Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-06
2001-05-15
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
06232065
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for characterizing the expression patterns of genes and gene families. Specifically, the present invention provides means to generate and monitor gene expression profiles resulting from cellular and physiological changes such that the expression patterns of individual genes or groups of genes can be readily identified and characterized.
BACKGROUND OF THE INVENTION
Developing methods to detect molecular alterations in biological samples is key to increasing our knowledge about the causes of diseases, the processes of cellular development and differentiation, and other physiological and cellular events, and in developing tools to detect, treat, alter, and monitor these conditions. Perhaps the most significant alteration that can occur in a cell is in its pattern of gene transcription, which exerts profound control on protein levels and activities. Thus, the detection of changes in mRNA levels in the thousands of genes expressed by a single cell is an important goal for many research programs.
With the extensive amount of cDNA sequence information available through the efforts of genome sequencing projects, as well as those of thousands of individual laboratories, it is becoming increasingly imperative to develop technologies that can utilize this information to study the patterns of gene expression in both development and disease. Most human cancers, for example, are the result of genetic changes that result in alterations in the profile of expressed genes within a cell. Methods that can rapidly and accurately measure the expression levels of thousands of genes will play an essential role in furthering our understanding of the causes and nature of progression of human cancers, detecting and monitoring cancers and others diseases, and identifying and developing treatment methods for the diseases.
Several approaches have been developed in recent years in an attempt to achieve reliable, economical measurement of patterns and levels of gene expression. These include sequencing-based methods such as expressed sequence tag (EST) databases (See e.g., Adams et al., Nature Genetics 4, 373 [19931]) and SAGE (See e.g., Velculescu et al., Science 270, 484 [1995]), PCR based methods such as differential display (See e.g., Liang et al., Cancer Res. 52, 6966 [1992]; and Liang and Pardee, Science 257, 967 [1992]), and methods based on hybridization to microarrays of EST clones or oligonucleotides (See e.g., Chee et al., Science 274, 610 [1996]; DeRisi et al., Nat. Genet. 14, 457 [1996]; Gress et al., Oncogene 13, 1819 [1996]; Maskos and Southern, Nucleic Acids Res. 21, 4663 [1993]; Pietu et al., Genome Res. 6, 492 [1996]; Schena et al., Science 270, 467 [1995]; and Schena et al., Proc. Natl. Acad. Sci. 93, 10614 [1996]) or by subtractive hybridization (See e.g., Diatchenko et al., Proc. Natl. Acad. Sci. 6025 [1996]). The strengths and weaknesses of each of these technologies is assessed below.
Partial sequencing of randomly selected cDNA clones directly from cDNA libraries (i.e., producing expressed sequence tags—ESTs) has been used as a means of identifying new genes and analyzing the expression pattern of tissues and cell lines (See e.g., Adams et al., Science 252, 1651 [1991]). In these methods, total mRNA is reverse transcribed to produce cDNA. The cDNA are hybridized to random primers and sequenced (typically with automated sequencers), with ESTs of longer than 150 bp providing the best data for comparison to sequence databases. The sequence information can be compared to available sequence databases to characterize the cDNA as being derived from a known or novel gene. However, sequencing ESTs is very labor intensive, time consuming, and expensive. As a means of monitoring gene expression, the value of the data depends on the extent to which sequence information is already available (i.e., the method may indicate that a previously identified gene is expressed in a given tissue but will not provide information about the expression of related genes that have yet to be identified and catalogued).
Serial analysis of gene expression (SAGE) provides another sequencing-based method to characterizes expression patterns (Velculescu et al., supra). In the SAGE technique, RNA is reverse transcribed to produce cDNA copies of the transcripts. The cDNA is then cleaved with a restriction enzyme that cuts each transcript at least once. The 3′ portion of the restriction products (containing the poly-A tail) are isolated using streptavidin beads. The samples are divided into two portions and the free restriction ends are ligated to one of two linkers containing a type IIS restriction site. IIS restriction enzymes cleave at a defined distance from their recognition sites (i.e., as opposed to cleaving directly at the recognition site). The linkers are designed to produce IIS cleavage products that contain only a short piece (i.e., the tag) of the original cDNA, ligated to the linker. Blunt ends are produced and the two pools are ligated together creating a “ditag” with the two types of linkers on either end and the short cDNA tags in the center. The ditags are then PCR amplified using primers that are complementary to sequence within the two linkers. The PCR products are then cloned and manually sequenced, before comparing to sequence databases or SAGE experiments from other samples. Although SAGE provides a means to compare gene expression patterns, its dependance on cloning and sequencing make it labor intensive. Furthermore, SAGE does not allow the study of specific genes or gene families, but instead screens all expressed transcripts.
A PCR-based approach for identifying gene expression differences between samples is the differential display of mRNAs using arbitrarily primed polymerase chain reaction (DDRT-PCR). The polymerase chain reaction is described by Mullis et al., in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference. Briefly, the PCR process consists of introducing a molar excess of two oligonucleotide primers to the cDNA mixture containing the desired target sequence (e.g., a poly-T primer that hybridizes to the poly-A tail of mRNAs and a random oligomer). The two primers are complementary to their respective strands of the double-stranded sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the primers are extended with a thermostable DNA polyerase so as to from complementary strands. The steps of denaturation, hybridization, and polymerase extension can be repeated as often as needed to obtain a relatively high concentration of a segment of the desired target sequence.
In the case of DDRT-PCR, the target is mRNA; the mRNA is, however, treated with reverse transcriptase in the presence of oligo(dT) primers to make cDNA prior to the PCR process. The PCR is carried out with random primers in combination with the oligo(dT) primer used for cDNA synthesis. In theory, since only cDNA (i.e., derived from mRNA) is amplified, only the expressed genes are amplified. Where two samples are to be compared, the amplified products are placed in side-by-side lanes of a gel; following electrophoresis, the products can be compared or “differentially displayed.”
DDTR-PCR has a number of drawbacks. The use of arbitrary random primers can cause faint banding at essentially every position of the gel, and there is usually a high level of false positives (See e.g, Bauer et al.,
PCR Methods and Applications
, Cold Spring Harbor Lab. Press, Plainview, N.Y., Supplement, pp. S97-S108 [1994]). Also, the process is generally biased toward high-copy number genes (See e.g., Bertioli et al., Nucleic Acids Res. 23, 4520 [1995]) and is often inappropriate for experiments where only a few genes vary in expression (See e.g., Sompayac et al., Nucleic Acids Res. 23, 4738 [1995]). Lastly, practitioners often complain a
Kung Hsing-Jien
Robinson Daniel R.
Case Western Reserve University
Whisenant Ethan
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