Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-01-13
1998-03-17
Campbell, Eggerton A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, C12Q 168, C12P 1934
Patent
active
057285241
DESCRIPTION:
BRIEF SUMMARY
This application is a National Phase filed under 35 USC .sctn.371 of PCT GB 93/01452 having priority to United Kingdom Patent No. 9214873.2 filed on Jul. 13, 1992.
This invention relates to a technique designed to facilitate study of the individual members of populations of nucleic acid sequences, particularly where the individual members of such populations may be present in widely varying amounts. This technique permits indexing of sequences.
Situations are increasingly arising in which it is necessary to study complex nucleic acid or polynucleotide populations. For example, it is now widely appreciated that an invaluable resource could be created if the entire sequence of the genomes of organisms such as man were determined and the information available. The magnitude of such a task should not, however, be underestimated. Thus, the human genome may contain as many as 100,000 genes (a very substantial proportion of which may be expressed in the human brain: Sutcliffe, Ann. Rev. Neurosci. 11:157-158 (1988)). Only a very small percentage of the stock of human genes has presently been explored, and this largely in a piecemeal and usually specifically targeted fashion.
There has been much public debate about the best means of approaching human genome sequencing. Brenner has argued (CIBA Foundation Symposium 149:6 (1990)) that efforts should be concentrated on cDNAs produced from reverse transcribed mRNAs rather than on genomic DNA. This is primarily because most useful genetic information resides in the fraction of the genome which corresponds to mRNA, and this fraction is a very small part of the total (5% or less). Moreover, techniques for generating cDNAs are also well known. On the other hand, even supposing near perfect recovery of cDNAs corresponding to all expressed mRNAs, some potentially useful information will be lost by the cDNA approach, including sequences responsible for control and regulation of genes. Nonetheless, the cDNA approach at least substantially reduces the inherent inefficiencies resulting from analysis of repeated sequences and/or non-coding sequences in an approach which depends upon genomic DNA sequencing.
Recently, the results of a rapid method for identifying and characterising new cDNAs has been reported (Adams, M. D. et al., Science 252, 1991, pp 1651-1656). Essentially, a semi-automated sequence reader was used to produce a single read of sequence from one end of each of a number of cDNAs picked at random. It was shown, by comparing the nucleic acid sequences of the cDNAs (or the protein sequences produced by translating the nucleic acid sequence of the cDNAs) to each other and to known sequences in public databases, that each of the cDNAs picked at random, could be unambiguously classified. The cDNAs could be classified as being either entirely new or as corresponding, to a greater or lesser extent, to a previously known sequence. cDNAs identified in this way were further characterised and found to be useful in a variety of standard applications, including physical mapping. Unfortunately, such a process is insufficient. The longer the process is pursued with any given population of cDNAs the less efficient it becomes and the lower the rate of identification of new clones. In essence, as the number of cDNAs which have already been picked rises, the probability of picking a particular cDNA more than once increases because of the wide range of abundancies at which different cDNAs are found, which abundancies can vary by several orders of magnitude. Thus, whereas some sequences are exceedingly rare, a single cDNA type may comprise as such as 10% of the population of cDNAs produced from a particular tissue (Lewin, B. Gene Expression, Vol. 2: Eucaryotic Chromosomes, 2nd ed., pp. 708-719. New York: Wiley, 1980). The need to avoid missing rarer species in any given population presents a considerable problem.
Various approaches have been tried in addressing the problem of increasing the efficiency of examination of a mixed nucleotide population, for example, such a population as is
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Campbell Eggerton A.
Medical Research Counsil
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