Methods for characterizing mRNA molecules

Details Methods for characterizing mRNA molecules Methods for characterizing mRNA molecules

2000-10-24

2003-12-30

Siew, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091510, C435S091520, C435S183000, C536S025400

Reexamination Certificate

active

06670121

ABSTRACT:

The present invention relates to methods for the qualitative and quantitative detection of differentially expressed mRNA molecules. The technique on which the invention is based is referred to hereinafter as DEPD (digital expression pattern display) for short.
According to current estimates, the genome of higher organisms comprises about 100,000 different genes, of which, however, only a comparatively small number is expressed in each cell of an organism and thus converted into polypeptides and proteins. It is assumed that substantially all processes and metabolic functions in the realm of living material depend on which genes are switched on or off at which time in which tissues. Thus, numerous findings indicate that cellular processes such as, for example, homeostasis, reactions to allergies, regulation of the cell cycle, ageing and entry of cells into programmed cell death (apoptosis) are based on the differential expression of particular genes or are connected thereto. Both the progress of normal development and the pathological manifestations leading to diseases such as, for example, cancer are essentially based on changes in the expression of genes.
Accordingly, there is a need for specific methods for detecting differently or differentially expressed mRNA molecules in order to identify differences in the expression of genes by comparison with suitable controls. Methods of this type would be of great importance both diagnostically and in the evaluation of therapeutic targets.
The novel method is used in particular for detecting as many as possible of the mRNA molecules present in a cell or in a tissue, and comparison thereof with other cells or tissues or with particular conditions (stages of disease or development) or treatment phases, in particular preferably both in qualitative and in quantitative respects. The novel method thus permits, for example, the construction of a comprehensive picture of the various mRNA molecules present in a defined mRNA population, and the subsequent use of the information obtained thereby, which is preferably digital, in database analyses. It is to be assumed that in the near future all human gene sequences will be available in appropriate databases. The present method therefore permits complete detection and characterization of cellular processes which are reflected by specific expression patterns at the level of the mRNA populations. It is possible thereby, for example, to identify quickly and reliably changes in the expression pattern of individual genes involved in a specific process. This makes it possible also to define novel targets for the essential action of active pharmaceutical ingredients. The resulting information can also be employed for producing the causal connection between known target genes and target proteins via comprehensible biochemical signal and synthesis routes.
The object stated above is known to the person skilled in the relative area. Various routes have been followed in the prior art to achieve the stated object.
For example, P. Liang and A. B. Pardee describe a method for separating individual mRNAs by a polymerase chain reaction (PCR) (P. Liang & A. B. Pardee 1992 Science 257, 967-971). This method was used in order to compare the mRNA populations expressed by two related cell types. Separation of the complex mixture of mRNA molecules into fractions each consisting of 50-100 genes of the total population was achieved by: 1) reverse transcription of the mRNA into single-stranded cDNA with 12 so-called 3′ anchor primers of the form T
11
VN (where T
11
=eleven consecutive Ts, V=A, C, G; N=A, C, G, T); 2) PCR amplification of each individual cDNA fraction with the appropriate 3′ anchor primer and an arbitrarily selected 5′ oligomer comprising 10 nucleotides in the presence of radiolabelled deoxyribonucleotides. The products were fractionated on sequencing gels, and 50-100 bands were observed in the 100-500 nucleotide size range. The bands resulted from the amplification of cDNAs which correspond to the 3′ ends of mRNAs which contained the complement of the 3′ anchor primer and of the arbitrarily selected 5′ oligomer. The patterns of the bands amplified from the two cDNAs were similar for each primer pair, and it was not possible to differentiate the intensities of about 80% of the bands. Certain bands appeared more strongly in one or other PCR mixture, while some were detectable in only one of the two mixtures.
If all the 50,000-100,000 different mRNAs expected for mammals were detectable using the arbitrarily selected 5′ primers (arbitrary primer), then a number of 80-95 such oligonucleotides and about 1000 PCRs would be necessary in order to detect with high probability about two thirds of these mRNAs. It has emerged from numerous investigations in recent years that the method described above leads to a high rate (up to 90%) of false-positive signals.
WO 95/13369 discloses a method (TOGA−TOtal Gene Expression Analysis) for simultaneous identification of differentially expressed mRNAs and for measurement of their relative concentrations. The method is based on the construction of double-stranded cDNA from isolated mRNA using a specific set of oligo (dT) primers. This entails employing a mixture of 12 anchor primers with the following structure: starting from 5′, a “stuffer” or “heel” fragment of 4-40 bases is followed by a recognition sequence for a restriction endonuclease (typically NotI), 7-40 dT nucleotides and finally two “anchor bases” V, N at the 3′ end of the primer. In this case, V is a deoxyribonucleotide of the group dA, dC or dG, while N defines the deoxyribonucleotides dA, dC, dG and dT. The cDNA obtained in this way is subsequently completely digested with a restriction enzyme which recognizes 4 bases as sequence for the cleavage site (for example MspI), cut with NotI and cloned into an appropriately treated plasmid vector. The orientation of the insert in this case is antisense relative to a vector-encoded, bacteriophage-specific promoter (typically T3). The ligations are transformed into an
E. coli
strain, whereby cDNA banks are generated. The plasmid DNA of these cDNA libraries is isolated and linearized by means of combination digestions by 6 different restriction enzymes which are different from those used above. The linearized cDNA is translated by T3 polymerase into cRNA and consequently transcribed into 16 subfractions of single-stranded cDNA. This entails use of a thermostable reverse transcriptase at high temperature and one of each of 16 different cRNA primers whose two 3′ nucleotides consist of a complete permutation of the 4 possible deoxyribonucleotides dA, dC, dG and dT. The products of the 16 cDNA fractions are employed as templates for PCR with use of a 3′ oligonucleotide which corresponds to a vector sequence near to the cloning site of the insert, and of a 5′ oligomer which corresponds to one of the 16 cDNA synthesis primers with addition of two 3′ nucleotides of the complete permutation of the 4 possible deoxyribonucleotides dA, dC, dG and dT. Up to 256 different pools are generated in this way, and the radiolabelled bands (35S-dATP or 32P-dCTP) thereof are analysed on polyacrylamide gels. It is said to be theoretically possible on the basis of the information obtained about the length and composition of the 8 identified bases in a labelled band to conclude the identity of the relevant gene in a complete database without cloning and sequencing steps.
The method described above gives rise to the following problems in connection with the high specificity, selectivity and reproducibility which are desired according to the invention:
1) potential loss of cDNA sequences through NotI digestion;
2) potential loss of cDNA sequences through vector ligation;
3) potential loss of cDNA sequences through transformation into
E. coli
and different amplification rates for different cDNA inserts;
4) contamination of the PCR templates with bacterial genomic DNA after plasmid amplificati

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