Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1994-12-21
2003-10-28
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S025300, C536S025400
Reexamination Certificate
active
06638715
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to intact extended and super-extended DNA, methods of producing the extended DNAs and to efficient and rapid mapping of genes and other sequences employing various forms of extended DNA and DNA probes, particularly for visual mapping such as where fluorescent hybridization (comparable to in situ hybridization techniques) is employed. The remarkable sensitivity and resolution of direct visualization detection employing extended DNA permits rapid detailed studies of DNA rearrangements, such as translocations, and select determination of order and distance between DNA segments. Further, the invention concerns methods of controlling DNA extension to tailor to particular needs including long and short range mapping of DNA sequences.
DESCRIPTION OF RELATED ART
A primary goal in developing a genome map is identification of genes and DNA sequences involved in disease states or disorders as well as in normal functions of the cell. The combined use of genetic and physical mapping of the human genome has proven useful in the placement of genes and/or molecular markers in reference to each other and in the cloning and identification of genes of biological and medical significance. This method of identification requires the use of genetic linkage data in a population to connect a disease or biological characteristic with molecular DNA markers. With this information, a physical map can be used to identify a gene of interest by its position in relation to the DNA markers.
The characterization of gene structure and genome organization has been greatly facilitated by the development of a number of physical mapping technologies. Among the first technologies developed were restriction mapping and DNA sequencing, which provide the highest resolution information, but are cumbersome when applied to large regions of DNA.
The development of pulsed-field gel electrophoresis and the use of rare cutting restriction enzymes has opened the door to restriction mapping in the megabase (Mb) size range. For the mapping of still larger regions of DNA, more recent developments of yeast artificial chromosomes (YACs) (Botstein et al., 1980) and radiation hybrid maps (Nakamura et al., 1987) have been useful. However, as the DNA regions to be mapped increase in size, fine structure resolution is generally sacrificed. Other techniques, such as the “fingerprinting” of repetitive elements (Litt et al. 1989), have been developed to overcome long range restriction mapping deficiencies.
A more global approach to mapping involves fluorescent in situ hybridization (FISH) to identify the position of probes on metaphase chromosomes (Weber et al. 1989). The use of this technique allows more rapid mapping of DNA probes with approximately 1 Mb resolution. For higher resolution mapping, the FISH technique has been applied to interphase nuclei (Evans et al. 1989). Since the DNA is less condensed in interphase nuclei than in metaphase chromosomes, resolution in the 50-100 kb range can be obtained. However, mapping the distance between two probes in three dimensional nuclei or compressed two dimensional nuclei is complex and requires large data sampling and probability calculations based on a random walk model (Coulson et al. 1988).
Long distance restriction maps of DNA regions have been generated using rare cutting restriction enzymes such as NotI (Poustka et al. 1987; Yagle et al. 1990; Barmeister et al. 1986). NotI linking clones, which encompass a NotI cleavable site, have been used to facilitate NotI mapping by identification of contiguous NotI fragments (Wallace et al. 1989). The use of frequent-cutting enzymes such as HindIII is not practical for mapping megabase-size DNA due to the complexity of the map.
Additional strategies for gathering physical linkage information on a still larger scale include the use of interspecific somatic cell hybrids, in which panels of rodent/human hybrid cell lines that retain various combinations of human chromosomes or parts thereof are used to localize probes to individual chromosomes or chromosomal regions (Ruddle et al. 1971). Radiation-induced hybrids, in which fragments of human chromosomes are retained in a rodent cell background (Goss et al. 1975) have also been employed.
Finally, fluorescent in situ hybridization (FISH) has become popular for determining approximate distances greater than 1 Mb between two or more probes on metaphase chromosomes (Lichter et al. 1990). FISH may also be used to a limited extent to determine the relative order of probes. For example, more closely linked probes along a 250 kb region have been hybridized to uncondensed DNA in interphase nuclei (Trask et al. 1989). While information concerning order may be obtainable with the method, there are some serious shortcomings; a major problem is that DNA in interphase nuclei is three dimensional. Labels that are not closely spaced or which are in reverse order to the observed order may be inaccurately determined because labels appear to be on top of one another, or because a twisted loop is viewed two-dimensionally.
A second shortcoming is the resolution. While improvements have been made, and the predicted resolution with FISH visualization is claimed to be about 10 kb, this range has not been substantiated, although resolution at 21 kbp has been reported (Heng et al. 1992). Higher resolution may be hampered because of the 3-D structure of the DNA, with lack of accessibility leading to poor resolution and difficulty in detection.
There have been reported attempts to reach levels of resolution around 10 kb; in one instance by expelling long 200 &mgr;m loops from the nucleus (Wiegant et al. 1992), releasing chromatin fibers (Heng et al. 1992) or creating nuclear “halos” by extending DNA from which histones have been extracted (Lawrence et al. 1992). Although believed possible to extend resolution below 10 kb, there do not appear to be published data demonstrating that such resolution has been achieved.
There is, therefore, a need to develop physical mapping techniques that eliminate the tremendous amount of time and effort needed for restriction mapping and Southern blot hybridization and to increase the resolution limitation associated with FISH methods. New methods would allow microscopic visualization of cosmids or other DNA probes hybridized to an uncondensed fully extended DNA molecule, such as mapping of specific probes with high resolution exceeding 5 kb on YAC DNA. Direct visual mapping of repetitive DNA elements along a DNA strand would provide a significant improvement and alternative to restriction mapping and fingerprinting techniques.
SUMMARY OF THE INVENTION
The present invention addresses one or more of the foregoing or other problems encountered in chromosome mapping, particularly the complexity and inadequate resolution of current techniques. The present invention involves methods of preparing and visualizing DNA strands in linearized, that is, straight-line extended or “super-extended” forms as a means of mapping small or large DNA segments or even gene clusters.
The invention includes a simple, effective method to produce linear, one-dimensional DNA. The method provides extended and super-extended forms of DNA, the latter stretched to a length beyond the calculated “unwound” length of native DNA. By comparison, the DNA from typical “spreads” (Lehninger, 1970) is two-dimensional in the sense of exhibiting two directions in a single plane (including curves and winding). The form of DNA disclosed in the present invention is virtually straight (linear), has no contour and little, if any, randomness. It is thus visualized as extending in only one direction in a single plane. The invention therefore provides a procedure to stretch DNA into novel, linear, one-dimensional forms up to and beyond 0.34 &mgr;m per kilobase pair. The DNA can be used as a target for hybridization of labelled probes to visually observe a high resolution map of probes along a single strand of DNA. Mapping resolutions as high as 1 kb are possible with the extended DNA. Super-extended D
CTRC Research Foundation
Fulbright & Jaworski L.L.P.
Marschel Ardin H.
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