Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-12
2002-04-23
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S455000, C435S471000, C536S023100, C536S025300, C530S350000
Reexamination Certificate
active
06376178
ABSTRACT:
TECHNICAL FIELD
The present invention relates, in general, to a process of enzymatically synthesizing nucleic acids containing nucleotides that are resistant to degradation. The invention further relates to methods of utilizing such nucleic acids in DNA and RNA amplification and sequencing, gene therapy and molecular detection protocols.
BACKGROUND
Cycle sequencing polymerase chain reaction (PCR) products has proven to be an effective alternative to the more traditional M13 sequencing technique. Advantages include the use of thermostable polymerases that allow high annealing and extension temperatures (Gyllenstein and Erlich,
Proc. Natl. Acad. Sci. USA
85:7652 (1988), Innis et al,
Proc. Natl. Acad. Sci. USA
85:9436 (1988)), as well as the ability to directly utilize double-stranded DNA for templates (Carothers et al,
Biotechniques
7:494; Sears et al,
Biotechniques
13:626 (1992)). High temperature annealing and extension reduce ambiguous sequencing data that arise because of mispriming and/or secondary structure of the template. The capability of sequencing double-stranded DNA allows PCR products to be sequenced immediately after amplification. However, a simpler method for sequencing PCR products is to incorporate sequence delimiters directly into the PCR amplification process.
A sequencing method based on the incorporation of 5′-&agr;-thiotriphosphates into PCR products has been demonstrated (Nakamaye et al,
Nucl. Acids Res.
16:9947 (1988)). 5′-&agr;-Thiotriphosphates were incorporated into DNA during PCR amplification and the positions of incorporated base-specific 5′-&agr;-thiotriphosphates were revealed by chemical degradation with either 2-iodoethanol or 2,3-epoxy-1-propanol. Also 5′-&agr;-thiotriphosphates have been used to sequence single stranded M13 DNA (Labeit et al,
Meth. Enzymol.
155:166 (1987)). After incorporation by Klenow into primer extension products, the positions of the 5′-&agr;-thiotriphosphates were revealed by exonuclease III digestion. However, attempts to combine the best features of both methods (PCR amplification and enzymatic digestion) to reveal the sequence delimiters have proven unsatisfactory because of uneven band intensity (Nakamaye et al,
Nucl. Acids Res.
16:9947 (1988); Olsen and Eckstein,
Nucl. Acids Res.
17:9613 (1989)). The present invention overcomes the problems of the art and provides a simple and accurate method of amplifying and sequencing nucleic acids in a single step. Furthermore, the method is fast and amenable to automation.
OBJECTS AND SUMMARY OF THE INVENTION
It is a general object of the invention to provide a method of enzymatically incorporating into a nucleic acid a modified nucleotide which, once incorporated, is stable to chemical or enzymatic degradation. It is a specific object of the invention to provide methods of nucleic acid amplification, including strand displacement amplification, nucleic acid sequencing, in vitro transcription and gene therapy. It is a further object of the invention to provide methods of probing molecular interactions of nucleic acids with other cellular components, which methods are based on the enzymatic incorporation into the nucleic acids of modified nucleotides, particularly, &agr;-boronated deoxyniucleoside triphosphates.
In one embodiment, the present invention relates to a method of synthesizing a nucleic acid fragment comprising:
contacting a nucleic acid template with:
i) a primer sufficiently complementary to a portion of the template to hybridize therewith,
ii) an enzyme that extends the primer so that a product complementary to the template is produced, and
iii) four different nucleotides at least one of which, once incorporated into the is nucleic acid, is resistant to enzymatic degradation,
the contacting being effected under conditions such that the at least one nucleotide is recognized by the enzyme and is thereby incorporated into the extension product of the primer.
In another embodiment, the present invention relates to a method of sequencing a nucleic acid comprising:
i) enzymatically amplifying the nucleic acid in the presence of (a) four nucleotides that, once incorporated into a product of the amplification, are susceptible to enzymatic degradation, and (b) a first modified nucleotide that is selectively incorporated into the product of the amplification in lieu of a first of the four nucleotides of (a), which first modified nucleotide, once incorporated into the product of the amplification is resistant to enzymatic degradation, and
repeating the enzymatic amplification in the presence of a second, third and fourth modified nucleotide, each of which second, third and fourth modified nucleotides is selectively incorporated into the product of the amplification in lieu of a second, third and fourth of the nucleotides of (a), each of the second, third and fourth modified nucleotides being resistant to enzymatic degradation once incorporated into the product of the amplification;
ii) treating the products of the amplification of step (i) containing the first, second, third and fourth modified nucleotides with an enzyme that digests the products in the 3′ to 5′ direction, the digestions terminating at the sites of incorporation of the modified nucleotides;
iii) separating the fragments resulting from the treatment of step (ii) and detecting the position of each of the modified nucleotides in the products of the amplification.
In a further embodiment, the present invention relates to a method of sequencing a nucleic acid comprising:
i) enzymatically amplifying the nucleic acid in the presence of (a) four nucleotides that, once incorporated into a product of the amplification, are susceptible to enzymatic degradation, and (b) a first modified nucleotide that is selectively incorporated into the product of the amplification in lieu of a first of the four nucleotides of (a), which first modified nucleotide, once incorporated into the product of the amplification is resistant to enzymatic degradation, and repeating the enzymatic amplification in the presence of a second, third and fourth modified nucleotide, each of which second, third and fourth modified nucleotides is selectively incorporated into the product of the amplification in lieu of a second, third and fourth of the nucleotides of (a), each of the second, third and fourth modified nucleotides being resistant to enzymatic degradation once incorporated into the product of the amplification;
ii) subjecting the products of the amplification of step (i) containing a first, second, third and fourth modified nucleotides to mass spectrometry and detecting the resulting fragment patterns and thereby the position of each of the modified nucleotides in the products of the amplification.
In yet another embodiment, the present invention relates to a method of producing a protein comprising introducing into a cell a nucleic acid sequence encoding the protein, which nucleic acid sequence includes a modified nucleotide that is resistant to enzymatic degradation, the introduction being effected under conditions such that the nucleic acid sequence is expressed and the protein thereby produced.
In yet another embodiment, the present invention relates to a method of amplifying a DNA sequence comprising:
i) contacting the DNA sequence with a primer having a first part and a second part,
the first part having a nucleotide sequence such that the first part hybridizes to a portion of the DNA sequence, and
the second part being 5′ to the first part, noncomplementary to the DNA sequence and having a nucleotide sequence that includes a restriction enzyme recognition site,
wherein the contacting is effected under conditions such that the first part of the primer hybridizes to the DNA sequence; and
ii) enzymatically extending the primer and the DNA sequence in the presence of a boronated deoxynucleoside triphosphate so that a boronated duplex extension product is produced, the boronated deoxynucleoside triphosphate being selected so that a boronated deoxynucleoside is inserted into t
Porter Kenneth W.
Ramsay Shaw Barbara
Sergueev Dmitri
Duke University
Nixon & Vanderhye P.C.
Wilder Cynthia B.
Zitomer Stephanie W.
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