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).

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

MUTATION IN THE FMO3 LOCUS AND ITS DISTRIBUTION IN A GROUP OF AYRSHIRE BREEDING BULLS USED IN FARMS OF THE KRASNODAR TERRITORY

2021 ◽  
pp. 10-15
Author(s):  
Н.В. КОВАЛЮК ◽  
Е.В. ШИРЯЕВА ◽  
Л.И. ЯКУШЕВА ◽  
Ю.Ю. ШАХНАЗАРОВА
Keyword(s):  
Polymerase Chain Reaction ◽  
Mutant Allele ◽  
Restriction Site ◽  
Fragment Length Polymorphism ◽  
Test System ◽  
Frequency Of Occurrence ◽  
Wild Type ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Рыбный привкус в коровьем молоке вызван наличием нонсенс-мутации (g.39523051C>T) в гене бычьего FMO3. Нами разработана тест-система для выявления FMO3- полиморфизма, основанная на полимеразной цепной реакции с последующим анализом полиморфизма длин фрагментов рестрикции с использованием эндонуклеазы TaqI. Фрагменты, которые амплифицировались с участка гена FMO3 «дикого» типа, расщеплялись эндонуклеазой TaqI  на 2 фрагмента: 136 и 99 пн. Фрагменты, амплифицированные с мутантного аллеля, сайта рестрикции не имели (их размер составлял  235 пн). Определена частота встречаемости носителей мутации в отечественной субпопуляции айрширского скота. Установлено, что среди айрширских быков-производителей (n=45), принадлежащих различным отечественным и зарубежным племорганизациям, частота встречаемости носителей мутации в гене FMO составила 9%. Учитывая, что выявленные носители мутации интенсивно используются и могут передавать эту аномалию значительному числу дочерей, генотипирование по локусу FMO3 должно стать обязательным для быков-производителей и групп быкопроизводящих коров племенных айрширских хозяйств. The fishy taste in cow's milk is caused by the presence of a nonsense mutation (g.39523051C>T) in the bovine FMO3 gene. We have developed a test system for detecting FMO3 polymorphism based on a polymerase chain reaction followed by analysis of restriction fragment length polymorphism using TaqI endonuclease. Fragments that were amplified from the wild-type FMO3 gene site were cleaved by TaqI endonuclease into 2 fragments: 136 and 99 bp. The fragments amplified from the mutant allele did not have a restriction site (their size was — 235 bp). The frequency of occurrence of mutation carriers in the domestic subpopulation of Ayrshire cattle was determined. It was found that among Ayrshire bulls (n=45) belonging to various domestic and foreign breeding organizations, the frequency of occurrence of carriers of the mutation in the FMO gene was 9%. Given that the identified carriers of the mutation are intensively used and can transmit this anomaly to a significant number of daughters, genotyping by the FMO3 locus should become mandatory for breeding bulls and groups of bull-producing cows of breeding Ayrshire farms.

scholarly journals Simple innovative adaptor to improve genome walking with convenient PCR

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Seyedeh-Samira Ashrafmansouri ◽  
Hossein Kamaladini ◽  
Fatemeh Haddadi ◽  
Marie Seidi
Keyword(s):  
Gene Sequence ◽  
Restriction Site ◽  
Restriction Enzymes ◽  
Phosphate Group ◽  
Genome Walking ◽  
Restriction Enzyme Digestion ◽  
Restriction Sites ◽  
Polymerase Chain ◽  
Adaptor Primer ◽  
Unknown Sequence

Abstract Background Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. Results The adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5′ phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5′ phosphate group. Conclusion PCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.

scholarly journals Novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Safia Akhtar ◽  
Silas A. Culver ◽  
Helmy M. Siragy
Keyword(s):  
Protein Expression ◽  
High Fat Diet ◽  
Normal Diet ◽  
Receptor Expression ◽  
Wild Type ◽  
High Fat ◽  
Renal Gluconeogenesis ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Renal Expression

AbstractRecent studies suggested that renal gluconeogenesis is substantially stimulated in the kidney in presence of obesity. However, the mechanisms responsible for such stimulation are not well understood. Recently, our laboratory demonstrated that mice fed high fat diet (HFD) exhibited increase in renal Atp6ap2 [also known as (Pro)renin receptor] expression. We hypothesized that HFD upregulates renal gluconeogenesis via Atp6ap2-PGC-1α and AKT pathway. Using real-time polymerase chain reaction, western blot analysis and immunostaining, we evaluated renal expression of the Atp6ap2 and renal gluconeogenic enzymes, PEPCK and G6Pase, in wild type and inducible nephron specific Atp6ap2 knockout mice fed normal diet (ND, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 8 weeks. Compared with ND, HFD mice had significantly higher body weight (23%) (P < 0.05), renal mRNA and protein expression of Atp6ap2 (39 and 35%), PEPCK (44 and 125%) and G6Pase (39 and 44%) respectively. In addition, compared to ND, HFD mice had increased renal protein expression of PGC-1α by 32% (P < 0.05) and downregulated AKT by 33% (P < 0.05) respectively in renal cortex. Atp6ap2-KO abrogated these changes in the mice fed HFD. In conclusion, we identified novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

scholarly journals A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes

1989 ◽  
Vol 86 (17) ◽  
pp. 6691-6695 ◽  
Author(s):  
B Davies ◽  
S Feo ◽  
E Heard ◽  
M Fried
Keyword(s):  
Ribosomal Protein ◽  
Recombinant Dna ◽  
Single Gene ◽  
Ribosomal Protein Gene ◽  
Pcr Primers ◽  
Pcr Product ◽  
Dna Libraries ◽  
Processed Pseudogenes ◽  
Polymerase Chain ◽  
Genomic Dna Sequence

We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns.

Detection ofErwinia amylovorain plant material using novel polymerase chain reaction (PCR) primers

2001 ◽  
Vol 29 (1) ◽  
pp. 35-43 ◽  
Author(s):  
R. K. Taylor ◽  
P. J. Guilford ◽  
R. G. Clark ◽  
C. N. Hale ◽  
R. L. S. Forster
Keyword(s):  
Polymerase Chain Reaction ◽  
Plant Material ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals BAGET 2.0: an updated web tool for the effortless retrieval of prokaryotic gene context and sequence

2021 ◽  
Author(s):  
Benjamin Hepp ◽  
Violette Da Cunha ◽  
Florence Lorieux ◽  
Jacques Oberto
Keyword(s):  
Gene Sequence ◽  
Single Gene ◽  
Daily Basis ◽  
Web Tool ◽  
Web Based ◽  
Internet Explorer ◽  
Prokaryotic Genomes ◽  
Exploration Tool ◽  
Genome Browsers ◽  
Archaeal Gene

Abstract Motivation The retrieval of a single gene sequence and context from completely sequenced bacterial and archaeal genomes constitutes an intimidating task for the wet bench biologist. Existing web-based genome browsers are either too complex for routine use or only provide a subset of the available prokaryotic genomes. Results We have developed BAGET 2.0 (Bacterial and Archaeal Gene Exploration Tool), an updated web service granting access in just three mouse clicks to the sequence and synteny of any gene from completely sequenced bacteria and archaea. User-provided annotated genomes can be processed as well. BAGET 2.0 relies on a local database updated on a daily basis. Availability and implementation BAGET 2.0 befits all current browsers such as Chrome, Firefox, Edge, Opera and Safari. Internet Explorer 11 is supported. BAGET 2.0 is freely accessible at https://archaea.i2bc.paris-saclay.fr/baget/

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