Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-19
2001-07-17
Jones, W. Gary (Department: 1566)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091520, C536S025320
Reexamination Certificate
active
06261770
ABSTRACT:
BACKGROUND OF THE INVENTION
The human body is comprised primarily of specialised cells performing different physiological functions organised into organs and tissues. All human cells contain DNA, arranged in a series of sub-units known as genes. It is estimated that there are approximately 100,000 genes in the human genome. Genes are the blueprints for proteins. Proteins may perform a wide variety of biological functions, for example messengers, catalysts and sensors. Such compounds are responsible for managing most of the physiological and biochemical functions in humans and all other living organisms. Over the last few decades, there has been a growing recognition that many major diseases have a genetic basis. It is now well established that genes play an important role in cancer, cardiovascular diseases, psychiatric disorders, obesity, and metabolic diseases. Significant resources are being focused on genomic research based on the notion that the nucleotide sequences of a particular gene and its predicted protein product will lead to an understanding of its function in healthy and malfunctioning cells or tissues. This understanding is expected, in turn, to lead to therapeutic and diagnostic approaches, focused on molecular targets associated with the gene and the protein it expresses. The first step on the way to the development of such applications is to identify the genes specifically involved in the different categories of diseases. Application of this knowledge can produce new and valuable markers, identifying regions producing major diseases to be used for diagnostic and therapeutic benefit.
Faced with the high complexity of the human genome, many approaches are being used to unravel the connection between primary gene structure and function. One well publicised approach is embodied in the Human Genome Mapping Project, where the sequence of all the individual genes in the entire human genome is painstakingly being determined. At the present, however, little information can be directly retrieved on the function of the identified genes and still less about temporal and spatial expression patterns of the developing or mature organism. Other approaches, such as random cDNA sequencing, involve the sequence determination of all genes expressed in a certain tissue, or developmental stage, of an organism. Like a number of other strategies, this is time consuming and prone to numerous problems.
Although the flood of data from large scale sequencing programmes is of enormous benefit to the scientific community, one of the major problems faced by such “shotgun” approaches is the lack of specific information that can be retrieved without significantly more work on the biology of each of the individual genes.
Several other approaches have been taken by molecular biologists to obtain more specific information on the genetic background of particular biological processes. Such approaches rely on a common concept. One gene, or a subset of genes, is switched on, initiating the healthy, pathological, or developmental status of an organ or cell type.
In a large number of experimental systems the isolation of genes, on the basis of their differential expression, has been applied successfully. Differential screening and subtractive hybridisation of cDNA libraries have become well established, cf. Zimmerman et al. (1980) and Davis et al. (1979). Differential library screening works well in practice for genes that are highly expressed, but mRNAs of low abundance are difficult to isolate. Subtractive hybridisation provides a more sensitive screening, but requires large amounts of RNA. More recently RNA fingerprinting methods (often referred to as differential display or DD/RT PCR) have been added to these tools, offering attractive new features for isolating genes. RNA fingerprinting methods are PCR based and therefore do not require large amounts of RNA for experiments. In addition to this, RNA fingerprinting methods allow a large number of RNA pools to be screened for specific mRNAs simultaneously. Investigation of a wide range of pathogenic developmental stages and their controls would be possible. To date, two methods of RNA fingerprinting have proven useful for isolating genes. In 1992 Liang et al. published a protocol (U.S. Pat. No. 5,262,311), soon after a protocol from Welsh et al. (1992) was presented. Both methods begin with cDNA synthesis from RNA using at least one arbitrary primer for the initiation of first and second strand synthesis.
Welsh et al. (1992) designed a protocol in which the same arbitrary 20-mer oligo is used for first and second strand synthesis. Using arbitrary primers only a subset of the mRNAs are transcribed to cDNA. The cDNA pools are then used for a standard PCR with the same primers. One of the dNTPs in the PCR mix contains a radioactive label (
35
S or
32
P) for visualisation of the PCR fragments with PAGE. The Liang and Welsh methods rely on at least one small arbitrary primer for selection of specific cDNAs. As a consequence annealing temperatures are low (~40° C.), and all amplified cDNA fragments originate from a certain degree of mismatch priming. Later several groups produced refinements and optimisations leading to a plethora of articles describing the usefulness of the method (Bauer and Warthoe et al. 1993; Warthoe et al. 1995; Liang and Warthoe et al. 1995; Rohde and Warthoe et al. 1996).
OBJECT OF THE INVENTION
It is an object of the present invention to provide new methods and means for investigating the expression patterns in cells, especially in eukaryotic cells. The results of such investigations may be used in drug development, gene discovery, diagnosis of diseases etc., and therefore such improved methods are highly desirable.
SUMMARY OF THE INVENTION
In its broadest scope, the invention pertains to a method for preparing a sub-divided library of amplified cDNA fragments from the coding region of mRNA contained in a sample, the method comprising the steps of
a) subjecting the mRNA derived from the sample to reverse transcription using at least one cDNA primer having the general formula
5-Con
1
-dT
n2
-V
n3
-N
n4
-3′
wherein Con
1
is any sequence between 1-100 nucleotides, dT is deoxythymidinyl, V is A, G or C, N is A, G, C or T, n2 is an integer ≧1, n3 is 0 or 1, if n3 is 0 then n4 is 0, and if n3 is 1 n4 is an integer ≧0, thereby obtaining first strand cDNA fragments,
b) synthesizing second strand cDNA complementary to the first strand cDNA fragments by use of the first strand DNA fragments as templates, and a second cDNA primer with the general formula
5′-Con
2
-N
x-
3′
wherein Con
2
is any sequence between 1-100 nucleotides and can be different or identical to con
1
, N
x
is A, G, T or C, and x is an integer ≧0, in a appropriate enzyme/-buffer solution which comprises the DNA pol I enzyme or the Klenow fragment of the DNA pol I enzyme, all four deoxyribonucleoside triphosphates and standard buffer and temperature conditions, thereby obtaining double stranded cDNA fragments,
c) subjecting the cDNA fragments obtained in step b) to a molecular amplification procedure so as to obtain amplified cDNA fragments, wherein is used a set of amplification primers having the general formula
5′-Con
3
-N
n1
-3′
wherein Con
3
is a sequence identical to either Con
1
or Con
2
or both, N is A, G, T or C, and n1 is an integer ≧0, wherein at least one set of primers has the general formula I where n>0, said at least one set being capable of priming amplification of any nucleotide sequence complementary in its 5′-end to Con
1
or Con
2
.
This method is advantageous for amplifying very small amounts of RNA. Using the method of the invention it is possible to perform gene-profile analysis from less than 100 cells equal to 10
−9
gram total RNA (10 pgram RNA per cell).
In a further aspect, the invention relates to a method for preparing a sub-divided library of amplified cDNA fragments from the coding region of mRNA (which may be of prokaryotic, Archae or eukaryotic origin) contain
Display Systems Biotech ApS
Frommer & Lawrence & Haug LLP
Jones W. Gary
Kowalski Thomas J.
Tung Joyce
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