Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-16
2001-04-24
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S023100, C536S024100, C536S024300, C536S024310, C536S024320, C536S025300, C536S025320
Reexamination Certificate
active
06221589
ABSTRACT:
BACKGROUND OF THE INVENTION
The growing demand for sequencing of unknown nucleic acid sequences has spurred the demand for rapid, inexpensive methods of sequencing large amounts of DNA. For example, the Human Genome Initiative will require the sequencing of about 4 billion base pairs of DNA. However, it is possible that current sequencing methodologies, such as Sanger or Maxam-Gilbert sequencing, are not capable of high enough throughput to allow a project of this magnitude to be completed in a reasonable time.
The attention of many researchers has turned to sequencing methods which process sequences in parallel, rather than the serial sequencing methods described above. The promise of parallel “sequencing by hybridization” (SBH) methods is that large amounts of information can potentially be obtained rapidly, in a single experiment. SBH involves the use of multiple probes disposed in an array format to bind to a sample of a target nucleic acid which has been cleaved into smaller fragments. Presently, however, SBH has been attempted on only small DNA targets and with small probe arrays.
Certain problems have arisen in attempts to implement SBH schemes. One serious difficulty is the need to correctly discriminate between target fragments that are perfectly matched to a probe sequence, and target fragments that are bound to a probe sequence despite one or more mismatched bases. This “mismatch discrimination” problem presents the possibility of misidentification of sequences. The problem is especially acute when attempting to differentiate between sequences which bind with significantly different binding energies. For example, in general, AT-rich sequences bind less strongly to their complementary probes than do GC-rich sequences, of the same length, to their respective complementary probes. Thus, it can be difficult to distinguish between perfectly-bound AT-rich sequences and partially mismatched GC-rich sequences. In view of these difficulties, hybridization of mismatched sequences is undesirable, as it makes the unambiguous determination of the target sequence harder to achieve.
SUMMARY OF THE INVENTION
This invention features methods of normalizing the melting temperatures of a plurality of nucleic acid duplexes.
In one aspect, the invention provides a method of normalizing the melting temperatures of at least two nucleic acid duplexes. The method includes the steps of contacting the at least two nucleic acid duplexes with a reaction mixture comprising a nucleic acid binding ligand which preferentially binds to one of the at least two nucleic acid duplexes; such that the melting temperatures of the at least two nucleic acid duplexes are normalized. In a preferred embodiment, a plurality of nucleic acid duplexes are provided in an array, e.g., a 96 well microtiter plate or a high density nucleic acid array, e.g., “gene chip”, such that modulating the stability of at least one of the nucleic acid duplexes in the array is effected by forming a reaction mixture comprising the plurality of nucleic acid duplexes and at least one base-preferring nucleic acid binding ligand.
In preferred embodiments, the nucleic acid binding ligand is a duplex-binding ligand. In preferred embodiments, the duplex-binding ligand is distamycin. In preferred embodiments, the reaction mixture comprises at least two nucleic acid binding ligands, and wherein each of the at least two nucleic acid binding ligands independently binds preferentially to one of the at least two nucleic acid duplexes. In preferred embodiments, the reaction mixture comprises at least two duplex-binding ligands. In preferred embodiments, at least one of the at least two nucleic acid binding ligands is a single-strand-binding ligand. In certain embodiments, the reaction mixture further comprises at least one nonspecific nucleic acid binding ligand. In certain embodiments, the reaction mixture further comprises a duplex denaturant, such as, e.g., urea.
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Benight Albert S.
Faldasz Brian D.
Lane Michael J.
DeConti Giulio A.
Lahive & Cockfield LLP
Riley Jezia
Tm Technologies, Inc.
Triano, III Nicholas P.
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