Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-01-18
2004-08-24
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06780593
ABSTRACT:
The present invention relates to a method for mapping a DNA molecule of the Happy Mapping type which comprises a step for ad infinitum amplification of the DNA of each panel. The present invention also relates to a kit for implementing this mapping method and to the use of the maps obtained for identifying genes imparting a phenotype of interest.
The emergence of a great number of genome sequencing projects and notably that of the human genome inevitably requires the development of novel fast and accurate mapping methods in order to carry out the assembling of raw data from systematic sequencing. Moreover, if the present trend of proceeding with shotguns of entire genomes is confirmed, detailed genomic maps will need to become available.
More than a dozen of microbial genomes have already been fully sequenced, as a result of a direct shotgun sequencing of their genome tigr.org/tdb/mdb.mdb.html). However, the latter have sizes from 580 kb to 4 Mb. The use of such an approach for sequencing larger genomes such as the human genome (3 000 Mb), as suggested by Weber and Myers, 1997, and announced by Venter et al., 1998 does however give rise to certain questions. Indeed, the large size of these genomes and the presence of many repeated sequences makes the assembling of the sequencing results difficult. Thus, it proves necessary to have detailed genomic maps in order to allow these data to be processed.
The drawing up of detailed genomic maps may also be very helpful for phylogenic studies. Indeed, studies on the evolution of genomes have clearly shown that the expression of many genes depends on their localization in a certain genetic context. Recent developments in genomics now allow the evolution of genomes to be studied in more detail on the basis of syntenic relationship changes. With detailed maps, obtained for two species, genetic links which have been maintained between these two species during evolution, may be assessed.
Another field for which the provision of detailed genetic maps would be of fundamental interest, is the localization of QTL (Quantitative Trait Loci). Indeed, most variations within a population or among different races, for example, are of a quantitative nature. Certain variations such as the size, the weight of individuals, the flowering date for plants or the amount of milk produced in mammals are not included in well-defined classes according to Mendelian proportions, but rather they operate in a continuous manner, according to a gradient from one extreme to the other. These variations preserved in the line of descent, are therefore transmitted genetically. The loci involved in the variation of quantitative phenotype traits are called QTL. Detection of links between a QTL and genetic markers provide a robust method for identifying these QTL. It is possible to localize a QTL by the so-called “interval mapping” (Lander et Botstein, 1989) method between two informative markers separated by more than 20 cM. However, such an interval makes the identification of the gene, problematical. Also, it is quite useful to be able to perform a zoom on the region of interest if a large number of markers are available, and to perform a fine mapping, in order to localize the sought-after gene specifically.
A conceivable mapping approach is the mapping by radiation hybrids (RH, Radiation Hybrid mapping) (Cox et al., 1990; Gyapay et al., 1996). It consists in irradiating cell lines, causing chromosomal random breaks. The different fragments of generated chromosomes are then integrated into the genome of the rodent cells. Thus, it is possible to determine the distance separating two markers by knowing that the closer they are, the more likely they will be incorporated within a same fragment and therefore be detected in a same line. However, this approach has certain drawbacks in addition to its cumbersomeness in the setting-up of a panel of radiation hybrids. Indeed, (i) certain loci which would not be cloned, cannot be integrated into a genome map; (ii) the interpretation of results may be confusing when the inserts are rearranged or ligated with each other; (iii) the presence of exogenous DNA, in this case the one of the hamster host cell, very often requires that a certain number of markers be set aside, those giving a positive response with this exogenous DNA.
With the more flexible Happy Mapping method, the different problems (Dear and Cook, 1993) may be circumvented. With this method, both events analyzed by cross-breedings of formal genetics, i.e. crossing-over and segregation, may be reproduced in vitro. In practice, crossing-over is mimicked by a random break of DNA into fragments, the size of which depends on the sought-after mapping. The markers are then segregated by a random distribution of these fragments into deposits of at least one equivalent of haploid genome per aliquot, then detected by PCR. Those which are genetically linked, tend to remain together in the same aliquot whereas those which are not linked, are randomly distributed. Their order and the distance which separate them may be inferred from the sequence of their co-segregation by a statistical calculation. It is important to remind here, that a panel which may be used for Happy mapping, is simple to produce as it only requires a few days or even a few weeks. In addition, it may be adapted to any resolution level, according to the size of the selected fragments and may even result in molecular cloning of fragments of interest for sequencing.
The Happy Mapping method comprises the following steps:
a) Genomic DNA is broken by irradiation,
b) About one equivalent of haploid genome is then placed in each well of a 96-well plate, which corresponds to about 60% of the initial genomic DNA (statistical distribution of the markers).
c) the DNA is amplified by PCR,
d) and the markers are then detected.
This mapping method has already proved to be reliable for genomes as different in size as the human chromosome 14-100 Mb—(Dear et al., 1998) or that of a parasite protozoan of the intestinal epithelium of many mammals, Cryptosporidium parvum—10 Mb—(Piper et al., 1998). However, for these two investigations, no satisfactory method was described for amplifying the initial panel, for mapping an unlimited number of markers and any kind of markers. In Dear et al., only a small portion of the total DNA, flanked by repeated sequences was able to be mapped. In Piper et al., the amplification level was not sufficient for providing direct detection of the markers by PCR. These authors had to proceed with nested PCR in order to view the markers to be mapped. Further, the amplification method used only allows a limited number of markers to be mapped, requiring a mapping panel to be reconstructed in order to localize further markers.
In order that the amplification method may be contemplated for Happy mapping, it should meet the following three criteria:
(i) A DNA amount close to one equivalent of haploid genome should be sufficient as a matrix;
(ii) the entire genetic information should be amplified;
(iii) the formed panel should be able to be re-amplified ad infinitum in order to provide mapping of an illimited number of markers.
Thus, the problem consists of amplifying the entire DNA in each well, whereby said amplification should not produce artefacts in the random distribution of markers. The objective is the development of an approach for total homogeneous and ad infinitum amplification of genomic DNA.
The conventional PCR technique has evolved, providing many amplification methods each having their own specificity. For example, it is possible to amplify several sequences simultaneously by using several pairs of primers in a same reaction tube, Apostolakos et al., (1993). However, the number of primer pairs rarely exceeds 3. Indeed, above, the amplifications lose their specificity. Other techniques, more or less derived from PCR, have been developed: LCR, Gap-LCR, ERA, CPR, SDA, TAS, NASBA. However, none of these amplification techniques seems to provide an adequate solution for total amplification of DNA.
The T-P
Cavaloc Yvon
Galibert Francis
Centre National de la Recherche Scientifique "CNRS"
Finnegan Henderson Farabow Garret & Dunner, L.L.P.
Horlick Kenneth R.
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