Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-16
2003-07-29
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06599703
ABSTRACT:
BACKGROUND OF THE INVENTION
Analysis of DNA with currently available techniques provides a spectrum of information ranging from the confirmation that a test DNA is the same or different than a standard sequence or an isolated fragment, to the express identification and ordering of each nucleotide of the test DNA. Not only are such techniques crucial for understanding the function and control of genes and for applying many of the basic techniques of molecular biology, but they have also become increasingly important as tools in genomic analysis and a great many non-research applications, such as genetic identification, forensic analysis, genetic counseling, medical diagnostics and many others. In these latter applications, both techniques providing partial sequence information, such as fingerprinting and sequence comparisons, and techniques providing full sequence determination have been employed (Gibbs et al.,
Proc. Natl. Acad. Sci USA
1989; 86:1919-1923; Gyllensten et al.,
Proc. Natl. Acad. Sci USA
1988; 85:7652-7656; Carrano et al.,
Genomics
1998; 4:129-136; Caetano-Anolles et al.,
Mol. Gen. Genet.
1992; 235:157-165; Brenner and Livak,
Proc. Natl. Acad Sci USA
1989; 86:8902-8906; Green et al., PCR Methods and Applications 1991; 1:77-90; and Versalovic et al.,
Nucleic Acid Res.
1991; 19:6823-6831).
DNA sequencing methods currently available require the generation of a set of DNA fragments that are ordered by length according to nucleotide composition. The generation of this set of ordered fragments occurs in one of two ways: chemical degradation at specific nucleotides using the Maxam Gilbert method (Maxam A M and W Gilbert,
Proc Natl Acad Sci USA
1977; 74:560-564) or dideoxy nucleotide incorporation using the Sanger method (Sanger F, S Nicklen, and A R Coulson,
Proc Natl Acad Sci USA
1977; 74:5463-5467) so that the type and number of required steps inherently limits both the number of DNA segments that can be sequenced in parallel, and the number of operations which may be carried out in sequence. Furthermore, both methods are prone to error due to the anomalous migration of DNA fragments in denaturing gels. Time and space limitations inherent in these gel-based methods have fueled the search for alternative methods.
Several methods are under development that are designed to sequence DNA in a solid state format without a gel resolution step. The method that has generated the most interest is sequencing by hybridization. In sequencing by hybridization, the DNA sequence is read by determining the overlaps between the sequences of hybridized oligonucleotides. This strategy is possible because a long sequence can be deduced by matching up distinctive overlaps between its constituent oligomers (Strezoska Z, T Paunesku, D Radosavljevic, I Labat, R Drmanac, R Crkvenjakov,
Proc Natl Acad Sci USA
1991; 88:10089-10093; Drmanac R, S Drmanac, Z Strezoska, T Paunesku, I Labat, M Zeremski, J Snoddy, W K Funkhouser, B Koop, L Hood, R Crkvenjakov,
Science
1993; 260:1649-1652). This method uses hybridization conditions for oligonucleotide probes that distinguish between complete complementarity with the target sequence and a single nucleotide mismatch, and does not require resolution of fragments on polyacrylamide gels (Jacobs, K A, R Rudersdorf, S D Neill, J P Dougherty, E L Brown, and E F Fritsch,
Nucleic Acids Res.
1988; 16:4637-4650). Recent versions of sequencing by hybridization add a DNA ligation step in order to increase the ability of this method to discriminate between mismatches, and to decrease the length of the oligonucleotides necessary to sequence a given length of DNA (Broude N E, T Sano, C L Smith, C R Cantor,
Proc. Natl. Acad. Sci. USA
1994;91:3072-3076, Drmanac R T,
International Business Communications
, Southborough, Mass.). Significant obstacles with this method are its inability to accurately position repetitive sequences in DNA fragments, inhibition of probe annealing by the formation of internal duplexes in the DNA fragments, and the influence of nearest neighbor nucleotides within and adjacent to an annealing domain on the melting temperature for hybridization (Riccelli P V, A S Benight,
Nucleic Acids Res
1993;21:3785-3788, Williams J C, S C Case-Green, K U Mir, E M Southern.
Nucleic Acids Res
1994;22:1365-1367). Furthermore, sequencing by hybridization cannot determine the length of tandem short repeats, which are associated with several human genetic diseases (Warren S T,
Science
1996; 271:1374-1375). These limitations have prevented its use as a primary sequencing method.
The base addition DNA sequencing scheme uses fluorescently labeled reversible terminators of polymerase extension, with a distinct and removable fluorescent label for each of the four nucleotide analogs (Metzker M L, Raghavaehari R, Richards S, Jacutin S E, Civitello A, Burgess K and R A Gibbs,
Nucleic Acids Res.
1994; 22:4259-4267; Canard B and R S Sarfati,
Gene
1994; 148:16). Incorporation of one of these base analogs into the growing primer strand allows identification of the incorporated nucleotide by its fluorescent label. This is followed by removal of the protecting/fluorescent group, creating a new substrate for template-directed polymerase extension. Iteration of these steps is designed to permit sequencing of a multitude of templates in a solid state format. Technical obstacles, include a relatively low efficiency of extension and deprotection, and interference with primer extension caused by single-strand DNA secondary structure. A fundamental limitation to this approach is inherent in iterative methods that sequence consecutive nucleotides. That is, in order to sequence more than a handful nucleotides, each cycle of analog incorporation and deprotection must approach 100% efficiency. Even if the base addition sequencing scheme is refined so that each cycle occurs at 95% efficiency, one will have <75% of the product of interest after only 6 cycles (0.95
6
=0.735). This will severely limit the ability of this method to sequence anything but very short DNA sequences. Only one cycle of template-directed analog incorporation and deprotection appears to have been demonstrated so far (Metzker M L, Raghavachari R, Richards S, Jacutin S E, Civitello A, Burgess K and R A Gibbs,
Nucleic Acids Res.
1994; 22:4259-4267; Canard B and R S Sarfati,
Gene
1994; 148:1-6). A related earlier method, which is designed to sequence only one nucleotide per template, uses radiolabeled nucleotides or conventional non-reversible terminators attached to a variety of labels (Sokolov B P,
Nucleic Acids Research
1989;18:3671; Kuppuswamy M N, J W Hoffman, C K Kasper, S G Spitzer, S L Groce, and S P Bajaj,
Proc. Natl. Acad Sci. USA
1991; 88:1143-1147). Recently, this method has been called solid-phase minisequencing (Syvanen A C, E Ikonen, T Manninen, M Bengstrom, H Soderlund, P Aula, and L Peltonen,
Genomics
1992; 12:590-595; Kobayashi M, Rappaport E, Blasband A, Semeraro A, Sartore M, Surrey S, Fortina P.,
Molecular and Cellular Probes
1995; 9:175-182) or genetic bit analysis (Nikiforov T T, R B Rendle, P Goelet, Y H Rogers, M L Kotewicz, S Anderson, G L Trainor, and M R Knapp,
Nucleic Acids Research
1994; 22:4167-4175), and it has been used to verify the parentage of thoroughbred horses (Nikiforov T T, R B Rendle, P Goelet, Y H Rogers, M L Kotewicz, S Anderson, G L Trainor, and M R Knapp,
Nucleic Acids Research
1994; 22:4167-4175).
An alternative method for DNA sequencing that remains in the development phase entails the use of flow cytometry to detect single molecules. In this method, one strand of a DNA molecule is synthesized using fluorescently labeled nucleotides, and the labeled DNA molecule is then digested by a processive exonuclease, with identification of the released nucleotides over real time using flow cytometry. Technical obstacles to the implementation of this method include the fidelity of incorporation of the fluorescently labeled nucleotides and turbulence created around the microbead to which the single molecule of DNA is attached (Dav
Hanley, Esq. Elizabeth A.
Horlick Kenneth R.
Lahive & Cockfield LLP
Lauro, Esq. Peter C.
University of Iowa Research Foundation
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