生物化学技术

Polymerase chain reaction (PCR) optimization and troubleshooting can consume considerable energy and resources because of the finicky and often unpredictable nature of the reactions. Small variations in any of the many variables in a given reaction can have a pronounced effect on the re ...

The ability to quantify nucleic acids accurately and rapidly is a prerequisite for many of the methods used in biochemistry and molecular biology. In the majority of situations this is carried out using spectrophotometry, which is nondestructive and allows the sample to be recovered for fur ...

The polymerase chain reaction (PCR) is a primer-mediated enzymatic amplification of specifically cloned or genomic DNA sequences (1). This PCR process, invented by Kary Mullis over 10 years ago, has been automated for routine use in laboratories worldwide. The template DNA contains the ta ...

There are several chemical methods for sequencing DNA. The classical method, introduced in 1977 by Maxam and Gilbert (1), uses four different chemical reactions to attack, modify, and open the heterocyclic bases at guanine (G), adenine and guanine (A + G), thymine and cytosine (T + C), and cytosine (C) in a r ...

The chemical method of sequencing DNA (1) has some advantages and some disadvantages compared with the enzymatic method (2). The major disadvantage is that it takes more time to produce the same amount of sequence. This is so for two main reasons. First, the DNA has to be end-labeled and then reisolated pr ...

The time and labor required for generating nucleotide sequence information has been significantly reduced since the development of the polymerase chain reaction (PCR). PCR allows the production of sufficient amounts of DNA template in vitro, and consequently the time-consuming cl ...

The polymerase chain reaction (PCR) is well known for being a rapid and versatile method for the amplification of defined-target DNA sequences. This technique can be applied to a variety of research areas, such as the identification and typing of single nucleotide substitutions of DNA sequen ...

Among the many techniques of cloning new genes, one approach involves degenerate primers (1–7). The approach usually requires three steps: 1. Using degenerate primers to amplify part of the gene of interest by PCR: The degenerate primers’ sequences may be designed from known pr

The analysis of RNA and DNA from clinical biopsy material for diagnostic and research purposes has become more and more important. Currently, available methods and kits are focusing on the extraction of only one kind of nucleic acid, but, as biopsy material is often limited, a method for the simulta ...

The use of magnetic particles in many fields of biochemistry, molecular biology, and medicine has been well documented and several magnetic particles are now available for diagnostic and cell separation purposes. The solid-phase approaches has improved robustness by the increased r ...

The advance of Tag-based polymerase chain reaction (PCR) technology (1–3) has had a tremendously positive impact on biomedical research. The combination of PCR and sequencing further revolutionized biological research (3–11). There are two general approaches for sequencing the DNA ...

The advent of direct sequencing of polymerase chain reaction (PCR) products has permitted extremely rapid analysis of DNA mutants and cDNA clones. However, direct PCR sequencing has been problematic for a number of technical reasons, including the presence of impurities and excess olig ...

Following the isolation of a clonal recombinant phage or plasmid after screening a cDNA library, the first analyses that are routinely performed are the determination of the insert size and the sequence of the 3′- and 5′-ends of the cloned fragment. Prior to the use of polymerase chain reaction (PCR), t ...

The dideoxy chain-termination method (1) involves enzymatic elongation of a oligonucleotide primer that is annealed to a single-stranded DNA template. Single-stranded DNA of bacteriophage M13 (2) or phagemid vectors (3) give the most consistently satisfactory sequencing data. H ...

In double-stranded DNA sequencing, the two DNA strands must first be separated to enable the primer to bind to the priming site. This may be done by treating the DNA with alkali. Conventionally, use of the alkali denaturation method involves neutralization of the sample by acid treatment followed ...

DNA sequencing involves a specific application of electrophoresis to resolve the linear single-stranded products of sequencing reactions. A 4–20% polyacrylamide gel is used, normally 0.4 mm thick and at least 40 cm in length.

The susceptibility of RNA to degradation by exogenous and endogenous RNase activity following cell lysis has been well documented (1,2). Moreover RNA usually occurs complexed with protein from which it must be released. Precautions to be taken against exogenous RNase include the use of pla ...

This chapter describes efficient procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. Phagemid vectors are used for placing randomly paired light (L) and heavy (H) ...

The storage of yeast artificial chromosome (YAC) libraries in ordered microtiter plates required a new approach to screening for clones containing specific DNA sequences. Screening libraries of some 60,000 clones by hybridization to filters prepared from individual 96-well micr ...

Yeast artificial chromosome (YAC) libraries stored in microtiter plates are available for screening as either complex PCR pools or hybridization filters generated from YACs gridded at high densities (see Chapter 57). Different libraries may be available as either PCR pools, hybridi ...