificity
4.
J Mol Evol ; 46(3): 277-87, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9493353

ABSTRACT

A long repetitive DNA sequence (OtY8) has been cloned from male chinook salmon and its genomic organization has been characterized. The repeat has a unit length of 8 kb and is present approximately 300 times per diploid male nucleus. All internal fragments within the 8-kb repeat segregate from father to son, suggesting that the entire repeat unit is located on the Y chromosome. The organization of this sequence into an 8-kb repeat unit is restricted to the Y chromosome, as are several male-specific repeat subtypes identified on the basis of restriction-site variation. The repeat possesses only weak internal sequence similarities, suggesting that OtY8 has not arisen by duplication of a smaller repeat unit, as is the case for other long tandem arrays found in eukaryotes. Based on a laddered pattern arising from partial digestion of genomic DNA with a restriction enzyme which cuts only once per repeat unit, this sequence is not dispersed on the Y chromosome but is organized as a head-to-tail tandem array. Pulse-gel electrophoresis reveals that the direct-tandem repeats are organized into at least six separate clusters containing approximately 12 to 250 copies, comprising some 2.4 Mb of Y-chromosomal DNA in total. Related sequences with nucleotide substitutions and DNA insertions relative to the Y-chromosomal fragment are found elsewhere in the genome but at much lower copy number and, although similar sequences are also found in other salmonid species, the amplification of the repeat into a Y-chromosome-linked tandem array is only observed in chinook salmon. The OtY8 repetitive sequence is genetically tightly associated with the sex-determination locus and provides an opportunity to examine the evolution of the Y chromosome and sex determination process in a lower vertebrate.


Subject(s)
DNA/genetics , Repetitive Sequences, Nucleic Acid , Salmon/genetics , Y Chromosome , Animals , Base Sequence , Chromosome Mapping , DNA Transposable Elements , Genetic Linkage , Male , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Sex Determination Processes , Species Specificity
5.
Genomics ; 13(2): 339-43, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1351867

ABSTRACT

We report the characterization of a dense cluster of CpG islands at D10S94 in proximal 10q11.2. D10S94 is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN 2A), a dominantly inherited tumor syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and/or parathyroid adenoma. To date, no recombinants between D10S94 and MEN2A have been identified. The gene(s) responsible for two additional dominantly inherited disorders involving cancer of the medullary thyroid, MEN 2B (MEN2B), and dominantly inherited MTC without additional clinical features (MTC1), also map to this region. The gene or genes responsible for these disorders may be located at or near the D10S94 locus. A 570-kb long-range restriction map has been generated by pulsed-field gel electrophoresis using probes developed during a 160-kb bidirectional cosmid walk at D10S94. Six CpG islands are clustered within a 180-kb region; five fall within a 145-kb NotI restriction fragment that is contained in its entirety in our cosmid contig. The SacII, SfiI, and NotI restriction maps for lymphoblast and cloned DNA are concordant. These CpG islands may represent the 5' ends of candidate genes for MEN2A, MEN2B, and/or MTC1. One gene designated mcs94-1, which is associated with one of the CpG islands in this cluster, has been isolated and characterized in detail.


Subject(s)
Chromosomes, Human, Pair 10 , Genes, Dominant , Genetic Markers , Multiple Endocrine Neoplasia/genetics , Base Sequence , Carcinoma/genetics , Chromosome Walking , Cosmids , Electrophoresis, Gel, Pulsed-Field , Humans , Hybrid Cells , Male , Restriction Mapping , Thyroid Neoplasms/genetics
6.
Genomics ; 13(2): 344-8, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1351868

ABSTRACT

We have identified a candidate for the gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) at D10S94 in proximal 10q11.2. An evolutionarily conserved sequence from D10S94 was used as a probe to isolate cDNAs corresponding to a gene that we have termed mcs94-1. The gene spans 11 kb and has an unmethylated CpG island at its 5' end. The mcs94-1 transcript is approximately 2.4 kb in length and is widely expressed. It encodes a putative 415-amino-acid polypeptide that is similar in sequence to nucleolin, an abundant nucleolar protein. Mcs94-1 was examined as a candidate for MEN2A through nucleotide sequence analysis of mcs94-1 exons from an MEN 2A chromosome and its wildtype homologue from an MEN 2A patient. The major portion of the expressed mcs94-1 sequence was examined. No differences in sequence were found between the two alleles.


Subject(s)
Genes, Dominant , Genetic Markers , Multiple Endocrine Neoplasia/genetics , RNA-Binding Proteins , Amino Acid Sequence , Animals , Base Sequence , Chromosomes, Human, Pair 10 , Cricetinae , Humans , Hybrid Cells , Mice/genetics , Molecular Sequence Data , Nuclear Proteins/genetics , Phosphoproteins/genetics , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Nucleolin
7.
Genomics ; 13(2): 409-14, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1351870

ABSTRACT

Repeat element-mediated PCR can facilitate rapid cloning and mapping of human chromosomal region-specific DNA markers from somatic cell hybrid DNA. PCR primers directed to human repeat elements result in human-specific DNA synthesis; template DNA derived from a somatic cell hybrid containing the human chromosomal region of interest provides region specificity. We have generated a series of repeat element-mediated PCR clones from a reduced complexity somatic cell hybrid containing a portion of human chromosome 10. The cloning source retains the centromere and tightly linked flanking markers, plus additional chromosome 10 sequences. Twelve new inter-Alu, two inter-L1, and four inter-Alu/L1 repeat element-mediated PCR clones were mapped by hybridization to Southern blots of repeat element-mediated PCR products amplified from somatic cell hybrid DNA templates. Two inter-Alu clones mapped to the pericentromeric region. We propose that a scarcity of Alu elements in the pericentromeric region of chromosome 10 contributed to the low number of clones obtained from this region. One inter-Alu clone, pC11/A1S-6-c23, defines the D10S94 locus, which is tightly linked to MEN2A and D10Z1.


Subject(s)
Chromosomes, Human, Pair 10 , Genetic Markers , Polymerase Chain Reaction/methods , Repetitive Sequences, Nucleic Acid , Animals , Base Sequence , Cricetinae , Cricetulus , Humans , Hybrid Cells , Mice , Molecular Sequence Data , Multiple Endocrine Neoplasia/genetics
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