Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-06
2004-07-13
Horlick, Kenneth (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024200, C536S024300
Reexamination Certificate
active
06762022
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of nucleic acid analysis. More particularly, it concerns the sequencing and mapping of double-stranded nucleic acid templates.
2. Description of Related Art
An aggressive research effort to sequence the entire human genome is proceeding in the laboratories of genetic researchers throughout the country. The project is called the Human Genome Project (HGP). It is a daunting task given that it involves the complete characterization of the archetypal human genome sequence which comprises 3×10
9
DNA nucleotide base pairs. Early estimates for completing the task within fifteen years hinged on the expectation that new technology would be developed in response to the pressing need for faster methods of DNA sequencing and improved DNA mapping techniques.
Currently physical mapping is used to identify overlapping clones of DNA so that all of the DNA in a particular region can be sequenced or otherwise studied. There are two basic techniques of physical mapping. First, all candidate overlapping clones can be restricted with a series of restriction enzymes and the restriction fragments separated by gel electrophoresis. Overlapping clones will share some DNA sequences and thus some common restriction fragments. By comparing the restriction fragment lengths from a number of clones, the extent of overlap between any two clones can be determined. This process is very tedious and can only evaluate a limited number of candidate clones. Second, if a large number of sequence tagged sites are known in the region studied, the DNA from those sequence tagged sites can be labeled and hybridized to the candidate clones. Clones that hybridize to the same sequence tagged sites are identified as overlapping. If many sequence tagged sites are shared between two clones, it is assumed that the overlap is extensive. Sequence tagged sites give a lot of information from a limited number of hybridization reaction, however, most regions of most genomes do not have extensive sequence tagged site resources. Both methods suffer from lack of direct correspondence between the sequence and the restriction sites or sequence tagged site locations.
Current DNA sequencing approaches generally incorporate the fundamentals of either the Sanger sequencing method or the Maxam and Gilbert sequencing method, two techniques that were first introduced in the 1970′s (Sanger et al., 1977; Maxam and Gilbert, 1977). In the Sanger method, a short oligonucleotide or primer is annealed to a single-stranded template containing the DNA to be sequenced. The primer provides a 3′ hydroxyl group which allows the polymerization of a chain of DNA when a polymerase enzyme and dNTPs are provided. The Sanger method is an enzymatic reaction that utilizes chain-terminating dideoxynucleotides (ddNTPs). ddNTPs are chain-terminating because they lack a 3′-hydroxyl residue which prevents formation of a phosphodiester bond with a succeeding deoxyribonucleotide (dNTP). A small amount of one ddNTP is included with the four conventional dNTPs in a polymerization reaction. Polymerization or DNA synthesis is catalyzed by a DNA polymerase. There is competition between extension of the chain by incorporation of the conventional dNTPs and termination of the chain by incorporation of a ddNTP.
The original version of the Sanger method utilized the
E. coli
DNA polymerase I (“pol I”), which has a polymerization activity, a 3′-5′ exonuclease proofreading activity, and a 5′-3′ exonuclease activity. Later, an improvement to the method was made by using Klenow fragment instead of pol I; Klenow lacks the 5′-3′ exonuclease activity that is detrimental to the sequencing reaction because it leads to partial degradation of template and product DNA. The Klenow fragment has several limitations when used for enzymatic sequencing. One limitation is the low processivity of the enzyme, which generates a high background of fragments that terminate by the random dissociation of the enzyme from the template rather than by the desired termination due to incorporation of a ddNTP. The low processivity also means that the enzyme cannot be used to sequence nucleotides that appear more than ~250 nucleotides from the 5′ end of the primer. A second limitation is that Klenow cannot efficiently utilize templates which have homopolymer tracts or regions of high secondary structure. The problems caused by secondary structure in the template can be reduced by running the polymerization reaction at 55° C. (Gomer and Firtel, 1985).
Improvements to the original Sanger method include the use of polymerases other than the Klenow fragment. Reverse transcriptase has been used to sequence templates that have homopolymeric tracts (Karanthanasis, 1982; Graham et al., 1986). Reverse transcriptase is somewhat better than the Klenow enzyme at utilizing templates containing homopolymer tracts.
The use of a modified T7 DNA polymerase (Sequenase™) was a significant improvement to the Sanger method (Sambrook et al., 1989; Hunkapiller, 1991). T7 DNA polymerase does not have any inherent 5′-3′ exonuclease activity and has a reduced selectivity against incorporation of ddNTP. However, the 3′-5′ exonuclease activity leads to degradation of some of the oligonucleotide primers. Sequenase™ is a chemically-modified T7 DNA polymerase that has reduced 3′ to 5′ exonuclease activity (Tabor et al., 1987). Sequenase™ version 2.0 is a genetically engineered form of the T7 polymerase which completely lacks 3′ to 5′ exonuclease activity. Sequenase™ has a very high processivity and high rate of polymerization. It can efficiently incorporate nucleotide analogs such as dITP and 7-deaza-dGTP which are used to resolve regions of compression in sequencing gels. In regions of DNA containing a high G+C content, Hoogsteen bond formation can occur which leads to compressions in the DNA. These compressions result in aberrant migration patterns of oligonucleotide strands on sequencing gels. Because these base analogs pair weakly with conventional nucleotides, intrastrand secondary structures during electrophoresis are alleviated. In contrast, Klenow does not incorporate these analogs as efficiently.
The use of Taq DNA polymerase and mutants thereof is a more recent addition to the improvements of the Sanger method (U.S. Pat. No. 5,075,216). Taq polymerase is a thermostable enzyme which works efficiently at 70-75° C. The ability to catalyze DNA synthesis at elevated temperature makes Taq polymerase useful for sequencing templates which have extensive secondary structures at 37° C. (the standard temperature used for Klenow and Sequenase™ reactions). Taq polymerase, like Sequenase™, has a high degree of processivity and like Sequenase 2.0, it lacks 3′ to 5′ nuclease activity. The thermal stability of Taq and related enzymes (such as Tth and Thermosequenase™) provides an advantage over T7 polymerase (and all mutants thereof) in that these thermally stable enzymes can be used for cycle sequencing which amplifies the DNA during the sequencing reaction, thus allowing sequencing to be performed on smaller amounts of DNA. Optimization of the use of Taq in the standard Sanger method has focused on modifying Taq to eliminate the intrinsic 5′-3′ exonuclease activity and to increase its ability to incorporate ddNTPs (EP 0 655 506 B1).
Both the Sanger and the Maxim-Gilbert methods produce populations of radiolabelled or fluorescently labeled polynucleotides of differing lengths which are separated according to size by polyacrylamide gel electrophoresis (PAGE). The nucleotide sequence is determined by analyzing the pattern of size-separated radiolabelled polynucleotides in the gel. The Maxim-Gilbert method involves degrading DNA at a specific base using chemical reagents. The DNA strands terminating at a particular base are denatured and electrophoresed to determine the positions of the particular base. By c
Langmore John P.
Makarov Vladimir L.
Fulbright & Jaworski L.L.P.
Horlick Kenneth
Kim Young J.
The Regents of the University of Michigan
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