Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-22
2004-04-20
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06723513
ABSTRACT:
The present invention relates to new methods of sequencing in which the information embodied by each base is effectively magnified, and methods which are particularly suitable for sequencing long nucleic acid molecules in which sequence information for portions of the sequence and details on the portions, positions within the sequence is combined, and kits for performing such methods.
Ever since Watson and Crick clarified the structure of the DNA molecule in 1953, genetic researchers have wanted to find fast and cheap ways of sequencing individual DNA molecules. Sanger/Barrell and Maxam/Gilbert developed two new methods for DNA sequencing between 1975 and 1977 which represented a major breakthrough in sequencing technology. All methods in extensive use today are based on the Sanger/Barrell method and developments in DNA sequencing in the last 23 years have more or less been modifications of this method.
In 1988, however, DNA sequencing technology acquired an entirely new focus. Led by the US, eighteen countries joined together in perhaps the largest individual project in the history of science, the sequencing of the entire human genome of 3×10
9
bp (the Human Genome Project, also called HGP), in addition to several other smaller genomes. As of today, the objective is to be finished during the year 2003. In spite of the fact that the project ties up large scientific resources and carries a large price tag, the gains of the project are considered sufficiently important to justify the cost.
An important part of the project is to develop new methods of DNA sequencing that are both reasonably priced and faster than current technology in principle, these can be divided into gel based (primarily new variants of the Sanger/Parrell method) and non gel-based techniques. The non gel-based techniques probably have a greater potential and mass spectrometry, flow cytometry, and the use of gene chips that hybridize small DNA molecules are some of the approaches that are being tested. Methods that are substantially better than current methods would result in a revolution not only for gene research but also for modern medicine since they would provide the opportunity for extensive patient gene testing and may play an important role in identification and development of drugs. The economic potential of such methods are naturally very great.
Using the currently known sequencing techniques, it has proved difficult to extend the length of the sequences that can be read for each sequencing reaction, and most methods used today are limited to about 7-800 base pairs per sequencing reaction. Nor is it possible to sequence more than one sequence per sequencing reaction with the methods widely used today.
To sequence many or long sequences, it is generally necessary to perform many parallel sequencing reactions (e.g. to sequence a diploid human genome of 6 billion base pairs, several million parallel sequencing reactions would be necessary). This is a considerable bottleneck because the total number of processes, the use of enzymes and reagents, the number of unique primers required, etc. are often directly proportional to the number of sequencing reactions that have to be performed. Furthermore, resources often have to be devoted to sequencing overlapping sequences. In addition, different types of organisation work must be performed, such as setting up and sorting a DNA library. It is also necessary to expend resources in order to isolate a possible target sequence if it is found among other sequences.
In order to illustrate the fundamental problems that limit the length of sequencing reactions, it is appropriate to divide the sequencing methods currently used and under development into two large groups (there are individual methods that fall outside this division, but they represent a small minority). In the first group, we have methods based on the size range of polynucleotides. The starting point is to make one or more polynucleotide ladders in which all molecules have one common and one arbitrary end. For example, the classical sequencing methods of Sanger and Maxam-Gilbert are based on four sequence ladders that represent each of the four bases A, C, G, and T.
The limiting factor with respect to the length of a sequencing reaction that can be read is that one must be able to distinguish between polynucleotides that only vary with one monomer. The longer the polynucleotides in the sequence ladder, the smaller the relative differences in size between the polynucleotides. Most of the methods for determining the size of molecules thus quickly reach a limit where it is not possible to distinguish between two adjacent polynucleotides.
In the other group the methods are based on a different principle. By identifying short pieces of sequences that are present in a target molecule, the target sequence can be reconstructed by utilising the overlaps between the sequence pieces.
Thus, in many sequencing methods target molecules are fragmented into smaller pieces, the composition of each fragment is deduced and by finding overlapping sequences the original sequence is constructed. For example, microarrays have been created with 65,536 addresses where each address contains unique octamers. All permutations with octamers (4
8
=65,536) are thus covered. If the target molecules then are tagged with fluorescence and hybridised with the octamers, the information about what sequence pieces are present in the target sequence can be obtained by registering the addresses that have been labelled with fluorescence.
An important limiting factor with respect to the length of sequencing reactions that can be read is the following combinatorial problem. The longer the sequencing reaction that is to be performed, the longer the sequence pieces must be in order to make reconstruction of the target sequence possible. However, the number of permutations that have to be tested increases exponentially with the length of the sequence pieces that are to be identified. This increases the need for unique addresses on the microarrays in a corresponding manner.
An alternative use for microarrays is resequencing of known sequences, e.g. by screening for gene mutations in a population. For this purpose, oligonucleotides can be adjusted to the known sequence so that the number of addresses required can be reduced and the length of the sequence pieces that are identified can be increased. However, designing microarrays for specific purposes is expensive and resource demanding, and at present there are only microarrays for a few DNA sequences. Since the human genome consists of somewhere between 100-140,000 genes, it would be very resource demanding to mass-sequence human genomes in this manner.
Another disadvantage with using microarrays is that the limitations of current construction technology (e.g. photolithography) does not make it possible to create pixels of less than about 10×10 micrometers. Thus, only a fraction of the resolution potential of the fluorescence scanner is utilised. Current fluorescence scanners are capable of distinguishing pixels of 0.1×0.1 micrometer, which means that microarrays can contain 10,000 times as much information as they currently contain.
It would therefore be advantageous to develop new methods/principles of identifying long sequence pieces where the combination problems mentioned above could be avoided. It would likewise be advantageous to develop new methods/principles that make it possible to sequence long target sequences without having the length of the sequence pieces that must be identified increase exponentially with the length of the target sequence.
Another sequencing method (for example as embodied in U.S. Pat. No. 5,714,330) that is based on identification of sequence pieces consists of distributing fragmented target DNA over a reading plate. Thereafter, the target DNA is treated so that a fluorescence signal representing one or several of the first base pairs is fixed to the target DNA. The fluorescence signals for each position is read before the procedure is repea
Lingvitae AS
Whisenant Ethan
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