Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-09-27
2001-02-20
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06190865
ABSTRACT:
FIELD OF THE INVENTION
The field of the present invention is methods of characterizing nucleic acid molecules. Specifically, the present invention concerns characterizing nucleic acid molecules by synthesizing DNA in the presence of a non-canonical deoxynucleoside triphosphate, excising the base portion of the non-canonical deoxynucleoside triphosphate, breaking the phosphodiester backbone of the DNA at the abasic sites, and analyzing the resulting DNA fragments.
BACKGROUND OF THE INVENTION
Methods of Characterizing Nucleic Acid Molecules
There are many reasons for characterizing nucleic acid molecules. For example, genes are rapidly being identified and characterized which are causative or related to many human, animal and plant diseases. Even within any particular gene, numerous mutations are being identified that are responsible for particular pathological conditions. Thus, although many methods for detection of both known and unknown mutations have been developed (e.g., see Cotton, 1993), our growing knowledge of human and other genomes makes it increasingly important to develop new, better, and faster methods for characterizing nucleic acids. Besides diagnostic uses, improved methods for rapidly characterizing nucleic acids will also be useful in many other areas, including human forensics, paternity testing, animal and plant breeding, tissue typing, screening for smuggling of endangered species, and biological research.
A variety of methods for characterizing DNA molecules are known in the art. For example, one can characterize DNA molecules by size based on their electrophoretic migration through an agarose or polyacrylamide gel. In these methods, the negatively charged DNA molecules move through a gel in the direction of the positively charged electrode. Provided that the percentage of agarose or polyacrylamide in the gel is appropriate for the size range of the DNA molecules being electrophoresed, smaller DNA molecules move through the pores of the gel more readily than larger DNA molecules. Because DNA molecules move in the gel at size-dependent rates, molecule sizes can be determined by staining and visualizing the DNA and then comparing the migration of the sample DNA molecules in the gel with the migration of marker DNA molecules of known size. Under the appropriate conditions, single-stranded DNA molecules differing in length by even a single nucleotide can be distinguished by denaturing polyacrylamide gel electrophoresis.
Another way to characterize DNA molecules is to treat each DNA molecule with one or more restriction endonucleases and then to determine the sizes of the various DNA fragments resulting from this treatment by agarose gel electrophoresis. Restriction endonucleases are enzymes that recognize specific sequences of bases in DNA (often 4, 5, 6 or sometimes 8 bases on each DNA strand) and then cut the phosphodiester bonds of the polynucleotide chains of DNA within to the recognition sequence. Because many restriction endonucleases with different recognition sequences are available, one can obtain a restriction map of an entire DNA molecule showing locations of restriction enzyme recognition sites and distances between them by determining which other restriction enzymes will cut each DNA fragment generated by any given restriction enzyme and what are the sizes of all of the resulting fragments. Such a restriction map is characteristic for a particular DNA molecule and can be used to obtain a rough identification of a particular sequence. Additionally, changes such as those caused by mutations in DNA may result in a loss or gain of a restriction site—a so-called “restriction fragment length polymorphism” (RFLP) (Kazazian, et al., 1989). An example of a diagnostically significant RFLP is a single base mutation in the beta-hemoglobin gene, the change from A to T which eliminates a Dde I restriction site, which results in sickle cell anemia (Kazazian, et al., 1989).
One of the most informative ways to characterize a DNA molecule is to determine its nucleotide sequence. One method for sequencing DNA (Maxam and Gilbert, 1977) is accomplished by treating each of four aliquots of one strand of a 5′- or 3′-end-labelled DNA molecule to be sequenced with one of four different chemical reagents. One chemical specifically modifies only the guanine base in the DNA, another modifies only cytosine, another modifies either guanine or adenine bases, and the last chemical modifies either thymine or cytosine bases. The chemical treatments are carried out under conditions so that only a small proportion of the total susceptible bases will actually be modified.
It is important that the chemical reactions are limited in order to generate a nested set of fragments differing by one base of a specific type. If all G residues, for example, were modified, the residues would all be susceptible to phosphodiester bond cleavage. Therefore, a collection of partially modified nucleic acids is required for sequencing. Subsequent treatment with piperidine results in cleavage of the phosphodiester bonds of the DNA molecule at the abasic sites, generating a mixture of all sizes of DNA molecules that are possible following chemical modification and loss of each one of the corresponding susceptible bases. The DNA molecules in each of the four reactions are then resolved by electrophoresis in adjacent lanes of a polyacrylamide gel and the pattern of bands is revealed by exposing the gel to X-ray film if the DNA molecules are labelled with a radioisotope. The sequence of the DNA is revealed by analyzing the exposed X-ray film. Alternatively, if the DNA molecules are labelled with a fluorescent, chemiluminescent or some other non-radioactive moiety, the sequence is revealed by an appropriate method known in the art.
The most commonly used method for sequencing DNA at this time (Sanger, et al., 1977) uses a DNA polymerase to produce differently sized fragments depending on the positions (sequence) of the four canonical bases (A=Adenine; C=Cytidine; G=Guanine; and T=Thymine) within the DNA to be sequenced. In this method, the DNA to be sequenced is used as a template for in vitro DNA synthesis. In addition to all four of the canonical deoxynucleotides (dATP, dCTP, dGTP and dTTP), a 2′,3′-dideoxynucleotide is also included in each in vitro DNA synthesis reaction at a concentration that will result in random substitution of a small percentage of a canonical nucleotide by the corresponding dideoxynucleotide. Thus, each DNA synthesis reaction yields a mixture of DNA fragments of different lengths corresponding to chain termination wherever the dideoxynucleotide was incorporated in place of the normal deoxynucleotide. The DNA fragments are labelled, either radioactively or non-radioactively, by one of several methods and the label(s) may be incorporated into the DNA by extension of a labelled primer, or by incorporation of a labelled deoxy or dideoxy nucleotide. By carrying out DNA synthesis reactions for each of the four dideoxynucleotides (ddATP, ddCTP, ddGTP or ddTTP), then separating the products of each reaction in adjacent lanes of a denaturing polyacrylamide gel, and detecting those products by one of several methods, the sequence of the DNA template can be read directly.
Cycle Sequencing is a variation of Sanger sequencing that achieves a linear amplification of the sequencing signal by using a thermostable DNA polymerase and repeating chain terminating DNA synthesis during each of multiple rounds of denaturation of a template DNA (e.g., at 95° C.), annealing of a single primer oligonucleotide (e.g., at 55° C.), and extension of the primer (e.g., at 70° C.).
Nucleic acid sequencing provides the highest degree of certainty as to the identity of a particular nucleic acid. Also, nucleic acid sequencing permits one to detect mutations in a gene even if the site of the mutation is unknown. Sequencing data may even provide enough information to permit an estimation of the clinical significance of a particular mutation or of a variation in the seq
Hoffman Leslie M.
Jendrisak Jerome J.
Smith Robert E.
Campbell Eggerton A.
Epicentre Technologies Corporation
Quarles & Brady LLP
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