DNA methyltransferase genotyping

Details DNA methyltransferase genotyping DNA methyltransferase genotyping

2000-06-09

2003-02-04

Fredman, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300, C536S024310, C536S024330

Reexamination Certificate

active

06514698

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to methods for DNA genotyping and gene mapping whereby nucleotide variations, mutations and polymorphisms are quickly and accurately detected by the enzymatic methylation of DNA using sequence-specific DNA methyltransferases. In particular, a novel method is. described in which DNA can be “painted ” at specific sites using radiometric or immunochemical detection. This novel methodology has been identified by the term “DNA Paint”. DNA methyltransferases can be used alone, or in combination with restriction endonucleases, to (a) diagnose diseases of plants, animals, or humans; (b) map genes; or (c) detect genetic mutations or polymorphisms.
BACKGROUND OF THE INVENTION
Starting in the early 1970's with the emergence of the recombinant DNA methods and rapid DNA sequence technologies that followed soon thereafter, scientists have relied upon restriction endonucleases to study the genetic makeup of plants and animals. The medical field has shown great advancements through the use of this molecular genetic revolution through the identification of nucleic acid variations, mutations, and polymorphisms within genes and over entire genomes. In addition, the ability to identify and recombine specific alleles for genes of interest allow researchers to create improved livestock or plant varieties much more quickly than traditional breeding methods have allowed.
The discovery of sequence-specific DNA cutting enzymes, restriction endonucleases, made the development of recombinant DNA technologies possible. Over 2,000 different sequence-specific endonucleases have been characterized, of which about 200 are available. R. J. Roberts & D. Macelis, “REBASE—Restriction enzymes and methylases”,
Nusleic Acids Res
., 24:223-235 (1996), incorporated herein by reference. These restriction endonucleases have been widely used in RFLP mapping, DNA fingerprinting, gene mapping, to detect mutations responsible for heritable human diseases and polymorphisms associated with traits of interest in animal and plant breeding programs, and to diagnose infectious disease agents or viral, bacterial, or fungal origin.
Gene mapping, DNA fingerprinting, and RFLP technologies routinely exploit the variations and mutations between the genomes of different individuals or species or varietal populations. When a point mutation, insertion, or deletion, alters the DNA sequence within a genome, the restriction endonuclease enzyme can detect these changes in nucleic acid sequence if the change creates or destroys a restriction endonuclease recognition sequence. Restriction endonucleases are sequence-specific DNA-cutting enzymes which recognize a specific nucleic acid sequence pattern and cleave DNA strands at specific locations. The restriction enzymes most often used in gene mapping and DNA fingerprinting technologies are Type II bacterial restriction enzymes, most of which recognize 4 to 6 base pair recognition sequences. Therefore, on average, most DNA restriction fragments are 4
4
to 4
6
(256 to 4096) base pairs (bp) long. When a mutation or variation occurs within one of these DNA sequence recognition sites, those restriction enzymes which cleave at that recognition sequence will no longer be able to cut at that site if the site is destroyed. Conversely, if a DNA sequence recognition site is created, the restriction enzyme for that site will be able to cleave the DNA where it could not previously. It is this variation in the presence and absence of restriction enzyme recognition sites within the genomes of different individuals that allows for DNA mapping and genetic fingerprinting using restriction enzymes. (Botstein et al., “Construction of a genetic linkage map in man using restriction fragment length polymorphisms”,
Am. J. Human Genetics
, 32:314-331 (1980); R. White and J. J. Lalouel, “Chromosome Mapping with DNA Markers”,
Scientific American
, 258:40-48 (1988).
While the advent of the restriction enzymes has revolutionized molecular genetics, it has not come without its inherent weaknesses. In order to visualize the changes in restriction endonuclease target sites (and to therefore define a restriction isotype or an allelic fingerprint), the DNA fragments that result from restriction endonuclease cleavage must be separated by size. In short, if DNA is cut into pieces then the resulting pieces must be separated. Differences in fragment size create different morphological patterns, hence polymorphisms, for each individual or trait of interest. These polymorphic patterns (DNA fingerprints) are best observed when run out by electrophoresis on agarose or polyacrylamide gels.
Another limitation using bacterial restriction enzymes in RFLP mapping or genotyping is that most mutations or polymorphic sites in DNA do not occur within these relatively rare (4 to 6 bp) endonuclease recognition sequences. The present invention overcomes this limitation through the use of sequence-specific DNA Methyltransferases (MTases) which can recognize short 2 to 4 bp sequence recognition sites as well as longer 4 to 8 bp sequence recognition sites. In particular, 75% of point mutations responsible for all known heritable human diseases occur at CG sites. D. Cooper & H. Youssoufin, “The CpG dinucleotide and human genetic disease”,
Human Genetics
78:151-165 (1988), incorporated herein by reference. Several DNA MTases recognize 2 bp CG sites, such as M.SspMQI, M.SssI, and M.DmtI of mouse (Okano, M., et al, “Cloning and Characterization of a Family of Novel Mammalian DNA (Cytosine-5) Methyltransferases”,
Nature Genetics
, Vol. 19, Jul. 1998, 219-220), human, Arabidopsis, and algal virus DNA MTases (Nelson et al., “DNA Methyltransferases and Site-specific Endonucleases Encoded by Chlorella Viruses”, in: G. P. Jost & H. P. Salusz HP, eds., DNA Methylation, Birkauser & Basel, pp. 186-211 (1993)). In contrast, no known restriction endonucleases recognize CG or any other dinucleotide sequences.
The present invention uses DNA MTases rather than restriction enzymes to overcome many of the above-mentioned problems. Each restriction endonuclease has a companion sequence-specific DNA methyltransferase (MTase) which has the same DNA recognition site. W. Arber & S. Linn, “DNA Modification and Restriction”,
Ann. Rev. Biochem
. 38:467-500 (1969), incorporated herein by reference. Several hundred of these DNA MTase specificities are known (McClelland et al., “Effect of site-specific modification on restriction endonucleases and DNA MTases”,
Nucleic Acids Res
. 22:3640-3659 (1994), incorporated herein by reference) and more are being discovered each year. In addition, as mentioned above, there are several DNA MTases which recognize 2 to 4 bp DNA sequences, such as the CG dinucleotide sequence, whereas there are no known restriction endonucleases with such short recognition sequences. As described in this patent application for the first time, it is possible to use methyltransferases rather than restriction enzymes to detect genetic polymorphisms and mutations at 2 to 8 bp sites within genomic DNA.
The present invention can utilize PCR amplification and sequence-specific DNA methylation to detect the presence or absence of specific DNA methyltransferase recognition sites.
DNA methyltransferases (MTases) catalyze the transfer of methyl groups from S-Adenosylmethionine (SAM) to specific sites in double-stranded DNA, yielding methylated DNA and S-Adenosylhomocysteine (SAH).
If radioactive
3
H-methyl-SAM is used as a substrate, then the number of methyl groups incorporated into DNA can be measured by trichloroacetic acid (TCA) precipitation of
3
H-methyl-DNA, followed by liquid scintillation counting.
This reaction is usually termed a “SAM-dependent DNA methyltransferase reaction”. However, it might be better termed a “DNA-dependent SAM methyl transfer reaction”, since the
3
H-methyl groups are incorporated into DNA only if a sequence-specific MTase recognition site is present. If one or more DNA MTase sequence recognition sites are present, then the number of such sites can be measured quantitatively

Affiliated with

Also associated with

Rating

DNA methyltransferase genotyping does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with DNA methyltransferase genotyping, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA methyltransferase genotyping will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3182928