Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-19
2002-01-22
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06340568
ABSTRACT:
FIELD OF INVENTION
The present invention relates to novel methods for analyzing the sequences of nucleic acid molecules using spectroscopic methods. In particular, the present invention relates to methods for gene expression analysis, sequence checking, mutation detection, and sequencing of nucleic acids. As such, the methods of the present invention broadly relate to molecular genetics and medical diagnostics.
BACKGROUND OF THE INVENTION
Knowledge of genetic information in the form of the nucleotide sequence of genes is critical to an understanding of various biological phenomenon such as cell development and differentiation, organism growth and reproduction, the underlying causes of disease, etc. For example, proteins serve a variety of structural and catalytic functions. These properties of proteins, however, are a function of the amino acid sequence of the protein, which in turn is encoded by nucleic acid sequences. Nucleic acids can also play a more direct role in cellular processes by functioning in the control and regulation of gene expression.
A variety of hybridization techniques have been developed to conduct various types of nucleic acid analyses to gain insight into how genetic information functions in these different types of biological processes. Typically, hybridization techniques involve the binding of certain target nucleic acids by nucleic acid probes under controlled conditions such that hybridization only occurs between complementary sequences. Using such hybridization techniques, it is possible to conduct gene expression studies, sequence checking studies and determine the sequence of nucleic acids of unknown sequence, as well as a variety of other types of analysis.
Gene expression studies are important because differential expression of genes has been shown to be associated with cell development, cell differentiation, disease states and adaptation to various environmental stimuli. For example, many diseases have been characterized by differences in the expression levels of various genes either through change in copy number of the genetic DNA or through alterations in levels of transcription. In certain diseases, infection is frequently characterized by elevated expression of genes from a particular virus.
Sequence checking refers to methods in which samples containing nucleic acid targets are analyzed to detect the presence of a sequence of interest. This type of analysis has utility in diverse applications, including research, clinical diagnostics, quality control, etc. One particular type of sequence checking which is particularly important is the identification of polymorphisms, which are variations in the genetic code. Often polymorphisms take the form of a change in a single nucleotide and are called single nucleotide polymorphisms (SNPs). In other instances, the polymorphism may exist as a stretch of repeating sequences that vary in length among different individuals. In those instances in which these variations exist in a significant percentage of the population, they can readily be used as markers linked to genes involved in mono- and polygenic traits. Thus, analysis of polymorphisms can play an important role in locating, identifying and characterizing genes which are responsible for specific traits. In particular, polymorphisms can be used to identify genes responsible for certain diseases. Similarly, diagnostic tests can also be developed to detect polymorphisms known to be associated with certain diseases or disorders.
Hybridization techniques can also successfully be used in sequencing nucleic acids of unknown sequence. Such methods typically are considerably faster than conventional sequencing techniques.
Chips to which nucleic acid probes are attached can be used to conduct nucleic acid analyses. Probes can be attached at specific locations on the chip; these locations are often referred to as elements or sites. In some applications, the chip may include many elements arranged in the form of an array. Genetic methods utilizing arrays on chips have the advantage of allowing for parallel processing that can dramatically increase the rate at which analyzes can be conducted as compared to conventional methods which often require laborious electrophoretic separations. However, the current nucleic acid methods using chips typically require complex labeling procedures in order to identify which nucleic acid probes have hybridized with a target molecule. Moreover, the methods frequently involve complicated stringency washes in order to minimize binding between probes and targets which are not fully complementary.
The present invention provides new methods for conducting various types of nucleic acid analysis in which hybridization of probe and target sequences can be detected directly, thereby allowing the analyses to be simplified relative to existing methodologies.
SUMMARY OF THE INVENTION
The present invention provides various methods of analyzing nucleic acids utilizing a system which is sensitive to the dielectric properties of molecules and binding complexes, such as hybridization complexes formed between a nucleic acid probe and a nucleic acid target. The methods include diagnostic methods which involve detecting the presence of one or more target nucleic acids in a sample, quantitative methods, kinetic methods, and a variety of other types of analysis such as sequence checking, expression analysis and de novo sequencing. The methods can detect binding between nucleic acids without the use of labels. Certain methods involve the use of arrays which allows for rapid throughput. Other methods involve the use of spectral profiles which makes it possible to distinguish between different types of hybridization complexes.
Some methods provided by the present invention involve contacting a nucleic acid probe that is electromagnetically coupled to a portion of a signal path with a sample containing a target nucleic acid. The portion of the signal path to which the nucleic acid probe is coupled typically is a continuous transmission line. A response signal is detected for a hybridization complex formed between the nucleic acid probe and the nucleic acid target. Detection may involve propagating a test signal along the signal path and then detecting a response signal formed through modulation of the test signal by the hybridization complex.
Certain diagnostic methods utilize this general approach and include using a nucleic acid probe which is complementary to a target of known sequence. A sample potentially containing the target of known sequence is contacted with the complementary probe. In some methods, the target and probe are allowed to hybridize and then the targets and probes are washed under stringent conditions. In other methods, the stringency wash is unnecessary. Detection of a response signal is indicative of the sample containing the target of known sequence. Such methods can be used in detecting a single nucleotide polymorphism (SNP). The nucleic acid target containing a polymorphic site includes a first or a second base at the polymorphic site. The nucleic acid probe is selected to be complementary to either a nucleic acid target wherein the polymorphic site includes the first base or is complementary to a nucleic acid target wherein the polymorphic site includes the second base. With knowledge of the sequence of the nucleic acid probe, detection of a response signal makes it possible to identify whether the target contains the first or second base at the polymorphic site.
In other aspects, the present invention provides a variety of methods which utilize spectral profiles to analyze nucleic acid hybridization complexes. A profile is a spectrum for a particular hybridization complex. It can include certain signals which are characteristic of the particular complex, thus making it possible to utilize signatures as a diagnostic tool and as a way to distinguish between different types of binding. Thus, certain methods include acquiring a spectrum for a hybridization complex formed between a nucleic acid probe and a nucleic acid target, wh
Horlick Kenneth R.
Signature BioScience, Inc.
Strzelecka Teresa
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