遗传学实验技术

相关专题 气相色谱仪由于结构复杂、条件设置多、恢复准备时间长等原因，在使用过程中经常会出现各种异常情况。如果不针对病因进行维护，会导致严重的后果。下面就简单介绍一下气相色谱仪在应用中易发生的异常情况及其检修办法。 一、气相色谱仪进样后不出色谱峰的故障： 气相色谱仪在进样后检测信号没有变化，也不出峰，输出仍为直线。遇到这种情况时，应从样品进样针、进样口。检测器至信号输出的顺序逐一检查。 1、首先检查注射器是否堵塞，如果没有问题， 2、再检 ...

The polymerase chain reaction (PCR) is a versatile, widely used method for the production of a very large number of copies of a specific DNA molecule (1,2). For some applications, it is advantageous to subclone the PCR product into a plasmid vector for subsequent replication in bacteria (3–6). Subcl ...

Among the many variations of polymerase chain reaction (PCR)-based mutagenesis procedures, the “megaprimer” method (1–5) is probably the simplest and most versatile. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cl ...

PCR amplification has become an extremely powerful and universally used technique for cloning and manipulating DNA segments. It is especially useful for the ability to alter the terminal sequences of an amplified product simply by using primers containing the desired changes. Howeve ...

Oligonucleotide-directed mutagenesis techniques are extensively used for studying gene regulation and DNA and protein structure/function relationships. A number of PCR-based mutagenesis methods have been developed recently (1–8). In general, these PCR-based approach ...

Flip-PCR was designed to allow the sequence inversion of small regions of DNA within a regulatory region in order to examine the importance of cis-acting elements on transcriptional activity (1). Flip-PCR, a modification of the two-step overlap extension method of site-specific mutagen ...

A number of mutagenesis methods allow the systematic survey of a region of transcriptional regulatory sequence for identifying functional elements. In these methods, clusters or blocks of point mutations are introduced at discrete locations that span a suspected regulatory region ...

PCR methodology is one of the fastest available procedures for site-directed mutagenesis (1,2). However, it has been criticized for a lack of reliability because of unwanted mismatches produced during the PCR reaction (3,4). In the present protocol, we describe an improvement on the effici ...

The examination of gene expression and the search for novel and/or tissue-specific genes has been facilitated by the presence of conserved domains among the members of gene families (e.g., ref. 1). As discussed in the previous chapters of this section, such a conserved domain can be exploited by des ...

Multigene families throw a particular twist into the problem of polymerase chain reaction (PCR) primer design. One frequently needs primers that will not only amplify the sequence of interest, but at the same time fail to amplify the set of very similar sequences that comprises the remaining me ...

The use of degenerate primers in the polymerase chain reaction (PCR) is an effective method for identifying related genes that share limited sequence similarity. Other methods, using oligonucleotide or heterologous probes, for example, may fail to identify genes that are not highly con ...

The invention of polymerase chain reaction (PCR) and its application to amplification of reverse transcribed cDNA copies of mRNA has opened new possibilities for the development of techniques for identification of changes in gene expression patterns. Two very similar variants of the s ...

Differential screening (1) is probably the most direct approach for the identification of new genes whose expression is associated with a change in physiological conditions. Traditionally, the approach involved the probing of duplicate plaque lifts of cDNA libraries with differe ...

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 ...

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 ...

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 ...

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, ...

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 ...

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 ...

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 ...