Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1982-06-17
1986-06-10
Tanenholtz, Alvin E.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
4351723, 435 91, 935 6, 935 78, 935 80, C12Q 168, C12N 1500, C12P 1934
Patent
active
045943184
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel process for isolating and identifying cloned DNA segments from defined regions of a chromosome.
BACKGROUND OF THE INVENTION
As stated in applicants' article entitled "Isolation and Localization of DNA Segments from Specific Human Chromosomes", Proc. Nat'l. Acad. SCI. USA, Vol. 77, No. 5, pp. 2829-2833, May 1980, there are two principal reasons for attempting to achieve as complete as possible fine-structure analysis of the human genome. First, delineation of the DNA sequence of individual genes and construction of the corresponding probes now can be used to detect the presence of human genetic disease. These procedures can be applied in utero so that birth of tragically defective babies can be prevented. Second, and perhaps of even greater importance to biomedical science, definition of DNA sequences over large chromosomal regions, including sequences specifying protein structure as well as the noncoding intervals within and between these sequences, appears to promise greater understanding of physiological and biochemical mechanisms of human gene regulation. The genetic data so obtained should illuminate many aspects of medicine and developmental biology including situations not usually considered to lie within the narrow scope of the term "genetic disease."
Mammalian gene mapping has made significant progress in recent years, particularly by means of cytogenetic identification of relatively gross regions on each chromosome and by development of powerful methods for determining DNA sequences. However, one of these techniques operates at the level of millions of base pairs whereas the other is limited to handling of thousands. Therefore, a large gap in revolving power exists which must be bridged before these techniques can be combined for high-resolution mapping of the human genome. The present invention demonstrates how this gap can be filled.
The recombinant DNA methods used previously isolated and characterized cloned DNA segments based on their ability to code for a specific mRNA. With the novel process described herein, cloned DNA segments can be isolated and characterized based on their genetic map position alone.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a diagram showing the various terminal deletions of human chromosome 11 in four cell hybrids; and
FIG. 2 is a schematic representation of human chromosome 11 with arrows indicating the breakpoints at which terminal deletions occurred in the four clones.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The object of the instant invention is to isolate DNA segments of a size permitting complete sequence analysis from a chromosome and to map these segments precisely on the chromosome.
This object has been achieved by:
(1) Preparing a series of cell hybrids from a first species and a second species, said cell hybrids containing a chromosome or deletion mutant of a chromosome as the only chromosomal component of the second species.
(2) Partially digesting the hybrid cell DNA with a restriction enzyme and then size fractionating the partially digesting DNA on a sucrose gradient in order to isolate DNA fragments 15-20 kb in length.
(3) Ligating the DNA fragments to a bacteriophage.
(4) Using an in vitro encapsidation procedure to obtain viable phage containing the ligated DNA.
(5) Propogating the phages to obtain a library of recombinant clones of DNA from the hybrid cell.
(6) Plaquing the library on culture plates.
(7) Transferring the plaques onto a medium which is capable of holding DNA in a covalent or non-covalent fashion.
(8) Hybridizing the medium of (7) to total, nicktranslated radioactive 2d species DNA.
(9) Collecting the radioactive 2d species plaques.
(10) Digesting the DNA from each corresponding phage clone with a restriction enzyme in order to produce subfragments.
(11) Fractionating the subfragments on an agarose gel.
(12) Staining the subfragments with ethidium bromide in order to visualize the DNA fragments.
(13) Transferring the subfragments onto a medium which is capable of holding DNA in a cov
REFERENCES:
Lewin, Benjamin, "Gene Expression", Eucaryotic Chromosomes, John Wiley & Sons, v. 2, 1980, pp. 244-252.
Wu, Ray, "Recombinant DNA", Methods in Enzymology, Academic Press, v. 68, 1979, pp. 389-395.
Hardman, Joel G., et al, "Hormone Action", Methods in Enzymology, Academic Press, v. XXXIX, 1975, pp. 122-128.
Gusella, James, et al, "Precise Localization of Human B-globin Gene Complex on Chromosome 11*", (DNA hybridization/hemoglobin B Chain/Hemoglobin o Chain/Regional Gene Mapping), Proc. Nat'l. Acad. Sci. USA, V. 76, No. 10, Oct. 1979, pp. 5239-5243.
Maniatis, Tom, et al, "The Isolation of Structural Genes from Libraries of Eucaryotic DNA", Cell, V. 15, Oct. 1978, pp. 687-701.
Varsanyi-Breiner, A., et al, "The Organization of a Nuclear DNA Sequence from a Higher Plant: Molecular Cloning and Characterization of Soybean Ribosomal DNA", Elsevier/North-Holland Biomedical Press, Amsterdam, Gene 7, (1979), pp. 317-334.
Blattner, F., et al., Reports, "Cloning Human Fetal .gamma. Globin and Mouse .alpha.-Type Globin DNA: Preparation and Screening of Shotgun Collections, Science, v. 202, AAAS, 1978, pp. 1279-1284.
Benton, W. David, et al., "Screening .lambda. gt Recombinant Clones by Hybridization to Single Plaques in Situ", Science, v. 196, Apr. 8, 1977, pp. 180-182.
Rigby, Peter W., et al., "Labeling Deoxyribonucleic Acid to High Specific Activity in Vitro by Nick Translation with DNA Polymerase I", J. Mol. Biol., v. 113, 1977, pp. 237-251.
Kao, Fa-Ten, et al., "Genetics of Somatic Mammalian Cells: Genetic, Immunologic, and Biochemical Analysis with Chinese Hamster Cell Hybrids Containing Selected Human Chromosomes*", Proc. Nat. Acad. Sci., USA, Genetics, v. 73, No. 1, Jan. 1976, pp. 193-197.
Kao, Fa-Ten, et al, "Genetics of Cell-Surface Antigens: Regional Mapping of Three Components of the Human Cell-Surface Antigen Complex, A.sub.L, On Chromosome 11.sup.1 ", Somatic Cell Genetics, v. 3, No. 4, 1977, pp. 421-429.
Davidson, Eric H., et al, "Regulation of Gene Expression: Possible Role of Repetitive Sequences", AAAS, Science, v. 204, Jun. 8, 1979, pp. 1052-1059.
Blattner, Frederick R., et al., "Charon Phages: Safer Derivatives of Bacteriophage Lambda for DNA Cloning", Laboratory of Genetics, Madison, WI, Apr. 8, 1977, pp. 161-169.
Britten, R. J., et al., "Repeated Sequences in DNA", Science, v. 161, No. 3841, Aug. 9, 1968, pp. 529-540.
Southern, E. M., "Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis", J. Mol. Biol. (1975) v. 98, pp. 503-517.
McConaughy, Betty L., et al, "Related Base Sequences in the DNA of Simple and Complex Organisms, VI. The Extent of Base Sequences Divergence Among the DNAs of Various Rodents", Biochemical Genetics, n. 4, 1970, pp. 425-446.
Thuring, R. W. J., et al., "A Freeze-Squeeze Method for Recovering Long DNA from Agarose Gels", Analytical Biochemistry, v. 66, 1975, pp. 213-220.
Gusella James F.
Housman David
Jones Carol
Kao Fa-Ten
Keys Cheryl
Massachusetts Institute of Technology
Tanenholtz Alvin E.
LandOfFree
Isolation and localization of DNA segments does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Isolation and localization of DNA segments, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Isolation and localization of DNA segments will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2406232