Characterizing DNA

Details Characterizing DNA Characterizing DNA

1999-12-29

2003-09-02

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S024300

Reexamination Certificate

active

06613511

ABSTRACT:

The present invention relates to a method for characterising DNA, especially cDNA, so that the DNA may be identified, for example, from a population of DNAs. The invention also relates to a method for assaying the DNA.
Analysis of complex nucleic acid populations is a common problem in many areas of molecular biology, nowhere more so than in the analysis of patterns of gene expression. Various methods have been developed to allow simultaneous analysis of entire mRNA populations, or their corresponding cDNA populations, to enable us to begin to understand patterns of gene expression in vivo.
Present methods, however, 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. Other methods such as ‘differential display’ and related ‘molecular indexing’ methods allow broader comparisons of gene expression between cell types but embodiments of these methods to date have been difficult to automate and are dependant on gel electrophoresis for analysis. 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.
The method of “subtractive cloning” (Lee et al, Proc. Nat. Acad. Sci. USA 88, 2825-2829) allows identification of mRNAs, or rather, their corresponding cDNAs, that are differentially expressed in two related cell types. One can selectively eliminate cDNAs common to two related cell types by hybridising cDNAs from a library derived from one cell type to a large excess of mRNA from a related, but distinct cell type. mRNAs in the second cell type complementary to cDNAs from the first type will form double-stranded hybrids. Various enzymes exist which degrade such ds-hybrids allowing these to be eliminated thus enriching the remaining population in cDNAs unique to the first cell type.
The method of “differential display” (Laing and Pardee, Science 257, 967-971, 1992) sorts mRNAs using PCR primers to selectively amplify specific subsets of an mRNA population. An mRNA population is sub-divided into aliquots, each of which is primed with a series of “anchored” poly-T primers to effect reverse transcription with normalisation of the length of the poly-A tail. A set of redundant gene specific primers, of maybe 10 nucleotides or so are used to amplify the reverse strand. Typically a set of 30 such primers are used. In this way mRNAs are characterised by the length of their amplification products. The resultant amplified sub-populations can then be cloned for screening or sequencing or the fragments can simply be separated on a sequencing gel. Low copy number mRNAs are less likely to get lost in this sort of scheme in comparison with subtractive cloning, for example, and it is probably marginally more reproducible. Whilst this method is more general than subtractive cloning, time-consuming analysis is required. Unfortunately with these methods each cDNA may have multiple amplification products. Furthermore, the methods are not quantitative and comparative information can only be determined for relatively closely related cell types, e.g. diseased and normal forms of a particular tissue from the same organism.
The method of serial analysis of gene expression (Velcelescu et al., Science 270, 484-487, 1995) allows identification of mRNAs, or rather, their corresponding cDNAs that are expressed in a given cell type. It gives quantitative information about the levels of those cDNAs as well. The process involved isolating a signature ‘tag’ from every cDNA in a population using adaptors and type IIs restriction endonucleases. A tag is a sample of a cDNA sequence of a fixed number of nucleotides sufficient to uniquely identify that cDNA in the population. Tags are then ligated together and sequenced. The method gives quantitative data on gene expression and will readily identify novel cDNAs.
Methods involving hybridisation grids, chips and arrays are advantageous in that they avoid gel methods for sequencing and are quantitative. They can be performed entirely in solution, thus are readily automatable. Such arrays of oligonucleotides are a relatively novel approach to nucleic acid analysis, allowing mutation analysis, sequencing by hybridisation and mRNA expression analysis. For gene expression analysis oligonucleotides complementary to and unique to known RNAs can be arrayed on a solid phase support such as a glass slide or membrane. Labelled cDNAs or mRNA are hybridised to the array. The appearance of labelled nucleic acid immobilised at a specific locus on the array is indicative of the presence of the corresponding mRNA to which the oligonucleotide at that locus is complementary. Methods of construction of such arrays have been developed, (see for example: A. C. Pease et al. Proc. Natl. Acad. Sci. USA. 91, 5022-5026, 1994; U. Maskos and E. M. Southern, Nucleic Acids Research 21, 2269-2270, 1993; E. M. Southern et al., Nucleic Acids Research 22, 1368-1373, 1994) and further methods are envisaged. Unfortunately, these methods require that the sequence of RNAs be known prior to construction of the array. This means that this approach is not applicable to organisms for which little or no information is known.
Immobilisation can be followed by partial sequencing of fragments by a single base method, e.g. using type IIs restriction endonucleases and adaptors. This particular approach is advocated by Brenner in PCT/US95/12678.
Arrays of oligonucleotides of N bp length can be employed. The array carries all 4
N
possible oligonucleotides at specific points on the grid. Nucleic acids are hybridised as single strands to the array. Detection of hybridisation is achieved by fluorescently labelling each nucleic acid and determining from where on the grid the fluorescence arises, which determines the oligonucleotide to which the nucleic acid has bound. The fluorescent labels also give quantitative information about how much nucleic acid has hybridised to a given oligonucleotide. This information and knowledge of the relative quantities of individual nucleic acids should be sufficient to reconstruct the sequences and quantities of the hybridising population. This approach is advocated by Lehrach in numerous papers and Nucleic Acids Research 22, 3423 contains a recent discussion. A disadvantage of this approach is that the construction of large arrays of oligonucleotides is extremely technically demanding and expensive.
The method of “molecular indexing” (PCT/GB93/01452) uses populations of adaptor molecules to hybridise to the ambiguous sticky-ends generated by cleavage of a nucleic acid with a type IIs restriction endonuclease to categorise the cleavage fragments. Using specifically engineered adaptors 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. However, time-consuming analysis is required and the methods disclosed in this patent application are difficult and expensive to automate.
The method of Kato (Nucleic Acids Research 23, 3685-3690, 1995) exemplifies the above molecular indexing approach and effects cDNA population analysis by 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 adaptors. The adaptors also carry primer sites which in conjunction with general poly-T primers allows 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 cho

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