Sequence analysis of the β-lactoglobulin locus in Holsteins identifies two new restriction fragment lengtn polymorphisms

1996 ◽  
Vol 76 (3) ◽  
pp. 299-303 ◽  
Author(s):  
J. F. Zhou ◽  
D. Zadworny ◽  
U. Kuhnlein
Keyword(s):  
Sequence Analysis ◽  
Restriction Fragment ◽  
Restriction Site ◽  
Allelic Frequency ◽  
Nucleotide Substitutions ◽  
Bull Calves ◽  
Length Polymorphisms ◽  
Restriction Sites ◽  
Polymerase Chain ◽  
Β Lactoglobulin

The polymerase chain reaction (PCR) was used to amplify and clone a region from the β-lactoglobulin (β-LG) locus that spanned exons IV and V (849 bp). The DNA was amplified from both AA and BB homozygous individuals and sequenced. Sequence analysis revealed that the intervening intron was 673 bp and that three nucleotide substitutions differentiated the A and B forms of β-LG. One of these substitutions was associated with the amino acid substitution (aspartic acid or glycine in the A and B variants, respectively), and the other two which were not reported previously were present in the intron sequence. These nucleotide substitutions resulted in restriction fragment length polymorphisms (RFLPs) that could be used to genotype individuals. The new restriction sites in the intron would result in a more accurate genotyping of the β-LG gene. Animals with B genotype were positive for the presence of two HaeIII restriction endonuclease sites, and type A animals were negative. Animals could also be genotyped on the basis of a polymorphism at a NlaIV restriction site. Genotyping of a random sample of 129 cows and 99 bull calves in Quebec revealed a frequency of 0.66 for the B allele. A comparison between bulls in current use by the artificial insemination industry (n = 114) and from the earliest years of the industry (n = 70) revealed frequencies of 0.58 and 0.56, respectively. Thus, it is unlikely that the sire selection program has affected the allelic frequency. Key words: β-lactoglobulin, Holsteins, DNA sequence, polymorphisms

Mitochondrial DNA variation and genetic relationships of Populus species

Genome ◽  
1993 ◽  
Vol 36 (1) ◽  
pp. 87-93 ◽  
Author(s):  
John W. Barrett ◽  
Om P. Rajora ◽  
F. C. H Yeh ◽  
Bruce P. Dancik ◽  
Curtis Strobeck
Keyword(s):  
Mitochondrial Dna ◽  
Restriction Fragment ◽  
Genetic Relationships ◽  
Mitochondrial Genomes ◽  
Nucleotide Substitutions ◽  
Dna Variation ◽  
Eastern Cottonwood ◽  
Length Polymorphisms ◽  
Populus Species ◽  
Atpase 6

We examined variation in and around the region coding for the cytochrome c oxidase I (coxI) and ATPase 6 (atp6) genes in the mitochondrial genomes of four Populus species (P. nigra, P. deltoides, P. maximowiczii, and P. tremuloides) and the natural hybrid P. × canadensis (P. deltoides × P. nigra). Total cellular DNAs of these poplars were digested with 16 restriction endonucleases and probed with maize mtDNA-specific probes (CoxI and Atp6). The only variant observed for Atp6 was interspecific, with P. maximowiczii separated from the other species as revealed by EcoRI digestions. No intraspecific mtDNA variation was observed among individuals of P. nigra, P. maximowiczii, P. × canadensis, or P. tremuloides for the CoxI probe. However, two varieties of P. deltoides were distinct because of a single site change in the KpnI digestions, demonstrating that P. deltoides var. deltoides (eastern cottonwood) and var. occidentalis (plains cottonwood) have distinct mitochondrial genomes in the region of the coxI gene. Populus × canadensis shared the same restriction fragment patterns as its suspected maternal parent P. deltoides. Nucleotide substitutions per base in and around the coxI and atp6 genes among the Populus species and the hybrid ranged from 0.0017 to 0.0077. The interspecific estimates of nucleotide substitution per base suggested that P. tremuloides was furthest removed from P. deltoides and P. × canadensis and least diverged from P. nigra. Populus maximowiczii was placed between these two clusters.Key words: mitochondrial DNA, poplars, phylogenetics, variation, restriction fragment length polymorphisms.

scholarly journals indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events

2017 ◽  
Author(s):  
Charles Hodgens ◽  
Zachary L. Nimchuk ◽  
Joseph J. Kieber
Keyword(s):  
Restriction Site ◽  
Genetic Manipulation ◽  
Pcr Primers ◽  
Null Alleles ◽  
Wild Type ◽  
Web Tool ◽  
Web Based ◽  
Restriction Sites ◽  
Polymerase Chain ◽  
Enzyme Recognition

AbstractGenetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be easily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

A robust strategy for screening and confirmation of familial defective apolipoprotein B-100

1993 ◽  
Vol 39 (1) ◽  
pp. 118-121 ◽  
Author(s):  
C D Mamotte ◽  
F M van Bockxmeer
Keyword(s):  
Apolipoprotein B ◽  
Annealing Temperature ◽  
Restriction Site ◽  
Pcr Primers ◽  
Test Methods ◽  
Taq Polymerase ◽  
Pcr Product ◽  
Restriction Sites ◽  
Pcr Products ◽  
Polymerase Chain

Abstract The diagnosis of familial defective apolipoprotein B-100 (FDB) has been facilitated by the use of mutagenic polymerase chain reaction (PCR) primers to introduce restriction sites at the FDB gene locus. We describe a two-test strategy for diagnosing FDB that overcomes the potential for error in single-test methods based on such techniques. We introduce an Sau96I restriction site for PCR products of the normal apolipoprotein B allele. Incomplete digestion of the PCR product with Sau96I suggests an FDB heterozygote, although a false-positive result due to nonideal digestion conditions remains a possibility. In such cases we use a second test that introduces an ScaI restriction site in PCR products of the FDB allele. The diagnosis is confirmed if half of this product can be digested with ScaI. Both tests use 0.25 units of Taq polymerase and are robust with respect to annealing temperature (31-58 degrees C) and to Mg2+ concentration (1.0-3.2 mmol/L).

Polymerase chain reaction - restriction fragment length polymorphisms for assessing and increasing biodiversity ofFrankiaculture collections

1999 ◽  
Vol 77 (9) ◽  
pp. 1261-1269 ◽  
Author(s):  
Erica Lumini ◽  
Marco Bosco
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Fragment ◽  
Fragment Length ◽  
Intergenic Spacer ◽  
Chain Reaction ◽  
Culture Collections ◽  
Length Polymorphisms ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Restriction Fragment Length

During the last few years, some Frankia culture collections that maintained a large number of unidentified and uncharacterized Frankia strains were closed because of funding shortages. To reduce the costs of maintenance, we evaluated the biodiversity of half of the Frankia strains from our collection, by polymerase chain reaction - restriction fragment length polymorphisms (PCR-RFLPs) of nifD-nifK intergenic spacer and 16S-23S rDNA intergenic spacer regions. In this way we were able to reduce the number of strains without reducing the biodiversity of the whole collection. In general the nifD-nifK target proved to be more polymorphic than the rrn target. From 51 isolates of Elaeagnus frankiae, PCR-RFLP results allowed us to detect 13 identical strains, and to predict that the genomic species P8 of Akimov and Dobritsa (1992) very likely agrees with genomic species 5 of Fernandez et al. (1989). Moreover, we revealed genomic groups not yet described, as well as intraspecific variability. For Alnus frankiae, the polymorphisms shown by both the nif and the rrn PCR-RFLPs revealed three host plant species-specific subgroups inside Frankia alni. An expandable data base was created to serve as reference for future biodiversity evaluations on both culture collections and unisolated Frankia populations. It will be accessible by Internet at the International Frankia Website (http://www.unifi.it/unifi/distam/frankia/international.html).Key words: Frankia, PCR-RFLP, nifD-nifK intergenic spacer, rrn 16S-23S intergenic spacer, biodiversity, culture collections.

Restriction Site Variation in the Zea Chloroplast Genome

Genetics ◽  
1987 ◽  
Vol 117 (1) ◽  
pp. 139-147
Author(s):  
J Doebley ◽  
W Renfroe ◽  
A Blanton
Keyword(s):  
Dna Analysis ◽  
Restriction Site ◽  
Sequence Divergence ◽  
Unique Sequence ◽  
Sequence Evolution ◽  
Nucleotide Substitutions ◽  
Restriction Site Variation ◽  
Chloroplast Genomes ◽  
Restriction Sites ◽  
Site Variation

ABSTRACT Nineteen accessions selected from the four species and three subspecies of the genus Zea and one accession from the related genus Tripsacum were surveyed for variation with 21 restriction endonucleases. In all, 580 restriction sites were assayed in each chloroplast (cp)DNA, this representing 2.2% of the genome. Twenty-four of the 580 sites were variable in one or more of the cpDNAs. The number of nucleotide substitutions per site (p) between Zea and Tripsacum (0.0056) approximates that between other closely related angiosperm genera. The range in values of p among Zea species (0.0003-0.0024) is on the lower end of the range reported for other angiosperm genera. Analysis of the distribution of restriction site mutations throughout the genome indicated that the inverted repeat evolves more slowly than either the small or large unique sequence regions. Parsimony phylogenetic analysis of the restriction site data produced a tree consistent with isoenzymatic and morphological measures of affinity among the species. Chloroplast DNA analysis was not useful in discriminating the subspecies within Zea mays. The lack of any detectable differences between the cpDNA of maize (Z. mays subsp. mays) and some teosintes (Z. mays subsps. mexicana and parviglumis) is consistent with the hypothesis that maize is a domesticated form of teosinte. Comparison of the degree of sequence divergence for Z. mays cpDNA and the Adh1 locus suggests the latter may be evolving at 10 times the rate of the former. Comparison of rates of sequence evolution for the mitochondrial and chloroplast genomes was inconclusive and could not clarify whether these two genomes have dissimilar rates of sequence evolution.

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