Sequencing by hybridization

Details Sequencing by hybridization Sequencing by hybridization

2000-11-09

2002-06-04

Whisenant, Ethan C. (Department: 1655)

Chemistry: molecular biology and microbiology

Apparatus

Including measuring or testing

C435S006120, C435S091100, C435S287100, C436S094000, C536S024300

Reexamination Certificate

active

06399364

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to the sequencing of nucleic acids by hybridization. line 5, replace the heading with the following new heading
2. Description of the Related Art
There are currently three formats for sequencing by hybridisation (SBH).
Format 1 SBH [1] attaches the nucleic acid to be analysed to a solid support and then sequentially hybridises labelled oligonucleotides. Format 2 SBH [2] attaches an array of positionally encoded oligonucleotides to a solid support and then hybridises the labelled nucleic acid to be analysed to the array. Format 3 SBH [3] attaches an array of positionally encoded oligonucleotides to a solid support and then hybridises the nucleic acid to be analysed to the array in the presence of is labelled oligonucleotides in free solution. A ligation reaction is then used in order to join the two oligonucleotides, giving greater specificity and information.
Format 1 SBH has been shown to work with short oligonucleotides [4]. 8 mers and even shorter oligonucleotides have been successfully employed [5]. Format 2 SBH requires the use of much longer oligonucleotides for success. 11 mer probes, or longer, are generally required. 20 mers are the norm [6], making the use of generic arrays of all N mers out of the question with current technology (an array of all 20 mers with the smallest pixels currently imaginable would be prohibitively large).
A difficulty with performing format 2 SBH arises because target nucleic acids often have secondary structure which sterically hinders some parts of the target from hybridising with oligonucleotides immobilised in an array. To overcome this problem it has been proposed to chop the target nucleic acid into shorter segments, e.g. of length comparable to the immobilised oligonucleotides. In practice such chopping has proved difficult to achieve in a reliable and uniform manner. The present invention can be seen as providing an indirect way of achieving the same effect. The invention permits the advantages of both format 1 and format 2 SBH to be combined in the same method. In particular, the use of a format 2 positionally encoded array of all N mers or a subset thereof is made possible with arrayed oligonucleotides of length less than 11 mers. This method allows the rapid and facile characterisation of sequence differences between two or more nucleic acid species. The method may be used in order to determine the existence or otherwise of point mutational differences between one or more test nucleic acids and a reference nucleic acid. The method may also be used in order to characterise sequence differences arising from either small deletions or insertions.
SUMMARY OF THE INVENTION
In one aspect the invention provides a method of analysing a target nucleic acid by the use of a mixture of labelled oligonucleotides in solution and an array of immobilised oligonucleotides, which method comprises the steps of:
a) incubating under hybridisation conditions the target nucleic acid with the mixture of labelled oligonucleotides.
b) recovering those labelled oligonucleotides that hybridised in a) with the target nucleic acid,
c) incubating under hybridisation conditions the recovered labelled oligonucleotides from b) with the array of immobilised oligonucleotides,
d) observing distribution of the labelled oligonucleotides on the array and using the information to analyse the target nucleic acid.
In another aspect the invention provides a method of determining differences between a target nucleic acid and a reference nucleic acid, by the use of a first mixture of oligonucleotides in solution labelled with a first label, a corresponding mixture of oligonucleotides in solution labelled with a second label distinguishable from the first label, and an array of immobilised oligonucleotides, which method comprises the steps of:
a) incubating under hybridisation conditions the target nucleic acid with the first mixture of labelled oligonucleotides; and incubating under hybridisation conditions the reference nucleic acid with the second mixture of labelled oligonucleotides,
b) recovering a mixture of those first labelled oligonucleotides and those second labelled oligonucleotides that hybridised in a) with the target nucleic acid or the reference nucleic acid,
c) incubating under hybridisation conditions the recovered mixture of first labelled oligonucleotides and of second labelled oligonucleotides from b) with the array of immobilised oligonucleotides,
d) observing distribution of first labelled oligonucleotides and of second labelled oligonucleotides on the array and using the information to determine differences between the target nucleic acid and the reference nucleic acid.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Preparation of Single Stranded Nucleic Acid
The target nucleic acids may be DNA, RNA, PNA [7], other nucleic acid mimetics or mixtures thereof. They may be single stranded or double stranded; linear, circular, relaxed or supercoiled. They may be of eukaryotic, prokaryotic or viral or archeabacterial origin and may range in size from oligomers to whole genomes.
The target nucleic acids are rendered single stranded. The most preferable method is to amplify the region of interest by PCR [8] and then capture one of the amplified strands using a solid support. Many methods will be obvious to those skilled in the art. The use of a biotinylated PCR primer followed by capture with streptavidin coated magnetic beads [9] is a preferred embodiment.
The PCR may be carried out either by using conventional dNTPs or dNTP analogues that impart altered properties to the PCR product—such as reduced intramolecular secondary structure and thus improved short oligonucleotide access to PCR product in single stranded form. Example nucleotide analogues include: dITP, 7-deaza-dGTP, 7-deaza-dATP, 7-deaza-dlTP, 5-hydroxymethyl-dUTP and 4-methyl-dCTP—either singly and in combination. Many other analogues will be obvious to those skilled in the art. Some of these analogues may require the use of lower PCR annealing temperatures and/or longer PCR extension times for optimal incorporation.
The method of the invention involves use of a mixture of labelled oligonucleotides in solution. This is preferably a mixture of all or a subset of N mers where N is from 5 to 10, preferably 8 or 9. The labelling moieties may be detected by means of fluorescence (emission, lifetime or polarisation), absorption, colour, chemiluminescence, enzymatic activity, radioactive emission, mass spectroscopy or refractive index effects (e.g. surface plasmon resonance).
The N mers in solution may be DNA, RNA, PNA, other nucleic acid mimetics or mixtures thereof. They may be single stranded or partially double stranded. The N mers may also contain bases such as 5-nitroindole, 3-nitropyrrole or inosine that pair with all four usual DNA bases—improving the hybridisation properties of the N mers without increasing the nucleic acid sequence complexity. The N mers may likewise contain bases such as 2-aminopurine and 5-methylcytosine that again improve the hybridisation properties without increasing the nucleic acid sequence complexity.
Structures that can only (or preferentially) form A helices are of particular interest as conditions may be found (e.g. R-loop conditions) where the N mer/PCR product complexes are more stable than the internal secondary structure within the PCR product.
The N mers could also be molecular beacon [10] type ‘panhandle’ structures with stems comprising 5-nitroindole, 3-nitropyrrole, inosine, isodC:isodG [11], dk:dX [12] or dk:dp [13] hairpins. Other such structures will be obvious to those skilled in the art.
The method of the invention also involves use of an array of immobilised oligonucleotides. Each oligonucleotide is immobilised at a spaced location on a surface of a support. The array is preferably of all possible N mer sequences or a subset thereof where N is prefe

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