遗传学实验技术

It is sometimes desirable to extract both RNA and DNA from the same sample, especially when the sample is small. This can be achieved by isolating a total nucleic acid fraction that is then divided into two portions, which are treated differentially with either Dnase I (to remove DNA and recover RNA) or with ...

There are two different methods of preparing tissue for histology: paraffin-embedding and freeze-embedding. Each has their advantages and drawbacks. Paraffin-embedded tissues (PET) produce optimum morphology but have comparatively poor molecular preservation and reco ...

Although individual microorganisms may well require a unique DNA extraction procedure, here we include robust techniques for the preparation of DNA from fungi, yeast, and bacteria, which yield DNA suitable for a PCR template.

The DNA extraction process represents one of the critical stages in the analysis of degraded or ancient DNA. If polymerase chain reaction (PCR) amplification starts from a poor extract containing low template quantities, stochastic variation in the amplification of individual alle ...

The successful extraction of viral RNA from biological material requires rapid transport and adequate storage of samples because of the unstable nature of RNA. Samples should be received and processed within 6 h and the relevant fractions stored at −70�C until testing. Also, it is difficult to a ...

In following any polymerase chain reaction (PCR)-based method, it is usual to identify the products of the reaction by some form of detection system. The majority of these still rely on size- and charge-based separation systems, although for some quantitative PCR applications, either direct ...

There are occasions where the only materiel available on a patient is stored plasma or serum samples. In normal individuals, the amount of DNA in these samples is very low but sufficient to serve as template for PCRs. Moreover, increased amounts of circulating DNA have been found in a variety of disorde ...

Although the best way of obtaining pure polymerase chain reaction (PCR) product will always be to optimize reaction conditions to yield only one product, there are still circumstances where DNA has to purifed from gels. Several good commercial products exist for the recovery of DNA from agaro ...

There are many differing protocols and a large number of commercially available kits used for the extraction of DNA from whole blood. This procedure is one we use routinely in both research and clinical service provision and is cheap and robust. It can also be applied to cell pellets from dispersed tis ...

The polymerase chain reaction (PCR) is a powerful method for fast in vitro enzymatic amplifications of specific DNA sequences. PCR amplifications can be grouped into three different categories: standard PCR, long PCR, and multiplex PCR. Standard PCR involves amplification of a single DNA ...

Based on the method of Chomczynski and Sacchi (1), this is an extremely reliable method without the requirement for centrifugation over CsCl gradients. As with any RNA protocol, extreme care should be taken to exclude RNAse contamination, the greatest source of which will be the sample itself. All ...

Determining the chromosomal origin of expressed sequence tags (ESTs) (1,2) lags far behind their identification in single-pass sequencing projects (1–10). Positional cloning of disease genes requires that previously uncharacterized transcripts be mapped to the smallest pos ...

The microsatellite repeat motifs (dC-dA) n are present in high abundance in the normal genome (1). If they were to occur at regular intervals, they could be as frequent as one in approximately every 30–40 kb of the human genome. Thus, the entire genome can be represented by a large number of dC-dA or dG-dT repeat se ...

Since the advent of the polymerase chain reaction (PCR), a variety of PCR-based procedures of mutagenesis have been developed through the use of synthetic primers encoding the mutation. Among these, the megaprimer method and related ones (1–5) remain some of the simplest and most versatile. Va ...

Labeling DNA during in vivo replication by the incorporation of exogenous thymidine and thymidine analogs has been a mainstay of DNA replication and repair studies for decades. Unfortunately, thymidine labeling does not work in fungi, because they lack the thymidine salvage pathway re ...

In metazoans, development and cell differentiation are known to affect various aspects of chromosomal organization at developmentally regulated gene loci (e.g., nuclear localization, locus accessibility, chromatin modifications, etc.). Recent evidence also indicates th ...

Plasticity is an inherent feature of chromosomal DNA replication in eukaryotes. Potential origins of DNA replication are made in excess, but are used (fired) in a partly stochastic, partly programmed manner throughout the S phase of the cell cycle. Since most origins have a firing efficiency b ...

The detection of breaks in mammalian cell DNA and the measurement of their repair represent primary endpoints for genotoxicity testing. Over the past three decades many techniques sensitive to the presence of DNA breaks have been developed: their availability significantly increas ...

Replication forks (RFs) frequently encounter barriers or lesions in template DNA that can cause them to stall and/or break. Efficient genome duplication therefore depends on multiple mechanisms that variously act to stabilize, repair, and restart perturbed RFs. Integral to at least so ...

New technologies such as DNA combing have led to the availability of large quantities of data that describe the state of DNA while undergoing replication in S phase. In this chapter, we describe methods used to extract various parameters of replication — fork velocity, origin initiation rate, fo ...