Problems of Replication

The replication process requires that each double-helical molecule of DNA produce two identical molecules of DNA. This means that wherever a G-C or A-T base pair occurs in the parental molecule, the identical base pair must occur in the progeny molecules. However, many factors interfere with accurate replication of DNA. If an A should pair with C or G with T as a result of tautomeriza-tion (Chapter 23), a point mutation (a change in one base pair) will result. Occasionally, a segment of DNA will be replicated more than once (duplication) or a segment may fail to be replicated (deletion). These and other aberrations in DNA replication do occur, but the mechanism of replication has evolved to minimize such mistakes. 

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DNA polymerase III in replication is described in detail. The special problems of replication arising from the amount of eukaryotic DNA in a cell and the structure of chromatin are introduced and the nature and functions of the telomeres and telomerase are described. 

An obvious approach to circumvent the problem of replicating plasmid-mediated inhibition of secondary metabolite production is to insert cloned DNA into the chromosome. A number of vectors are now available that are capable of site-specific integration into the chromosome. Table 4 lists some of these along with their salient features. 

Before a procedure can provide useful analytical information, it is necessary to demonstrate that it is capable of providing acceptable results. Validation is an evaluation of whether the precision and accuracy obtained by following the procedure are appropriate for the problem. In addition, validation ensures that the written procedure has sufficient detail so that different analysts or laboratories following the same procedure obtain comparable results. Ideally, validation uses a standard sample whose composition closely matches the samples for which the procedure was developed. The comparison of replicate analyses can be used to evaluate the procedure s precision and accuracy. Intralaboratory and interlaboratory differences in the procedure also can be evaluated. In the absence of appropriate standards, accuracy can be evaluated by comparing results obtained with a new method to those obtained using a method of known accuracy. Chapter 14 provides a more detailed discussion of validation techniques. 

Replication avoids the problem of sample deterioration in the instrument, but it is destructive in that reaction of the material cannot be continued after the replica has been prepared. Transitory features cannot be detected unless a series of preparations are examined corresponding to increasing progress of the reaction considered. The textures of replicas have been shown [220] to be in satisfactory agreement with those of the original surface as viewed in the scanning electron microscope. The uses and interpretations of observations made through sample replication procedures are illustrated in the studies of decomposition of metal carboxyl-ates by Brown and co-workers [97,221—223]. 

Even If the cost of estimation error cannot be quantified as this model requires, effective allocation of resources may be possible when detailed knowledge of sources of variation Is available. In this case, a replication strategy can be based on variance component and cost Information. For example, consider the problem of deciding how many samples to collect and how many analyses to perform on each sample Let... 

There are a number of other problems relating to the manipulation and interpretation of data that cause difficulty. The most common are (i) uncertainty about the number of replicate results required for proper comparison of the certified reference value, and (2) the actual analytical result and how gross outlier results should be handled. These issues and how to deal with data that falls outside the confidence limit are reviewed in detail by Walker and Lumley (1999), who conclude that whilst customer requirements may provide answers the judgement of the analyst must always be the final arbiter in any decision ... 

The method will now be tested in a ringtest by 8 dfferent laboratories in Europe. A preliminary range finding will be done in Vienna in October 1999. Test oganisms will be Stapylococus aureus and Pseudomonas aeruginosa, exposure time will be 5 minutes. The test surfaces will be stainless steel disks (2 cm diameter) with a specific finish. The problem of a standardized drying procedure was discussed. There was no decision about the number of replicates of the test. 

Gautier There are two possible explanations. The trivial one is that it is a problem of dose, in that in the embryo the right number of breaks is not reached, because it is a fairly large amount of cytoplasm and the signalling is too weak. The other option is that there is no replication checkpoint. Blocking replication in the embryo will therefore not affect the G2/M transition. The embryo will still divide even though it doesn t replicate. 

Gautier There is replication. We have looked at the amount of DNA, and there is replication. I suspect it is more likely to be a problem of dose. We need to reach a threshold of DNA ends in order for activation to occur. 

The basic problem of virus replication can be simply put the virus must somehow induce a living host cell to synthesize all of the essential components needed to make more virus particles. These components must then be assembled into the proper structure and the new virus particles must escape from the cell and infect other cells. The various phases of this replication process in a bacteriophage can be categorized in seven steps ... 

Notice that the common feature of many evolutionary transitions identified by Maynard Smith and Szathmary involves a change in the status of replicators. Once the replication of a class of entities becomes dependent on a larger whole, they do not become independent replicators again. So new replicators at the emergent level must exist and replicate independently in order for there to be a still higher level transition. Given the nature of replicators as analyzed by both Dawkins and Hull, there is a problem with this characterization of the common feature only one or possibly two evolutionary transitions are at all likely to have occurred in the history of life. 

A hypercycle is a more complex organisation form. Its precondition is the presence of several RNA quasi-species which are able to amalgamate chemically with certain proteins (enzymes or their precursors). If such a protein is linked to a quasi-species, the resulting duo favours the replication of a second quasispecies. According to Dyson, the linked populations get stuck in a stable equilibrium. Problems occur at this level Any theory on the origin of replication has the central problem that the replication process must occur perfectly in order to ensure survival . If there are replication errors, these will increase from generation to generation, until the system collapses the error catastrophe has then occurred ... 

Since all DNA polymerases require a primer and work only in the 5 to 3 direction, there s a problem with replicating the 5 ends of the DNA. If an RNA primer has to be laid down and later removed, these ends can t get replicated. For bacteria with a circular genome, this isn t a problem. Eukaryotes have specialized structures called telomeres at the... 

A set of replicate results should number at least twenty-five if it is to be a truly representative statistical sample. The analyst will rarely consider it economic to make this number of determinations and therefore will need statistical methods to enable him to base his assessment on fewer data, or data that have been accumulated from the analysis of similar samples. Any analytical problem should be examined at the outset with respect to the precision, accuracy and reliability required of the results. Analysis of the results obtained will then be conveniently resolved into two stages - an examination of the reliability of the results themselves and an assessment of the meaning of the results. 

An analyst in the true sense encounters a serious problem when the number of replicates at his disposal is SMALL. Firstly, the divergent result shows a distinct and significant effect upon the mean value (x) and... 

The standard deviation in measurements, however, can vary with the analyte concentrations. On the other hand, RSD, which is expressed as the ratio of standard deviation to the arithmetic mean of replicate analyses and is given as a percentage, does not have this problem and is a more rational way of expressing precision ... 

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