A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors

1989 ◽  
Vol 67 (4-5) ◽  
pp. 205-209 ◽  
Author(s):  
Cynthia R. D'Souza ◽  
Ken V. Deugau ◽  
John H. Spencer
Keyword(s):  
Genomic Dna ◽  
Restriction Site ◽  
Genomic Library ◽  
Restriction Endonuclease Site ◽  
Dna Libraries ◽  
Target Dna ◽  
Restriction Sites ◽  
Enzyme Cleavage ◽  
Simplified Procedure ◽  
Genomic Cdna

The properties and characteristics of oligonucleotide adaptors for use in a simplified procedure for the construction of cDNA and genomic DNA libraries are described. The adaptors are suitable for joining to blunt ended cDNA or sheared genomic DNA, and then to the cohesive ends of restriction sites in vectors. Each adaptor consists of two oligonucleotides with complementary but nonpalindromic sequences that include an internal restriction site, a 5′ phosphorylated blunt end, and an overlapping or staggered 5′ hydroxylated end corresponding to a restriction endonuclease site in a vector of choice. Ligation of the blunt end to high molecular weight target DNA proceeds efficiently and there is no tandem concatenation of the adaptor. Insertion into the appropriate vector only requires ligation of the cohesive ends. There is no requirement for methylation, restriction enzyme cleavage, G-C tailing, or denaturation after ligation of the adaptor to the target DNA, all characteristics of other procedures.Key words: library, genomic, cDNA, oligonucleotides, adaptors.

scholarly journals Molecular Analysis and Cloning of Malus Ribosomal DNA

1992 ◽  
Vol 117 (1) ◽  
pp. 164-168 ◽  
Author(s):  
C.J. Simon ◽  
N.F. Weeden
Keyword(s):  
Population Dynamics ◽  
Genetic Markers ◽  
Ribosomal Dna ◽  
Genomic Dna ◽  
Restriction Site ◽  
Intergenic Spacer ◽  
Ribosomal Genes ◽  
Intergenic Spacer Region ◽  
Restriction Sites ◽  
Crab Apple

The ribosomal genes of the two crab apple (Malus) genotypes White Angel' and `Robusta 5' were characterized to determine the extent of between- and within-genotype heterogeneity. Initial investigations with a cloned sequence of soybean rDNA failed to detect some Malus intergenic spacer region fragments. An alternative probing method that used electrophoretically purified Malus rDNA was developed. Double-digests of total genomic DNA with combinations of 13 restriction endonucleases identified the positions of 35 restriction sites. Restriction site polymorphism was observed both between and within the crab apple genotypes. Ribosomal DNA from White Angel' was cloned in phage and plasmid vectors and mapped with 11 enzymes. The region of the spacer causing length heterogeneity was identified. These clones should be useful as genetic markers and for examining population dynamics and systematic of Malus and closely related taxa.

scholarly journals Absence of Replication-Competent Human-Tropic Porcine Endogenous Retroviruses in the Germ Line DNA of Inbred Miniature Swine

2004 ◽  
Vol 78 (5) ◽  
pp. 2502-2509 ◽  
Author(s):  
Linda Scobie ◽  
Samantha Taylor ◽  
James C. Wood ◽  
Kristen M. Suling ◽  
Gary Quinn ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Germ Line ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
Miniature Swine ◽  
Dna Libraries ◽  
Porcine Endogenous Retroviruses ◽  
Perv Transmission

ABSTRACT The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol. 76:3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long env open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.

scholarly journals Naturally occurring variation in the restriction map of the amy region of Drosophila melanogaster.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 619-629
Author(s):  
C H Langley ◽  
A E Shrimpton ◽  
T Yamazaki ◽  
N Miyashita ◽  
Y Matsuo ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Restriction Site ◽  
Natural Populations ◽  
Alpha Amylase ◽  
Low Frequencies ◽  
Linkage Disequilibria ◽  
Restriction Sites ◽  
Site Variation ◽  
Transcriptional Units ◽  
Polymorphic Restriction

Abstract The restriction maps of 85 alleles of the Amy region of Drosophila melanogaster from natural populations were surveyed. A subset of these were also scored for allozyme phenotype and adult enzyme activity of alpha-amylase. Large insertions were found in 12% of the alleles in a 15-kb region surrounding the two transcriptional units of the duplicated Amy locus. The low frequencies at which each of these large insertions were found are consistent with earlier reports of variation in other loci. Four small deletions were found in the region 5' to the Amy genes. Each was also rare in the population. Restriction site variation provided an estimate of per nucleotide heterozygosity of 0.006. Several statistically significant linkage disequilibria were observed between four polymorphic restriction sites and the allozymes. Adult alpha-amylase activity was correlated with the allozymes and with the polymorphism at one restriction site close to the transcriptional units.

Molecular and cytogenetic analysis of repetitive DNA in pea (Pisum sativum L.)

Genome ◽  
2001 ◽  
Vol 44 (4) ◽  
pp. 716-728 ◽  
Author(s):  
Pavel Neumann ◽  
Marcela Nouzová ◽  
Jirí Macas
Keyword(s):  
Pisum Sativum ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Tandem Repeats ◽  
Genomic Library ◽  
Chromosome Morphology ◽  
Plant Genome ◽  
Genomic Repeats ◽  
Dispersed Repeats

A set of pea DNA sequences representing the most abundant genomic repeats was obtained by combining several approaches. Dispersed repeats were isolated by screening a short-insert genomic library using genomic DNA as a probe. Thirty-two clones ranging from 149 to 2961 bp in size and from 1000 to 39 000/1C in their copy number were sequenced and further characterized. Fourteen clones were identified as retrotransposon-like sequences, based on their homologies to known elements. Fluorescence in situ hybridization using clones of reverse transcriptase and integrase coding sequences as probes revealed that corresponding retroelements were scattered along all pea chromosomes. Two novel families of tandem repeats, named PisTR-A and PisTR-B, were isolated by screening a genomic DNA library with Cot-1 DNA and by employing genomic self-priming PCR, respectively. PisTR-A repeats are 211–212 bp long, their abundance is 2 × 104 copies/1C, and they are partially clustered in a secondary constriction of one chromosome pair with the rest of their copies dispersed on all chromosomes. PisTR-B sequences are of similar abundance (104 copies/1C) but differ from the "A" family in their monomer length (50 bp), high A/T content, and chromosomal localization in a limited number of discrete bands. These bands are located mainly in (sub)telomeric and pericentromeric regions, and their patterns, together with chromosome morphology, allow discrimination of all chromosome types within the pea karyotype. Whereas both tandem repeat families are mostly specific to the genus Pisum, many of the dispersed repeats were detected in other legume species, mainly those in the genus Vicia.Key words: repetitive DNA, plant genome, retroelements, satellite DNA, Pisum sativum.

Isolation and characterization of six different chicken actin genes

1984 ◽  
Vol 4 (11) ◽  
pp. 2498-2508
Author(s):  
K S Chang ◽  
W E Zimmer ◽  
D J Bergsma ◽  
J B Dodgson ◽  
R J Schwartz
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Genomic Library ◽  
Smooth Muscle Actin ◽  
Actin Gene ◽  
Muscle Actin ◽  
Repeated Dna ◽  
Alpha Smooth Muscle Actin ◽  
Actin Genes ◽  
Actin Isoform

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

scholarly journals Computational design of probes to detect bacterial genomes by multivalent binding

2020 ◽  
Vol 117 (16) ◽  
pp. 8719-8726 ◽  
Author(s):  
Tine Curk ◽  
Chris A. Brackley ◽  
James D. Farrell ◽  
Zhongyang Xing ◽  
Darshana Joshi ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Bacterial Infections ◽  
Bacterial Genome ◽  
Computational Design ◽  
Diagnostic Methods ◽  
Detection Sensitivity ◽  
Target Sequence ◽  
Inappropriate Use ◽  
Single Target ◽  
Target Dna

Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target–probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.

Section 3 update: Isolation of high molecular weight genomic DNA from soil bacteria for genomic library construction

2008 ◽  
pp. 2741-2751
Author(s):  
Mark R. Liles ◽  
Lynn L. Williamson ◽  
Jo Handelsman ◽  
Robert M. Goodman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Genomic Dna ◽  
Soil Bacteria ◽  
Genomic Library ◽  
Library Construction

Construction of Genomic DNA Libraries in Cosmid Vectors

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4000
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  
Genomic Dna ◽  
Dna Libraries

scholarly journals An approximately 300 bp deletion involving part of the 5' beta-globin gene region is observed in members of a Turkish family with beta- thalassemia

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 583-586 ◽  
Author(s):  
JC Diaz-Chico ◽  
KG Yang ◽  
A Kutlar ◽  
AL Reese ◽  
M Aksoy ◽  
...  
Keyword(s):  
Promoter Region ◽  
Genomic Dna ◽  
Restriction Site ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Sequences ◽  
Beta Globin Gene ◽  
Turkish Family ◽  
Thalassemia Trait

Abstract Detailed gene mapping analyses of genomic DNA from two Turkish subjects with a beta-thalassemia trait demonstrated an approximately 300 bp deletion, which is located between the Rsa I restriction site 128 bp 5′ to the Cap site and the Acc I restriction site 284 bp 3′ to the same Cap site; it includes the 5′ beta promoter region, the first exon, and (part of) the IVS-I. Heterozygotes for this and two other beta- thalassemia types, which are also caused by deletions involving 5′ beta promoter sequences, appear to have higher hemoglobin (Hb) A2 levels, perhaps because the loss of this promoter results in an increased transcription of the delta globin gene, as delta and beta promoters may be influenced by the same enhancing sequences 3′ to the beta globin gene.

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