Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-01
2003-03-04
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024100
Reexamination Certificate
active
06528258
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the analysis of polymer molecules, and particularly of polynucleotides. Polynucleotides are polymeric molecules comprising repeating bases of nucleosides bound together in a linear fashion. Examples of polynucleotides are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the genetic material of living organisms. It is the molecule that stores and transmits the code of life. Segments of DNA known as genes act as the templates for the formation of proteins, which are the molecules that comprise the structure and function of all living matter.
DNA polymers are made up of strings of four different nucleotide bases known as adenine (A), guanine (G), cytosine (C), and thymine (T). The particular order, or “sequence” of these bases in a given gene determines the structure of the protein encoded by the gene. Furthermore, the sequence of bases surrounding the gene typically contains information about how often the particular protein should be made, in which cell types, etc. Knowledge of the DNA sequence in and around a gene provides valuable information about the structure and function of the gene, the protein it encodes, and its relationship to other genes and proteins.
The complete nucleotide sequence of all DNA polymers in a particular individual is known as that individual's “genome”. Whereas most bacteria have genomes on the order of a few million bases long, the human genome contains more than 3.5 billion bases. In recent years, both government and private organizations have expended enormous resources attempting to build a complete, detailed map of the human genome. In particular the Human Genome Project, a government-funded effort directed by the National Institutes of Health, has promised to deliver a complete human sequence by the year 2003 at a cost that is expected to exceed $3 billion. Private corporations have also entered into the race. For example, Celera, Inc. a Rockville, Md. company, has spent over $300 million dollars with the purpose of sequencing the genome by 2001.
There are significant reasons that so much effort and money is focused on sequencing the human genome. First, there is the obvious scientific merit associated with having a detailed map of mankind's genetic template. The information embodied by such a map will allow scientists to better understand the relationship between our genetic code and the functions of the nearly 100,000 proteins that make up our bodies. It is already known that there is a direct relationship between particular DNA sequences and certain-disease states. This fact has encouraged many pharmaceutical companies to invest heavily in the field of genomics research in the hope of discovering the underlying genetic nature of these diseases.
Another reason that sequence information is important is the expected ability to determine an individual's susceptibility to particular diseases based on his or her genetic sequence. The field of genetic diagnostics is dedicated to identifying nucleotide sequence elements whose presence in a genome correlates with development of a particular disorder or feature. The more information is available about genomic sequence elements observed in the population the more powerful this field becomes. Furthermore, the more rapidly information about the prevalence and penetrance of sequence elements in the general population, as well as the presence of particular such elements in the genomes of particular individuals being tested, the more effective the analysis becomes.
Yet another reason that sequence information is valuable is that a number of pharmaceutical companies seek to develop drugs that are custom-tailored to an individual's genetic profile. The hope is to provide targeted, potent drugs, possibly with decreased dosage levels appropriate to the genetic characteristics of the particular individual to whom the drug is being administered.
Current Sequencing Technology
Most currently available nucleotide sequencing technologies determine the nucleotide sequence of a given polynucleotide strand by generating a collection of complementary strands of different lengths, so that the collection includes molecules terminating at each base of the target sequence and ranging in size from just a few nucleotides to the full length of the target molecule. The target molecule's sequence is then determined by analyzing the truncated complementary strands and determining which terminate with each of four DNA nucleotides. A “ladder” is constructed by arranging the truncated molecules in order by length, and the terminal residue of each rung is read off to provide the complement of the target polynucleotide sequence.
The most popular DNA sequencing systems generate the collection of truncated complementary molecules by performing a template synthesis reaction in the presence of low concentrations of modified versions of each of the four natural nucleotides. These modified compounds can be added to a polynucleotide chain but cannot be extended. Furthermore, each one is labeled with a different fluorescent dye, so that chains terminated with different nucleotides can be distinguished from one another by the color of fluorescence they emit (see, for example, Smith et al., U.S. Pat. No. 5,821,058; Smith et al., U.S. Pat. No. 5,747,249; Kaiser et al.,
Methods Enzymol
218:122-153, 1992;
Automated DNA Sequencing Chemistry Guide
, PE Applied Biosystems, A division of Perkin-Elmer (1998); each of which is incorporated herein by reference). The sequence of the target molecule is then determined by reading the sequence of fluorescent colors emitted by the arranged rungs of the molecular ladder of complementary strands.
These DNA sequencing methods have been automated, and machines that perform them are available in the commercial marketplace. The most advanced of these machines are capable of carrying out the above sequencing process in parallel reactions (up to 96 at a time). Under certain conditions the output from one machine may exceed 300,000 bases per day. Using large numbers of such machines, some organizations claim sequencing rates as high as 100 million bases per day (The Economist 347(8068):87-88 May 16 1998; incorporated herein by reference).
Currently-available DNA sequencing systems are very powerful. However, they are limited by their speed, their complexity, and their cost. Because of these problems, their use is not widespread in clinical environments. For example, even the most sophisticated genetic diagnostic procedures involve the analysis of only very short regions of sequence (often not by direct sequencing but rather by indirect methods that probe the underlying sequence). Large scale sequencing of patient DNA is simply not performed in the clinic.
The speed of currently available automated sequencers is limited by the inability of the machines to analyze more than several hundred (typically around 600) nucleotides of sequence at a time. Allowing for the overlaps needed to piece together correctly strands less than 1000 bases longs, the standard sequencing process may have to be performed as many as 70 million times in order to determine the human genome sequence (Technology Review 102(2):64-68 1999 Mar/Apr; incorporated herein by reference). As has been noted, it takes many 600 s to fit into 3 billion and in practice it takes many more than that to make a full sequence because the individual fragments have to be linked together by matching up their overlaps. (
The Economist
347(8068):87-88 May 16 1998; incorporated herein by reference). At a theoretical rate of even 100 million bases per day it will take at least a year to sequence the human genome once. With these techniques, large-scale sequencing cannot become a clinical tool. For genetic diagnostics to become practical in a clinical setting, the sequencing rate will have to be increased by at least three to five orders of magnitude.
The complexity of current sequencing technology arises from the need to amplify and modify the genetic molecules being seque
Baker C. Hunter
Choate Hall & Stewart
Herschbach Jarrell Brenda
LifeBeam Technologies, Inc.
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