Baths – closets – sinks – and spittoons – Flush closet – Tank only
Reexamination Certificate
1999-03-18
2002-06-11
Campbell, Eggerton A. (Department: 1656)
Baths, closets, sinks, and spittoons
Flush closet
Tank only
C435S091200
Reexamination Certificate
active
06401267
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to the field of molecular biology. The invention particularly provides novel methods and compositions to enable highly efficient sequencing of nucleic acid molecules. The methods of the invention are suitable for sequencing long nucleic acid molecules, including chromosomes and RNA, without cloning or subcloning steps.
2. Description of the Related Art
Nucleic acid sequencing forms an integral part of scientific progress today. Determining the sequence, i.e. the primary structure, of nucleic acid molecules and segments is important in regard to individual projects investigating a range of particular target areas. Information gained from sequencing impacts science, medicine, agriculture and all areas of biotechnology. Nucleic acid sequencing is, of course, vital to the human genome project and other large-scale undertakings, the aim of which is to further our understanding of evolution and the function of organisms and to provide an insight into the causes of various disease states.
The utility of nucleic acid sequencing is evident, for example, the Human Genome Project (HGP), a multinational effort devoted to sequencing the entire human genome, is in progress at various centers. However, progress in this area is generally both slow and costly. Nucleic acid sequencing is usually determined on polyacrylamide gels that separate DNA fragments in the range of 1 to 500 bp, differing in length by one nucleotide. The actual determination of the sequence, i.e., the order of the individual A, G, C and T nucleotides may be achieved in two ways. Firstly, using the Maxam and Gilbert method of chemically degrading the DNA fragment at specific nucleotides (Maxam & Gilbert, 1977), or secondly, using the dideoxy chain termination sequencing method described by Sanger and colleagues (Sanger et al., 1977). Both methods are time-consuming and laborious.
More recently, other methods of nucleic acid sequencing have been proposed that do not employ an electrophoresis step, these methods may be collectively termed Sequencing By Hybridization or SBH (Drmanac et al., 1991; Cantor et al., 1992; Drmanac & Crkvenjakov, U.S. Pat. No. 5,202,231). Development of certain of these methods has given rise to new solid support type sequencing tools known as sequencing chips. The utility of SBH in general is evidenced by the fact that U.S. Patents have been granted on this technology. However, although SBH has the potential for increasing the speed with which nucleic acids can be sequenced, all current SBH methods still suffer from several drawbacks.
SBH can be conducted in two basic ways, often referred to as Format 1 and Format 2 (Cantor et al., 1992). In Format 1, oligonucleotides of unknown sequence, generally of about 100-1000 nucleotides in length, are arrayed on a solid support or filter so that the unknown samples themselves are immobilized (Strezoska et al., 1991; Drmanac & Crkvenjakov, U.S. Pat. No. 5,202,231). Replicas of the array are then interrogated by hybridization with sets of labeled probes of about 6 to 8 residues in length. In Format 2, a sequencing chip is formed from an array of oligonucleotides with known sequences of about 6 to 8 residues in length (Southern, WO 89/10977; Khrapko et al., 1991; Southern et al., 1992). The nucleic acids of unknown sequence are then labeled and allowed to hybridize to the immobilized oligos.
Unfortunately, both of these SBH formats have several limitations, particularly the requirement for prior DNA cloning steps. In Format 1, other significant problems include attaching the various nucleic acid pieces to be sequenced to the solid surface support or preparing a large set of longer probes. In Format 2, major problems include labelling the nucleic acids of unknown sequence, high noise to signal ratios that generally result, and the fact that only short sequences can be determined. Further problems of Format 2 include the secondary structure formation that prevents access to some targets and the different conditions that are necessary for probes with different GC contents. Therefore, the art would clearly benefit from a new procedure for nucleic acid sequencing, and particularly, one that avoids the tedious processes of cloning and/or subcloning.
SUMMARY OF THE INVENTION
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing new methods and compositions for the sequencing of nucleic acids. The novel techniques described herein have been generally termed Format 3 by the inventors and represent marked improvements over the existing Format 1 and Format 2 SBH methods. In the Format 3 sequencing provided by the invention, nucleic acid sequences are determined by means of hybridization with two sets of small oligonucleotide probes of known sequences. The methods of the invention allow high discriminatory sequencing of extremely large nucleic acid molecules, including chromosomal material or RNA, without prior cloning, subcloning or amplification. Furthermore, the present methods do not require large numbers of probes, the complex synthesis of longer probes, or the labelling of a complex mixture of nucleic acids segments.
To determine the sequence of a nucleic acid according to the methods of the present invention, one would generally identify sequences from the nucleic acid by hybridizing with complementary sequences from two sets of small oligonucleotide probes (oligos) of defined length and known sequence, which cover most combinations of sequences for that length of probe. One would then analyze the sequences identified to determine stretches of the identified sequences that overlap, and reconstruct or assemble the complete nucleic acid sequence from such overlapping sequences.
The sequencing methods may be conducted using sequential hybridization with complementary sequences from the two sets of small oligos. Alternatively, a mode described as “cycling” may be employed, in which the two sets of small oligos are hybridized with the unknown sequences simultaneously. The term “cycling” is applied as the discriminatory part of the technique comes from then increasing the temperature to “melt” those hybrids that are non-complementary. Such cycling techniques are commonly employed in other areas of molecular biology, such as PCR, and will be readily understood by those of skill in the art in light when reading the present disclosure.
The invention is applicable to sequencing nucleic acid molecules of very long length. As a practical matter, the nucleic acid molecule to be sequenced will generally be fragmented to provide small or intermediate length nucleic acid fragments that may be readily manipulated. The term nucleic acid fragment, as used herein, most generally means a nucleic acid molecule of between about 10 base pairs (bp) and about 100 bp in length. The most preferred methods of the invention are contemplated to be those in which the nucleic acid molecule to be sequenced is treated to provide nucleic acid fragments of intermediate length, i.e., of between about 10 bp and about 40 bp. However, it should be stressed that the present invention is not a method of completely sequencing small nucleic acid fragments, rather it is a method of sequencing nucleic acid molecules per se, which involves determining portions of sequence from within the molecule—whether this is done using the whole molecule, or for simplicity, whether this is achieved by first fragmenting the molecule into smaller sized sections of from about 4 to about 1000 bases.
Sequences from nucleic acid molecules are determined by hybridizing to small oligonucleotide probes of known sequence. In referring to “small oligonucleotide probes”, the term “small” means probes of less than 10 bp in length, and preferably, probes of between about 4 bp and about 9 bp in length. In one exemplary sequencing embodiment, probes of about 6 bp in length are contemplated to be particularly useful. For the sets of oligos to cover all combinations of sequences for the length of pr
Campbell Eggerton A.
Marshall Gerstein & Borun.
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