RNA fingerprinting to determine RNA population differences

Details RNA fingerprinting to determine RNA population differences RNA fingerprinting to determine RNA population differences

1995-06-09

1997-09-09

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

935 77, 935 80, C12Q 168

Patent

active

056655444

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND TO THE INVENTION

The total number of expressing genes in a typical mammalian cell is unknown, although current estimates range from 10000 to 20000 (1, 2). As many as 70-90% of these genes may perform `household` functions and are likely to always be expressed by different cell types irrespective of their origin (2). In total, there are perhaps 50000-100000 genes expressed in all the different cell types from a given mammal, based on the fact that approximately 3% of the haploid genome corresponds to genes (2).
High resolution 2-dimensional protein gel electrophoresis is commonly used for visualising large numbers of individual gene products expressed simultaneously in a given cell type. At present, the sensitivity of most available 2-dimensional gel techniques limits their use to providing quantitative (and qualitative) analysis of only those 1000-2000 proteins that comprise more than 0.001% of the total protein from a typical mammalian cell type (3). This approach may, however, allow the identification of phenotype specific proteins, involved, for example, in human disease, carcinogenesis, cell differentiation, proliferation and ageing, which can then be microsequenced to provide information so that cDNAs coding for such proteins can eventually be isolated (4).
Unfortunately, to date, an efficient, well developed technique for determining differences in poly(A).sup.+ RNA composition and for monitoring changes in gene activity by the simultaneous quantitative and qualitative analysis of several hundred individual mRNAs has not been available. The use of a technique based on RNA analysis would not only generate additional information, to complement that obtained using 2 dimensional protein analysis, but would also provide a more convenient means for the cloning/sequencing of differentially expressed genes. RNA transcripts of DNA can be sequenced using suitable primers, in chain termination reactions (Mierendorf et al, Methods in Enzymology (1987) 152, 563-566). For the past 15 years, differential screening of cDNA libraries has been widely applied for the isolation of differentially expressed genes (5,6). This technique is, however, rather inefficient and labour intensive, requiring several hundred micrograms of poly(A).sup.+ RNA and several months work to isolate a small number of useful cDNA clones (7). Also, the use of `enriched` cDNA probes/libraries or the application of PCR technology for amplification of such libraries only partially solves the problem of cloning low abundance, differentially expressed, cDNAs (8). Amplification of DNA/cDNA using PCR, with pairs of arbitrary sequence primers (18-40 nucleotides in length), has been used for the identification and characterization of mutations, resulting from nucleotide substitutions, insertions or deletions (9) or for the analysis of cDNA composition differences (9-11). However, this revealed only qualitative, rather than quantititative differences between two different RNA populations. This was mainly because short (8-10 nucleotide) primers, when used for cDNA analysis, are relatively inefficient in the PCR process (9), and the degree of amplification is also more sensitive to both the primary structure of the template DNA and to the reaction conditions used (12). Described herein is a new method for determination of the poly(A).sup.+ RNA composition (pattern) of a given cell or tissue type.


SUMMARY OF THE INVENTION

The present invention provides a method of producing an RNA fingerprint of a poly(A).sup.+ RNA preparation, comprising adding to the poly(A).sup.+ RNA preparation at least one oligonucleotide primer and a supply of nucleotides of which one is a terminator nucleotide, synthesising cDNA products from the RNA template and separating the cDNA products to produce an RNA fingerprint. In another approach, this invention can be used to obtain a similar fingerprint by utilizing single-stranded cDNA, transcribed from the poly(A).sup.+ RNA, as the template in the above process.
An RNA fingerprint is therefore defined herein as a patter

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R.C. Mierendorf et al., Methods in Enzymology, vol. 152, New York (1987), pp. 563 to 566.

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