Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-30
2001-08-21
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06277571
ABSTRACT:
SUMMARY OF THE INVENTION
Proteins are comprised of polypeptide domains which share amino acid sequence identity and/or homology. The same domains can be identified in a variety of different proteins and protein families, generally possessing the same or similar functions. One explanation for the existence of shared domains is evolutionary. In this theory, the occurrence of common polypeptide domains among proteins suggests that the genetic information encoding them has been spread through the genome and dispersed into many different proteins. Thus, recombination and shuffling of preexisting polypeptide domains may lead to the evolution of new proteins. See, e.g., Doolittle,
Sci. Am.,
253(4):88-99, 1985; Gilbert,
Science,
228: 823-824. 1985.
There are many examples in which genes coding for different proteins contain homologous domains. For example, proteins as diverse as the epidermal growth factor and the low density lipoprotein receptor share homologous polypeptide domains. See, e.g., Sudhofet al.,
Science,
228:893-895, 1985. Proteins can also share more than one common domain. Proteins targets for the signaling protein tyrosine kinases share several different polypeptides domains. For instance, p85 contains SH3, BCR, SH2 (Src Homology 2), and SH2 domains arranged linearly from 5′ to 3′. GAP120, on the other hand, contains a 5′ to 3′ arrangement of SH2, SH3, SH2, and PH (pleckstrin homology domain). See, e.g.,
Exploring Genetic Mechanisms
, Singer and Berg, 1997, especially, pages 209-237.
The fact that conserved functional polypeptide domains are widespread throughout the genomes of living organisms has led to the discovery of protein sequence motifs which can be used to define and identify such functional domains in proteins. Moreover, consensus oligonucleotides can be designed, based on these motifs, and utilized to identify known and/or novel genes for a variety of different purposes.
A novel aspect of the present invention exploits the ordinal arrangement of these functional domains in genes and gene families to identify known and/or novel genes and/or analyze their expression and/or organization in the genome. In a preferred method, a set of consensus sequence oligonucleotides (e.g., more than two), each designed to a different polypeptide domain within a protein, are used to select, and divide into subsets, segments of DNA which contain the designated domains. The DNA segments are preferably selected from a heterogenous nucleic acid pool by nucleic acid amplification techniques, such as polymerase chain reaction. Since the domains are organized consecutively in both the proteins and the DNA which encode them, any differences in the organization of the domains within the DNA will be reflected in the amplification products. Thus, the size and/or absence of the selected segments discriminates between different genes and their expression patterns. This information can be useful to assess the health and vitality of living organisms.
For instance, consider performing a method of the invention on one DNA pool containing p85 and a second DNA pool containing p85 and GAP120, utilizing consensus sequence oligonucleotides based on SH2 and SH3 functional domains. If a first step of amplification (as a way of selection) is performed using the SH2 oligonucleotides, a segment of DNA would be identified from the first and second pool, each having a different size. A sequential step of amplification, using SH2 and SH3 consensus oligonucleotides, would again result in segments of different sizes, providing further information about the content of the DNA pools. The extraction of such information is useful, for example, to diagnose cancer and other cell cycle diseases.
As discussed above, in a first step of a preferred method according to the present invention, a first segment of nucleic acid is selected from a sample nucleic acid containing a mixture of different nucleic acid sequences. An example of a nucleic acid sample can be a cDNA pool or library which is prepared from a desired source, such as normal tissue, a tumor, or a part of the body in which a diffuse malignancy exists. Since cells exhibit a complex pattern of gene expression, they typically possess a heterogenous pool of mRNAs which, when transcribed into cDNA, results in an assortment of different sequences. The total cDNA mixture can be referred to as a sample DNA.
To characterize the sample, a subset of nucleic acids is selected on the basis of nucleotide sequences present in the subset but, either absent or less frequently represented, in other members of the sample. The nucleotide sequences utilized to perform the selection step are preferably consensus sequences designed on the basis of functional polypeptide domains or protein motifs. Preferably, the selection step is accomplished by nucleic acid amplification, e.g., using the polymerase chain reaction. For example, a pair of oligonucleotides primers can be chosen which contain functional domains present in a subset(s) of DNAs (see, e.g.,
FIG. 1A
, containing domains CSR, ZBR, and PLR) whose selection is desired, but, absent in other DNAs. The DNA subset(s), whose selection is desired, can be referred to as the “target DNA.” A target DNA can be comprised of one or more functional domains which share varying amounts of similarity, e.g., 100% sequence identity or less than 100% identity (i.e., sequence homology). Consequently, the oligonucleotide primers can comprise a variety of sequence types, including: sequences which are perfectly complementary to a selected domain; degenerate sequences; sequences which are less than perfectly complementary to a selected domain; consensus sequences for a desired domain or region shared by two or more nucleic acids; or even arbitrary or random sequences.
A reaction is performed on the sample nucleic acid under conditions in which amplification is achieved. Such conditions include, e.g., effective concentrations of oligonucleotides, of nucleic acid polymerase, of salt and cofactors, temperatures to achieve hybridization, etc. These conditions can be selected routinely or performed according to the conventional protocols known in the art. See, e.g., PCR Protocols:
A Guide to Methods and Applications
, Innis et al., eds., Academic Press, New York, 1990.
The result of the amplification is the production of a first segment of double-stranded nucleic acid. The segment is defined at its 5′ and 3′ ends by the oligonucleotide pairs utilized in the amplification reaction. By the term “segment,” it is meant, e.g., a portion, part, or component of the target nucleic acid, i.e., less than the whole target nucleic acid. Thus, amplification with the oligonucleotide pair results in the production of a fragment of the target nucleic acid comprising only a part of an entire nucleotide sequence. In
FIG. 1C
, for instance, a degenerate 5′ CSR primer is used with an oligo-dT primer in a polymerase chain reaction to produce a set of DNA segments defined at their 5′ end by the CSR domain and their 3′ end by oligo-dT. Since multiple different genes exist which possess the CSR domain, the reaction (e.g., when separated on a gel or by other means) will result in a ladder of differently sized DNA segments. In a preferred aspect of the invention, the first segment of nucleic acid is present in more than one type of nucleic acid, e.g., the segment is present in at least two cDNA types, each of which codes for a different but related polypeptide. The polypeptides can be members of the same and/or different class of proteins.
In certain aspects of the present invention, for instance, when expression patterns of two different tissue types are being compared (e.g., for diagnostic and treatment purposes), the first segment of double-stranded DNA may be present in one sample tissue type, but not in another. This result can be readily observed by separating the segment on the basis of size, e.g., by gel electrophoresis or on a sizing column. Alternatively, one or more segments can be present in both sam
Broaddus William
Fillmore Helen
Gillies George
Millen White Zelano & Branigan
Virginia Commonwealth University Intellectual Property Foundatio
Whisenant Ethan
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