Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-27
2004-12-28
Nguyen, Nam (Department: 1753)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C204S451000, C204S456000
Reexamination Certificate
active
06835537
ABSTRACT:
BACKGROUND OF THE INVENTION
Resolution of DNA sequences by the method of Sanger et al. (1977) (the publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References) becomes difficult in regions of dyad symmetry with high G+C content. These regions form secondary structures within the dideoxy-terminated product resulting in gel compressions during electrophoresis. Replacing deoxyguanosine triphosphate (dGTP) with deoxyinosine triphosphate (dITP) alleviates this problem (Tabor and Richardson, 1987). dGTP-dCTP forms three hydrogen bonds whereas dITP-dCTP forms two hydrogen bonds (Mills and Kramer, 1979). A number of other compounds have also been used to remove compressions. These include 7-deaza-2′-deoxyguanosine-5′-triphosphate (Mizusawa et al.,
1986
) (which like dITP is also used in place of dGTP) and N-4-methyl-2′-deoxycytidine-5′-triphosphate, which is used in place of deoxycytidine triphosphate (dCTP) (Li et al.,
1993
). Using 7-deaza-ATP with 7-deaza-GTP has also been shown to help reduce compressions (Jensen et al., 1991). However, these components in sequencing reactions have not been as effective as dITP in reducing compressions or have resulted in appearance of banding artifacts caused by premature termination (Li et al., 1993; Ausubel et al., 1999).
Non-biochemical methods have also been suggested in alleviating compressions and have resulted in a lesser degree of success than have biochemical means. The most common non-biochemical method is to add formamide to sequencing gels in order to promote denaturation of secondary structures as dideoxy-terminated molecules migrate through the gel (Rocheleau et al., 1992). A second method for removing compressions requires running a sequencing gel at higher temperatures than the temperature at which they are commonly run (Ausubel et al., 1999). However, this approach is not widely used as glass gel plates crack at higher temperatures and resolution suffers. Another method, not widely used, is to modify the C residues chemically so they can no longer form GC base pairs. This can be accomplished by treating the sequencing products with bisulfite or a mixture of bisulfite and methoxyamine (Ambartsumyan and Mazo, 1980; Hayatsu, 1976).
The best candidate for removal of compressions is dITP which is widely used. A number of problems have been associated with use of dITP and solutions have been suggested in the literature. First, the rate of dITP incorporation is slower than is the rate of dGTP incorporation causing the reaction to terminate quickly thereby resulting in inability to resolve bases far from the primer (McCrea et al., 1993). To overcome this problem, ddGTP concentration can be reduced in the mixture. A second problem associated with dITP is that while the sequencing enzyme uses dITP efficiently, it has a tendency to stall in sequencing reactions and produce sequencing ladders that have a higher frequency of bands in all four lanes (as in the case of use of radioactive labels). Adding terminal deoxynucleotidyl transferase to the reaction can eliminate this problem (Fawcett and Bartlett, 1990).
Another significant and yet unresolved problem associated with the use of dITP is the frequent decrease in amplitude of G peaks following an A peak (FIG.
1
). Also, the amplitude of G in a string of two or more Gs is low. This is most noticeable when the first G is preceded by an A. This results in problems in resolution of G peaks far from the primer and in reactions where the overall signal intensity is low and could result in erroneous base calling. This phenomenon has been observed both in dye terminator and dye primer sequencing chemistries. An explanation for this result could be that dITP gets incorporated at a higher frequency than does ddGTP after dATP has been incorporated.
SUMMARY OF THE INVENTION
In DNA sequencing reactions which utilize dITP to minimize band compressions on gels, ddITP or a combination of ddITP and ddGTP as chain terminators in sequencing reactions is used to increase the amplitude of G peaks following A peaks. This use of ddITP or combination of ddITP with ddGTP results in the G peaks which follow A peaks being of greater amplitude than they are in the absence of using ddITP and more similar to the amplitude of neighboring peaks thereby allowing longer and more accurate reads of each sequencing gel.
REFERENCES:
patent: 4962020 (1990-10-01), Tabor et al.
patent: 5006210 (1991-04-01), Yueng et al.
patent: 6013488 (2000-01-01), Hayashizaki
patent: 6030814 (2000-02-01), Jendrisak
S. Tabor and C. Richardson “DNA sequence analysis with a modified bacteriophage DNA polymerase” Proceedings of the National Acadeny of Sciences (USA), (Jul. 1987), vol. 84, No. 14, 4767-4771.*
Ambartsumyan et al., Elimination of the Secondary Structure Effect in Gel Sequencing of Nucleic Acids, FEBS Lett 114(2):265-8, 1980.
Ausubel, “DNA Sequencing by the Dideoxy Method,” Chapter 7, DNA Sequencing, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1999. (20 pages).
Bankier and Barrell, Shotgun DNA Sequencing, Techniques in Life Sciences, B5:Nucleic Acid Biochemistry (R.A. Flavell, ed.), pp. 1-34, Elsevier/North Holland, Limerick, Ireland, 1983.
Boyer et al., “Analysis of Mutations at Position 184 in Reverse Transcriptase of Human Immunodeficiency Virus Type 1,” Antimicrobial Agents and Chemotherapy 39(7):1624-1628, Jul. 1995.
Hayatsu, “Bisulfite Modification of Nucleic Acids and Their Constituents,” Prog Nucleic Acid Res Mol Biol 16:75-124, 1976.
Jensen et al., “Improvements in the chain-termination method of DNA sequencing through the use of 7-deaza-2′-deoxyadenosine,” J. DNA Seq 1(4):233-9, 1991.
Li et al., “Elimination of band compression in the sequencing gels by the use of N4-methyl-2′-deoxycytidine 5′-triphosphate,” Nucleic Acids Res. 21(11):2709-14, Jun. 11, 1993.
McCrea et al., “Gel Compressions and Artifact Banding Can Be Resolved in the Same DNA Sequence Reaction,” Biotechniques 15(5):843-844, 1993.
Mills et al., “Structure-independent nucleotide sequence analysis,” Proc. Natl. Acad. Sci. USA 76(5):2232-5, May 1979.
Mizusawa et al., “Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP,” Nucleic Acids Res. 14(3):1319-24, Feb. 11, 1986.
Rochleleau et al., “Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis,” Electrophoresis 13(8):484-6, Aug. 1992.
Strahl et al., “The effects of nucleoside analogs on telomerase and telomeres in Tetrahymena,” Nucleic Acids Res 22(6):893-900, Mar. 25, 1994.
Tabor et al., “DNA sequence analysis with a modified bacteriophage T7 DNA polymerase,” Proc. Natl. Acad. Sci. USA 84(14):4767-71, Jul. 1987.
Bhatnagar Satish K.
Pruss Dmitry
Shen Min-Jui Richard
Tusneem Nadeem
Myriad Genetics Inc.
Nguyen Nam
Starsiak Jr. John S.
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