生物化学技术

After transformation, cells containing parental or recombinant plasmids are usually selected by taking advantage of an antibiotic resistance marker present on the vector plasmid. Cells containing recombinant plasmids can often be identified as containing recombinant plas ...

The Klenow fragment of DNA polymerase I is a proteolytic fragment obtained by the treatment of DNA polymerase I with subtilisin. The fragment still has the polymerase and 3′–5′ exonuclease activity, but lacks the 5′–3′ exonuclease activity of the holoenzyme (1). Radiolabeling of DNA fragments is ...

In contrast to the end labeling methods described in chapters 39 and chapters 41, end labeling with terminal transf erase can be used for DNA fragments with 3′ protruding ends, 3′ recessed ends, and blunt ends. Terminal deoxynucleotidyl transferase (terminal transferase) adds ribonucleo ...

The pentose phosphate pathway plays an important role in several cellular processes including biosynthesis and catabolism of five-carbon sugars and generation of reducing power through NADPH synthesis. Although the pentose phosphate metabolic reaction network has been mapp ...

Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentra ...

In this chapter, we describe a method for the quantitative analysis of glycolytic intermediates using ion chromatography-mass spectrometry (IC-MS) and gas chromatography (GC)-MS as complementary methods. With IC-MS-MS, pyruvate, glucose-6-phosphate, fructuse-6-phospha ...

In recent years, bile acids (BAs) have received much attention as signaling molecules as well as biosurfactants for lipophilic nutrients. To understand exact BA behavior, the precise distribution of BAs in vivo must be determined. However, to date, it has been difficult to know the precise roles ...

The determination of purines and pyrimidines in biofluids is useful for the clinical–biochemical characterization of acute and chronic pathological states that induce transient or permanent alterations of metabolism. In particular, the diagnosis of several inborn errors of m ...

The analysis of organic acids in urine is commonly included in routine procedures for detecting many inborn errors of metabolism. Many analytical methods allow for both qualitative and quantitative determination of organic acids, mainly in urine but also in plasma, serum, whole blood, am ...

With the development of new technologies for simultaneous analysis of many genes, transcripts, or proteins (the “omics” revolution), it has become common to outsource parts of the experimental work. In order to maintain the integrity of the research projects, it is important that the interp ...

MicroRNAs (miRNAs) are small 20–22 nt long RNAs which function as post-transcriptional regulators altering the expression of genes either by blocking translation or by destabilizing mRNAs (for recent reviews see, e.g., Zhang et al. (J Cell Physiol, 210:279–289) and Engels and Hutvagner (On ...

Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. ...

Electrophoretic mobility shift assay, or EMSA, is a well-established technique for separating macromolecules under native conditions based on a combination of shape, size, and charge. The use of EMSA can provide both general and specific information concerning the interaction betw ...

The post-transcriptional operon provides a means of synexpression of mRNAs encoding interrelated proteins. The coordination of gene expression may be achieved by a trans-acting RNA-binding protein attaching to similar cis-elements in different, yet functionally clustered, ...

Post-transcriptional regulation of gene expression plays an important role in complex cellular processes. Just like transcription factors regulate gene expression through combinatorial binding to multiple, physically dispersed cis elements, mRNA binding proteins can r ...

A post-transcriptional operon is a set of monocistronic mRNAs encoding functionally related proteins that are co-regulated by a group of RNA-binding proteins and/or small non-coding RNAs so that protein expression is coordinated at the post-transcriptional level. The post-trans ...

Poly(A) tail length plays an important role in mRNA stability and translational control. Poly(A) fractionation is a very powerful technique to separate mRNAs according to the length of the poly(A) tail. Poly(A) fractionation can be used to detect small changes in poly(A) tail length or to prepare sa ...

Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membrane is a ...

Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full ...

Like protein coding sequences, functional motifs in RNA elements are frequently conserved, but this conservation is most often at the structure level rather than sequence based. Proper characterization of these structural RNA motifs is both the key and the limiting step to understandi ...