Categorising nucleic acids

Details Categorising nucleic acids Categorising nucleic acids

2000-04-10

2001-10-02

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S025300

Reexamination Certificate

active

06297017

ABSTRACT:

This invention relates to methods of analysing heterogeneous populations of nucleic acids. More specifically this application relates to improved methods of analysing populations of complementary DNA derived from poly-adenylated messenger RNA.
Present methods suffer from numerous drawbacks. The simplest methods such as ‘subtractive cloning’ allow crude comparative information about differences in gene expression between related cell types to be derived, although these methods have had moderate success in isolating rare cDNAs, they are not suited to high throughput applications where large numbers of samples need to be analysed at low cost whilst still generating detailed data on the expression of a large number of genes, if not all genes simultaneously. Other methods such as ‘differential display’ and related ‘molecular indexing’ methods such as Sibson (PCT/GB93/0145) or Kato (EP 0 735 144 A1) allow generation of gene expression data for large numbers of genes in a single experiment but embodiments of these methods to data have not been fully automated and are dependant on gel electrophoresis for analysis. These methods are thus too labourious and expensive for high throughput applications. Still more informative methods have arrived recently such as SAGE, Serial Analysis of Gene Expression, which give quantitative data on gene expression without prior knowledge and can readily and specifically identify cDNAs expressed in a given cell type but at the cost of excessive sequencing for a high resolution gene expression profile.
Sibson, D. R., PCT/GB93/01452 provides methods for gene expression analysis using populations of adapter molecules to identify the ambiguous sticky-ends generated by cleavage of a nucleic acid with a type IIs restriction endonuclease to categories the cleavage fragments. Using specifically engineered adapters one can specifically immobilise or amplify or clone specific subsets of fragments in a manner similar to differential display but achieving a greater degree of sorting and control. The methods disclosed in this patent are difficult and expensive to automate.
Kato, Nucleic Acids Research 23, 3685-3690, 1995 and EP 0 735 144 A1 provides a method for analysing patterns of gene expression in a tissue which comprise sorting terminal cDNA fragments into sub-populations followed by selective amplification of specific subsets of cDNA fragments. Sorting is effected by using type IIs restriction endonucleases and adapters. The adapters also carry primer sites which in conjunction with general poly-T primers allow selective amplification of terminal cDNA fragments as in differential display. It is possibly more precise than differential display in that it effects greater sorting; only about 100 cDNAs will be present in a given subset and sorting can be related to specific sequence features rather than using primers chosen by trial and error. The subsets can then be analysed by gel electrophoresis to separate the fragments by length and generate a profile of mRNAs in a tissue. This method is dependant on PCR amplification which distorts the frequencies of each cDNA present. Furthermore the methods of analysis used so far have been dependant on gel electrophoresis.
The Gene Profiling technology described in patent PCT/GB97/02403 provides a further method of molecular indexing for the analysis of patterns of gene expression in a cell by sampling each cDNA within the population of that cell. In one embodiment, the sampling system takes two samples of 4 bp from each cDNA in a population and determines their sequence with respect to a defined reference point. The methods of this invention are amenable to automation but require many steps to derive signature information.
It is an object of this invention to provide a method of gene expression profiling that is amenable to high throughput and automation which has great sensitivity. In this way should be possible to avoid the need for exponential amplification of cDNAs which distorts the frequencies of the cDNAs which is essential information in interpreting changes in gene expression patterns between different states of a given tissue and between different tissues of the same organism which have differentiated differently. This invention provides methods to derive a signature for each cDNA in a library which require fewer steps than many conventional approaches hence reducing sample loss and distortion of measurements of quantities of each mRNA.
Accordingly, this invention provides a method for categorising one or more nucleic acids, which method comprises immobilising double-stranded nucleic acids on a solid phase support, cleaving the immobilised nucleic acids with an endonuclease such that each cleaved nucleic acid has a double-stranded portion, denaturing the cleaved nucleic acids to form single-stranded cleaved nucleic acid, hybridising one or more oligonucleotide sequences to the resulting single-stranded cleaved nucleic acid, each oligonucleotide sequence comprising a pre-determined recognition sequence situated such that it recognises a sequence which was part of the double-stranded portion of the nucleic acid and a label specific to the recognition sequence, extending correctly hybridised oligonucleotide sequences along the single-stranded portion of the immobilised nucleic acid to form an extended strand, denaturing the extended strand from the immobilised strand and characterising the immobilised nucleic acid by identifying the size of the extended strand and the identity of the recognition sequence.
This invention also provides a kit for categorising a nucleic acid, which kit comprises one or more adaptors and one or more sets of oligonucleotide sequences, wherein the adaptors comprise nucleic acid having a double-stranded primer portion of a known sequence and a single-stranded portion of a pre-determined length, either each single-stranded portion of each nucleic acid in the adaptors having the same pre-determined sequence or all possible sequences of the single-stranded portion being represented in the adaptors, and wherein each oligonucleotide sequence comprises a first sequence, a second sequence attached to the first sequence and a third sequence attached to the second sequence, in which the first sequence is complementary to the sequence of the primer portion of the adaptor, the second sequence is the same sequence as the single-stranded portion of the adaptors or all possible second sequences of the same length as the single-stranded portion of the adaptors are represented within the set of oligonucleotides, and the third sequence comprises a pre-determined recognition sequence.


REFERENCES:
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patent: 5508169 (1996-04-01), Deugau et al.
patent: 5919626 (1999-07-01), Shi et al.
patent: 0 667 393 (1995-08-01), None
patent: 735144A1 (1996-10-01), None
patent: WO 93/23562 (1993-11-01), None
patent: WO 94/01582 (1994-01-01), None
patent: WO 95/04160 (1995-02-01), None
Kato, Nucleic Acids Research, vol. 23, No. 18, 1995, May 11, 19991, pp. 2251-2259, XP000204316.
Wong et al., Nucleic Acids Research, vol. 19, No. 9, May 11, 1991, pp. 2251-2259, XP000204316.
Guilfoyle et al., Nucleic Acids Research, vol. 25, No. 9, May 1, 1997, pp. 1854-1858, XP002076198.
Kato, Nucleic Acids Research, vol. 23, No. 18, 1995, pp. 3685-3690, XP-002053720.

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