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丁香实验推荐阅读
Generation of a PCR-Renewable Source of Subtractive cDNA

Differential (+/-) first-strand cDNA screening methods identify clones corresponding to mRNAs that are expressed at a higher level in one of a pair of phenotypically different cells. This approach is limited by the fact that screening of libraries with labeled first-strand cDNAs synthes ...

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Subtractive cDNA Cloning Using Magnetic Beads and PCR

Subtractive cloning is a method that facilitates the isolation of nucleotide sequences present in a test sample but absent or present at much lower levels, in a reference sample. A variety of situations lend themselves to this methodology and it has been used, for example, to characterize changes ...

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Normalization of cDNA Sequence Representation by Molecular Selection

Consider the situation in which one would like to clone sequences that are differentially expressed between two developmental stages. An experiment could be set up in which cDNA from one developmental stage is taken as reference and cDNA from a later developmental stage is taken as target. The ob ...

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Generation and PCR Screening of Bacteriophage Sublibraries Enriched for Rare Clones (the Sublibrary Method)

The simple sublibrary method described in this chapter allows the detection and rapid isolation of rare clones from bacteriophage λ libraries. The method is based on the ability of PCR to detect clones present in a library at very low frequencies. Clones present at frequencies as low as one in 10,000, ...

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Use of PCR for cDNA Library Screening

cDNA cloning from a library is now a routine laboratory practice. Hybridization screening with either radiolabeled or nonradiolabeled probes, which is laborious and time-consuming, has been commonly used. Application of the polymerase chain reaction (PCR) is surprisingly expan ...

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Rapid Directional Walk Within DNA Clones by Step-Out PCR

Genomic segments of many model experimental organisms are now available as segments within cosmid, P1, or YAC vectors (1–3). Even cDNA portions of these segments are becoming available. Preliminary analyses with any of these clones would include sequencing and mapping (either on the genome ...

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Anchoring a Defined Sequence to the 5 Ends of mRNAs: The Bolt to Clone Rare Full-Length mRNAs

Among numerous applications, the PCR (1,2) provides a convenient means to clone 5′ ends of rare messengers and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods (e.g., screening of cDNA libraries). Basically, the amplification of cDNAs by ...

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An End-Trimming Method and Its Application to Amplify Adjacent cDNA and Genomic DNA Fragments by PCR

An end-trimming method was developed to amplify adjacent cDNA fragments by PCR (1,2). In an attempt to clone a novel cDNA, the 5′ and/or 3′ end are often missing. A well-known PCR technique for cloning the missing sequence is a method of rapid amplification of cDNA ends (RACE) (3). Although the missing 5′ and 3′ ends ...

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Amplification of Gene-Regulating Regions with Single-Sided Specificity

The isolation of genomic 5′ regulating regions of genes starting from a suitable sequence (either a cDNA or an amino acid derived oligonucleotide) can be achieved in several ways. One of these, although long and labor-intensive, is the screening of a genomic library, followed by the isolation, sub ...

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Using the SELEX Combinatorial Chemistry Process to Find High Affinity Nucleic Acid Ligands to Target Molecules

The SELEX (Systematic Evolution of Ligands by EXponential enrichments) combinatorial chemistry process is a procedure by which nucleic acid ligands of high affinity can be isolated against a molecular target (1). A wide variety of target molecules have been used successfully in the SELEX ...

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Isolation of Coding Sequences from Yeast Artificial Chromosome (Yac): Clones by Exon Amplification

Exon amplification is a technique designed to address a central problem in mammalian molecular genetics—how to extract coding sequences from large tracts of genomic DNA. As shown in Fig. 1, the technique (also known as exon trapping) exploits the ability of the eukaryotic splicing machinery ...

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Rapid Amplification of Gene Ends (RAGE) from Gene Libraries by Anchored PCR

Isolation of a full-length gene on the basis of limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically, nucle ...

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Inverse PCR: An Efficient Approach to Cloning cDNA Ends

Since the first report on cDNA cloning in 1972 (1), this technology has developed into a powerful and universal tool in isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. However, the conventional methods of cDNA cloning require much effort to generate a lib ...

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Sequence-Based High Resolution Chromosomal Comparative Genomic Hybridization (CGH)

We aimed to devise an appropriate method to directly link the fluorescence profile of chromosomal copy number alterations detected by chromosomal comparative genomic hybridization (cCGH) or any other hybridization or staining information with the genome sequence data. Our goal w ...

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Maternal mRNA Knock-down Studies: Antisense Experiments Using the Host-Transfer Technique in Xenopus laevis and Xenopus tropicalis

The ability to inhibit the activity of maternally stored gene products in Xenopus has led to numerous insights into early developmental mechanisms. Oocytes can be cultured and manipulated in vitro and then implanted into the body cavity of a host female to make them competent for fertilizati ...

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Genetic Analysis of Xenopus tropicalis

The pipid frog Xenopus tropicalis has emerged as a powerful new model system for combining genetic and genomic analysis of tetrapod development with robust embryological, molecular, and biochemical assays. Its early development closely resembles that of its well-understood rela ...

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Forward Genetic Screens in Xenopus Using Transposon-Mediated Insertional Mutagenesis

The class II DNA “cut-and-paste” transposons have been used to efficiently modify the Xenopus genome for transgenesis applications. Once integrated, the transposon is an effective substrate for excision and re-integration (remobilization) elsewhere in the genome by simply supp ...

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Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides

Here we present a protocol, which allows loss-of-function studies in Xenopus embryos using antisense morpholino oligonucleotides (MOs). Gene knockdown studies provide a critical method for assessing gene function in vitro and in vivo. Such studies are currently performed in Xenopus ...

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Targeted Gene Disruption with Engineered Zinc-Finger Nucleases (ZFNs)

Reverse genetics in Xenopus has been limited to knockdown strategies using antisense morpholino oligonucleotides (MOs). Recently, engineered zinc-finger nucleases have been used to induce targeted mutations resulting in null alleles. Zinc-finger nuclease (ZFN) technolo ...

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Chemical Genetics and Drug Discovery in Xenopus

Chemical genetics uses small molecules to modulate protein function and has the potential to perturb any biochemical event in a complex cellular context. The application of chemical genetics to dissect biological processes has become an attractive alternative to mutagenesis scr ...

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