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An Introduction to PCR Primer Design and Optimization of Amplification Reactions

PCR optimization is usually performed in order to obtain maximum specificity and yield. In some applications for which the amount of template may be limiting, or when there is a large amount of nontarget sequences, the sensitivity is maximized. Nonoptimized conditions promote artifactu ...

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Amplification of Short Tandem Repeat Loci Using PCR

Short tandem repeat (STR) loci consist of repetitive elements of 3-7 nucleotides. The STR loci, which are numerous in the human genome, are highly polymorphic in length and may also vary in the sequences of the repetitive elements. The polymerase chain reaction (PCR) makes it possible to analyze ve ...

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17 Genotyping STR Loci Using an Automated DNA Sequencer

This introduction is intended to inform the forensic scientist of the historical development of STR typing on automated sequencers, as these precedents will have a bearing on the techniques and equipment used in the present-day laboratory. The section then concludes with a brief outline of ...

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Manual Electrophoretic Methods for Genotyping Amplified STR Loci

This chapter describes the manual typing of STR markers, detailing the electrophoretic techniques used to separate amplified products, and the relevant methods for their detection.

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19 Amplification and Sequencing of Mitochondrial DNA in Forensic Casework

Mitochondrial DNA (mtDNA) typing is increasingly used for the forensic identification of human remains (1-9). This is especially true when only limited quantities of sample are present, such as when the sample has undergone extensive degradation and nuclear-typing methods are ineffe ...

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Separation of PCR Fragments by Means of Direct Blotting Electrophoresis

For separation of alleles of PCR-dependent short tandem repeat (STR) polymorphisms, high resolution with the power to discriminate fragments differing by one base pair is often desired. Furthermore, sequencing reactions also require unambiguous one base-pair separation. For bo ...

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Recovery of High-Molecular-Weight DNA from Blood and Forensic Specimens

The isolation of genomic deoxyribonucleic acid (DNA) is a crucial step in the process of DNA profiling. The success of all subsequent genetic-typing procedures depends on the availability of sufficient amounts of highly purified DNA from biological crime stains as well as from reference b ...

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Transformation of Oats and Its Application to Improving Osmotic Stress Tolerance

Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic stress due to drought and/or salinity. To genetically transform the available commercial oat cultivars, a genotype-independent and efficient regeneration system from shoot apical merist ...

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Transgenic Wheat, Barley and Oats: Production and Characterization

Ever since the first developments in plant transformation technology using model plant species in the early 1980s, there has been a body of plant science research devoted to adapting these techniques to the transformation of crop plants. For some crop species progress was relatively rapid, ...

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Selection of Transformed Plants

The low frequency and randomness of transgene integration into host cells, combined with the significant challenges of recovering whole plants from those rare events, makes the use of selectable marker genes routine in plant transformation experiments. For research applications ...

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Biolistics Transformation of Wheat

We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used meth ...

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Reporter Genes

Reporter genes have been widely used in plant molecular biology, typically to discern patterns of gene expression, but also as markers of transformed cells during stable transformation procedures. The ideal marker gene would be expected to display characteristics such as ease and chea ...

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Transposon-Based Mutagenesis Generates Diverse Adeno-Associated Viral Libraries with Novel Gene Delivery Properties

The engineering of novel properties and functions into viral vectors for improved gene delivery remains a barrier to the development of efficient, customized gene delivery vehicles. Rational methods for designing improved viral vectors are often experimentally challenging and ...

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Photochemical Enhancement of DNA Delivery by EGF Receptor Targeted Polyplexes

Photochemical internalization (PCI) is a physico-chemical targeting method that enables light directed delivery of nucleic acids into cells. The technology is based on photosensitizers that localize in the membranes of endocytic vesicles. A light activation of the photosensit ...

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Reducing the Genotoxic Potential of Retroviral Vectors

The recent development of leukemia in gene therapy patients with X-linked severe combined immunodeficiency disease because of retroviral vector insertional mutagenesis has prompted reassessment of the genotoxic potential of integrating vector systems. In this chapter, va ...

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Evaluation of Promoters for Use in Tissue-Specific Gene Delivery

Vectors used in gene therapy require an expression cassette. The expression cassette consists of three important components: promoter, therapeutic gene and polyadenylation signal. The promoter is essential to control expression of the therapeutic gene. A tissue-specific prom ...

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Adenovirus-Mediated Transduction of Auto- and Dual-Regulated Transgene Expression in Mammalian Cells

Transduction of therapeutic transgenes using multiply attenuated viral vectors is considered an essential technology for gene therapy scenarios. While first-generation viral transduction systems were engineered for constitutive expression of a single therapeutic tr ...

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Regulated Expression of Adenoviral Vectors-Based Gene Therapies: Therapeutic Expression of Toxins and Immune-Modulators

Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expr ...

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Liver-Directed Gene Therapy Using the Sleeping Beauty Transposon System

Sleeping Beauty (SB) is a transposon system genetically reconstructed from teleost fish that mediates chromosomal integration of DNA sequences by a cut-and-paste mechanism. SB has been shown to mediate transposition in a variety of cells and tissues, has been used for the generation of tra ...

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Generation and Functional Analysis of Zinc Finger Nucleases

The recent development of artificial endonucleases with tailored specificities has opened the door for a wide range of new applications, including the correction of mutated genes directly in the chromosome. This kind of gene therapy is based on homologous recombination, which can be sti ...

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