Table 1. Restriction enzymes for inverse PCR and plasmid rescue

2009 ◽  
Vol 2009 (4) ◽  
pp. pdb.tab195199-pdb.tab195199
Keyword(s):  
Restriction Enzymes ◽  
Inverse Pcr ◽  
Plasmid Rescue

Recovery of DNA Sequences Flanking P-Element Insertions in Drosophila: Inverse PCR and Plasmid Rescue

2009 ◽  
Vol 2009 (4) ◽  
pp. pdb.prot5199-pdb.prot5199 ◽  
Author(s):  
A. M. Huang ◽  
E. J. Rehm ◽  
G. M. Rubin
Keyword(s):  
Dna Sequences ◽  
P Element ◽  
Inverse Pcr ◽  
Plasmid Rescue

scholarly journals Here and there: the double-side transgene localization

2021 ◽  
Vol 25 (6) ◽  
pp. 607-612
Author(s):  
P. A. Salnikov ◽  
A. A. Khabarova ◽  
G. S. Koksharova ◽  
R. V. Mungalov ◽  
P. S. Belokopytova ◽  
...  
Keyword(s):  
Gene Annotation ◽  
Restriction Enzymes ◽  
Inverse Pcr ◽  
Pcr Analysis ◽  
Regulatory Sequences ◽  
Chromosome Engineering ◽  
Position Effects ◽  
Transposon Insertion ◽  
Flanking Regions ◽  
Screening Approaches

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identification of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact filtration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s flanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two flanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the field of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to efficiently localize genomic transposon integrations, which was confirmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

Isolation of the Promoter Region of a Pollen-specific Gene PSG076 by Inverse-PCR

2012 ◽  
Vol 30 (3) ◽  
pp. 309
Author(s):  
Ling CHEN ◽  
Pei-Pei SU ◽  
Han-Wen TONG ◽  
Yi-Ke LIU ◽  
Zhan-Wang ZHU ◽  
...  
Keyword(s):  
Promoter Region ◽  
Inverse Pcr ◽  
Specific Gene

AFLP-derived, Codominant Markers for Locus-specific Applications

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 514e-514
Author(s):  
James M. Bradeen ◽  
Philipp W. Simon
Keyword(s):  
Linkage Mapping ◽  
Large Scale ◽  
Pcr Primers ◽  
Inverse Pcr ◽  
Sequence Information ◽  
Pcr Assay ◽  
Specific Primers ◽  
Simultaneous Evaluation ◽  
Feral Populations ◽  
Diversity Assessment

The amplified fragment length polymorphism (AFLP) is a powerful marker, allowing rapid and simultaneous evaluation of multiple potentially polymorphic sites. Although well-adapted to linkage mapping and diversity assessment, AFLPs are primarily dominant in nature. Dominance, relatively high cost, and technological difficulty limit use of AFLPs for marker-aided selection and other locus-specific applications. In carrot the Y2 locus conditions carotene accumulation in the root xylem. We identified AFLP fragments linked to the dominant Y2 allele and pursued conversion of those fragments to codominant, PCR-based forms useful for locus-specific applications. The short length of AFLPs (≈60 to 500 bp) precludes development of longer, more specific primers as in SCAR development. Instead, using sequence information from cloned AFLP fragments for primer design, regions outside of the original fragment were amplified by inverse PCR or ligation-mediated PCR, cloned, and sequenced. Differences in sequences associated with Y2 vs. y2 allowed development of simple PCR assays differentiating those alleles. PCR primers flanking an insertion associated with the recessive allele amplified differently sized products for the two Y2 alleles in one assay. This assay is rapid, technologically simple (requiring no radioactivity and little advanced training or equipment), reliable, inexpensive, and codominant. Our PCR assay has a variety of large scale, locus-specific applications including genotyping diverse carrot cultivars and wild and feral populations. Efforts are underway to improve upon conversion technology and to more extensively test the techniques we have developed.

Gene Frequency of CYP2D6*4 and *10 Variants in Karachi Population

2020 ◽  
Vol 17 ◽  
Author(s):  
Tamkeen Fatima ◽  
Farah Zeb ◽  
A. Dar Farooq
Keyword(s):  
Antipsychotic Drugs ◽  
Screening Program ◽  
Restriction Enzymes ◽  
Normal Subjects ◽  
Control Group ◽  
Poor Metabolizer ◽  
Cyp2d6 Polymorphism ◽  
Wild Allele ◽  
Schizophrenic Patients ◽  
Intermediate Metabolizer

Background: CYP2D6 is to be considered the most pronounced gene in pharmacegenetic field which is involved in metabolizing ~25% of all clinically used neuroleptic drugs and other antidepressants. We designed a study to evaluate differential expression of CYP2D6*4 and CYP2D6*10 variants which are very prevalent in Asian countries and exhibit variation in drug metabolizing ability that affect therapeutic responses. Objective: The purpose of this study is to determine the genotypic frequencies of CYP2D6 *1 (normal metabolizer), *4 (poor metabolizer) and *10 (intermediate metabolizer) variants among schizophrenic subjects and compared with control group from a sub-set of Karachi population. Method: Genomic deoxyribonucleic acid (DNA ) was extracted and amplified with CYP2D6*4 and *10 primers using polymerase chain reaction (PCR) and digested by Bacillus stereothermophilus (BstN1) and Hemophilus parahemolyticus (Hph1) restriction enzymes. The digested bands were identified as wild type or mutants and their genotypic frequencies were estimated statistically by Hardy-Weinberg equation (HWE) and analyzed further under non-parametric Chi-square test. Results: The results mentioned the frequencies of CYP2D6*1 wild allele (57%) which produces functional enzyme in normal subjects but CYP2D6*4 variant (9%) that produces non-functional enzyme and CYP2D6*10 allele (70%) produces altered enzyme with reduced activity that was most prevalent in schizophrenic patients. Conclusion : Genotyping of CYP2D6 alleles among schizophrenic patients indicated prevalence of *4 and *10 variants in Karachi population producing non-functional and reduced functional drugs metabolizing enzymes respectively that increases the incurability rate of schizophrenia. Therefore, CYP2D6 gene screening program should be conducted routinely in clinical practice to help clinicians to prescribing appropriate doses according to patient’s genotype and minimize the sufferings of schizophrenia. Discussion: In last, drug response is a complex phenomenon that is dependent on genetic and environmental factors. CYP2D6 polymorphism may un-cured the schizophrenia due to improper drug metabolism and protein-proteins interaction that may alter the antipsychotic drugs metabolism among patients with variable drug resposes. Gene testing system need to establish for analyzing maximum patient’s genotypes predicted with poor metabolizer, intermediate metabolizer and ultrarapid metabolizer for the adjustment of antipsychotic drugs.

scholarly journals Molecular variation in chloroplast DNA regions in ancestral species of wheat.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 883-889 ◽  
Author(s):  
N T Miyashita ◽  
N Mori ◽  
K Tsunewaki
Keyword(s):  
Chloroplast Dna ◽  
Restriction Enzymes ◽  
Triticum Dicoccoides ◽  
The Other ◽  
Major Type ◽  
Aegilops Speltoides ◽  
Triticum Timopheevi ◽  
Restriction Sites ◽  
D Values ◽  
Chloroplast Dna Regions

Abstract Restriction map variation in two 5-6-kb chloroplast DNA regions of five diploid Aegilops species in the section Sitopsis and two wild tetraploid wheats, Triticum dicoccoides and Triticum araraticum, was investigated with a battery of four-cutter restriction enzymes. A single accession each of Triticum durum, Triticum timopheevi and Triticum aestivum was included as a reference. More than 250 restriction sites were scored, of which only seven sites were found polymorphic in Aegilops speltoides. No restriction site polymorphisms were detected in all of the other diploid and tetraploid species. In addition, six insertion/deletion polymorphisms were detected, but they were mostly unique or species-specific. Estimated nucleotide diversity was 0.0006 for A. speltoides, and 0.0000 for all the other investigated species. In A. speltoides, none of Tajima's D values was significant, indicating no clear deviation from the neutrality of molecular polymorphisms. Significant non-random association was detected for three combinations out of 10 possible pairs between polymorphic restriction sites in A. speltoides. Phylogenetic relationship among all the plastotypes (plastid genotype) suggested the diphyletic origin of T. dicoccoides and T. araraticum. A plastotype of one A. speltoides accession was identical to the major type of T. araraticum (T. timopheevi inclusively). Three of the plastotypes found in the Sitopsis species are very similar, but not identical, to that of T. dicoccoides, T. durum and T. aestivum.

scholarly journals A cookbook for DNase Hi-C

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Maria Gridina ◽  
Evgeniy Mozheiko ◽  
Emil Valeev ◽  
Ludmila P. Nazarenko ◽  
Maria E. Lopatkina ◽  
...  
Keyword(s):  
Rational Design ◽  
Restriction Enzymes ◽  
Nucleotide Exchange ◽  
Computational Tools ◽  
3 Dimensional ◽  
Cultured Human Cells ◽  
Dna Ends ◽  
By Products ◽  
Robust Protocol

Abstract Background The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation. Results In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming. Conclusions We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.

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