基因表达差异显示实验

The availability of whole genomic sequences provides a great framework for biologists to address a broad range of scientific questions. However, functions of most mammalian genes remain obscure. The forward genetics strategy of insertional mutagenesis uses DNA mutagens such as ret ...

SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. Combined with the ultrahigh-throughput sequencing technologies, SuperSAGE enables analysis of millions of transcripts with lower cost and redu ...

MicroRNAs (miRNAs) are ∼22 nucleotide regulatory RNA molecules that play important roles in controlling developmental and physiological processes in animals and plants. Measuring the level of miRNA expression is a critical step in methods that study the regulation of biological fu ...

Alu PCR is a rapid and easy-to-perform “DNA fingerprinting” technique based on the simultaneous analysis of many genomic loci flanked by Alu repetitive elements, which allows the detection of genetic polymorphisms and mutations in human and primate genomes. In the protocol described in t ...

Asynchronous PCR (aPCR) is a new PCR method that directs an ordered and sequential amplification of the + and − strands of DNA amplicons. There are several unique characteristics of aPCR that generate new application opportunities. The melting temperature (Tm) of the forward and reverse aPCR p ...

The ability to amplify genetic material using PCR has transformed the field of diagnostics. Now any organism can be detected by identifying the presence of specific nucleic acids. However, there still remain areas in which traditional PCR cannot easily be applied. In this chapter, we describe a ...

“Universal” or group-specific PCR primers have a tendency to predominately hybridise with the common sequences in samples with mixed templates. The result is that the rarer sequences are seldom retrieved by cloning or sequencing. The use of a blocking oligonucleotide (oligo) designed to ...

The loading of amplicons onto solid supports such as beads during multiplex PCR or emulsion PCR conventionally has been performed by use of Solid Phase PCR or asymmetric Solid Phase PCR. These approaches are restrictive with respect to amplification efficiency and degree of amplicon load ...

Overlap extension PCR (OE-PCR) has been widely used in site-directed mutagenesis. The original OE-PCR included two rounds of PCRs and required tedious steps to purify the first-round PCR product. By combining asymmetric PCR and overlap extension, a novel asymmetric overlap extension PCR ...

The selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works e ...

Nucleotide analogues are used increasingly in medicine and biotechnology to effect DNA sequence change, principally via clastogenic and transcriptional effects. This article, however, discusses the use of mutagenic nucleotide analogues to improve the sequencing of recalci ...

The cap analysis of gene expression (CAGE) technology has been established to detect transcriptional starting sites (TSSs) and expression levels by utilizing 5′ cDNA tags and PCR. It has been reported that the amount of templates is proportional to the amplification efficiency of PCR. CAGE h ...

In vivo footprinting and ligation-mediated PCR (LMPCR) are well-established methods for the examination of the chromatin structure of eukaryotic genes. Here, we describe an improved method (pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR) that overcomes the ...

The experimental measurement of haplotype requires the determination of two or more genotypes on the same DNA molecule. Because such measurements are much more complicated than measurements of genotypes, haplotypes are typically inferred using population data for linkage diseq ...

Multiplex Ligation-dependent Probe Amplification (MLPA) is a PCR-based technique that was developed for identifying deletions and duplications in genomic DNA. The simplicity and sensitivity of this approach has led to it being implemented in many laboratories around the world. Si ...

As DNA methylation analysis enters the mainstream of biomedical research, there is increasing interest in methodologies that can be used for rapid analysis of a wide variety of biological and clinical samples. The methods that have been commonly used for methylation analysis are usually t ...

We describe here a protocol to faithfully amplify global cDNAs from single cells. The amplified cDNAs retain their sense–antisense orientation and can be easily applied to template preparation for quantitative high-density oligonucleotide microarray analyses. The amplific ...

While PCR primer design for the amplification of known sequences is usually quite straightforward, the design, and successful application of primers aimed at the detection of as yet unknown genes is often not. The search for genes that are presumed to be distantly related to a known gene sequence, s ...

We describe a single nucleotide polymorphism (SNP) typing protocol developed for the NanoChip electronic microarray. The NanoChip array consists of 100 electrodes covered by a thin hydrogel layer containing streptavidin. An electric currency can be applied to one, several, or all ele ...

Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and miniseque ...