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丁香实验推荐阅读
PCR Fluorescence Differential Display

Differential display of mRNA via polymerase chain reaction (DD-PCR) has become a powerful procedure for the quantitative detection of differentially expressed genes in distinct cell populations (1–4).

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AU-Differential Display, Reproducibility of a Differential mRNA Display Targeted to AU Motifs

AU-rich elements (AREs) are found in 3′ untranslated regions (3′ UTR) of many highly unstable mRNAs for mammalian early-response genes. The minimal AU sequence core within the ARE is the heptamer WAUUUAW, although from a functional point of view, several pentanucleotides clustered in close p ...

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Differential Display: A Technical Overview

Since the completion of the human genome-sequencing project, scientists are now able to read the code of all human genes stored on the 46 chromosomes of the human genetic library. However, we are far from reaching an understanding of the functional relationships existing between more than a tiny f ...

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Quantitation of Multiple RNA Species

Real-time quantitative polymerase chain reaction (PCR) methods (1) can be further optimized for various purposes by performing quantitative PCR for multiple RNA species in one sample (2). Advantages of this method not only include the elimination of differences in reaction mix volumes ...

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Quantitative PCR for cAMP RI Alpha mRNA: Use of Site-Directed Mutation and PCR Mimics

Precise and accurate determination of mRNA expression levels in tissues and model systems is a central methodology in a wide range of research applications. Expression of many genes is currently assessed by northern blotting, RNAse protection assays, Serial Analysis of Gene Expressi ...

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Ultrasensitive Quantitative PCR to Detect RNA Viruses

The use of quantitative polymerase chain reaction (qPCR) to detect RNA viruses has become increasingly important as a prognostic marker and in patient management, for example, in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. Drug therapies can be monitored by r ...

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Ultrasensitive PCR Detection of Tumor Cells in Myeloma

Chromosomal aberrations, such as translocations or inversions, described for a growing number of malignancies, are now widely used to detect tumor cells by polymerase chain reaction (PCR). However, in multiple myeloma (MM), no such ubiquitous PCR marker exists. Therefore, other means ha ...

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Qualitative and Quantitative PCR: A Technical Overview

The nature of the polymerase chain reaction (PCR) process lends itself well to qualitative determinations. It transforms very small quantities of analyte into the realms of bucket chemistry, allowing specific gene portions to be directly visualized with ethidium bromide and ultrav ...

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Long PCR Methodology

In this chapter, we detail protocols of long polymerase chain reaction (PCR) and long RT-PCR, which we have found to be versatile, sensitive, and straightforward to optimize. We have used these protocols with success on several different templates, including lambda phage DNA, HAV, HBV, HCV (1), to ...

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Long PCR Amplification of Large Fragments of Viral Genomes: A Technical Overview

The polymerase chain reaction (PCR) has become an essential and ubiquitous tool for biological research and laboratory diagnostic applications. Until recently, reliable and sensitive amplification of large templates (several kb) was difficult to achieve. However, in 1994, an imp ...

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Direct PCR from Serum: Application to Viral Genome Detection

Nucleic acids used for polymerase chain reaction (PCR) assays usually are extracted by the phenol-chloroform method or an alternative rapid purification. The acid-guanidinium thiocyanate-phenol-chloroform method for RNA extraction and proteinase K digestion-phenol-c ...

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Microsphere-Based Single Nucleotide Polymorphism Genotyping

Single nucleotide polymorphisms (SNPs) are single base differences in genomic DNA (1). These single-base mutations, estimated to occur every 1000 bases, are thought to represent the most common form of genetic variation in the human genome (2). Several million SNPs have been identified (3). H ...

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Randomly Amplified Polymorphic DNA Fingerprinting: The Basics

The study of genetic polymorphism among diverse populations of organisms is a complex task. However, this can be accomplished by using newer tools, such as randomly amplified polymorphic DNA (RAPD). RAPD is a polymerase chain reaction (PCR) technique that relies on the generation of amplifi ...

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Subcycling PCR for Long-Distance Amplifications of Regions with High and Low Guanine-Cystine Content: Amplification of the Intron 22 Inversion of the

Hemophilia A is an X-linked disorder caused by mutations in the factor VIII gene. Around 50% of all patients with severe hemophilia A share a common mutation. This intron 22 inversion results from homologous recombination of a sequence within intron 22 of the factor VIII gene and identical sequence ...

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Nested RT-PCR in a Single Closed Tube

There are several methods now in widespread use for detecting and characterizing specific RNA targets. These methods include in situ hybridization, Northern blotting, dot or slot blot, RNase protection assay, and reverse transcription coupled to polymerase chain reaction (RT-PCR). ...

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Mutation and Polymorphism Detection: A Technical Overview

Analysis of DNA variation (polymorphism and mutations) is one of the most common challenges faced by molecular biologists. Studies of polymorphisms and mutations as molecular markers of or underlying causes of disease have confirmed the importance of mutation and polymorphism dete ...

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RACE and RAGE Cloning in Parasitic Microbial Eukaryotes

Many gene-cloning strategies and gene survey often provide partial sequence data. To exploit the information from these partial sequences numerous PCR-based approaches have been developed to clone full-length open reading frames. These approaches can be successful using small q ...

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Positive Selection Scanning of Parasite DNA Sequences

Parasites successfully exist within the host as a result of highly specific genetic adaptations. Therefore, detecting genes that contain relevant adaptive mutations can provide a guide to biological processes that are potentially essential to the parasite. Random genetic mutat ...

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Expressed Sequence Tags: Analysis and Annotation

Expressed sequence tags (ESTs) present a special set of problems for bioinformatic analysis. They are partial and error-prone, and large datasets can have significant internal redundancy. To facilitate analysis of small EST datasets from in-house projects, we present an integrated “ ...

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Expressed Sequence Tags: Medium-Throughput Protocols

Generating expressed sequence tags is a simple, cheap, and efficient way to sample the genome of a target organism. An expressed sequence tag (EST) is a single-pass sequence derived from a single complementary DNA (cDNA) clone, and the sequence serves to identify the gene from which it derives. We pre ...

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