Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1997-06-18
2001-10-02
Alexander, Lyle A. (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S082080, C436S086000, C436S094000, C436S096000, C250S461200, C435S006120
Reexamination Certificate
active
06296810
ABSTRACT:
1. INTRODUCTION
Considerable interest has been developing in the past few years to sequence the entire human genome (i.e., all of the genetic material in a human cell). The task, however, is enormous because it involves the sequencing of at least 3,000,000,000 base pairs, an effort which is likely to take ten or more years and cost $3,000,000,000 if undertaken using conventional technology (1993 Edgington, Bio/Technology 11:39-42, which is incorporated herein by reference).
The Committee on Mapping and Sequencing the Human Genome of the National Research Council in their 1988 report entitled, Mapping and Sequencing the Human Genome (which is incorporated herein by reference), stated that, “No foreseeable technology will be able to automate DNA sequencing comprehensively.” The present invention is a method and apparatus for comprehensively automating this effort with substantial improvements in speed and cost. The invention is applicable to the sequencing of genetic material from any source, human or otherwise.
2. BACKGROUND OF THE INVENTION
2.1. DNA and RNA
Deoxyribonucleic acid (DNA) is the primary genetic material of most organisms. Ribonucleic acid (RNA) is the primary genetic material in certain viruses. Additionally, a form of RNA known as messenger RNA (mRNA) is found in all cells and comprises copies of portions of the primary genetic information found in the DNA. In its natural state, DNA is found in the form of a pair of complementary chains of nucleotides which are interconnected as a double helix (see FIG.
1
). A nucleotide in turn is composed of a nitrogenous base (see FIGS.
2
and
3
), which identifies the nucleotide, linked by an N-glycosidic bond to a five-carbon sugar. RNA differs from DNA in that in DNA the nucleotide sugar is deoxyribose, while in RNA, the sugar is ribose. A phosphate group serves to link the nucleotides together, forming the backbone of a single strand of DNA (see FIG.
2
). Normally, the nitrogenous base is one of the following: adenine, guanine, thymine and cytosine (respectively denoted A, G, T, and C), or uracil (U) in place of thymidine in RNA (see FIG.
3
). The order of the four nucleotides, A, G, T and C, in the chain is often referred to as the sequence of the DNA and can be specified simply by setting down the symbols A, G, T and C in the order in which these four nucleotides appear in the DNA strand.
The two chains (or strands) of a DNA double helix are held together by hydrogen bonding between the nitrogenous bases of their individual nucleotides. This hydrogen bonding is specific in that adenine in one strand must pair with thymine (or uracil in RNA) in the other strand, and guanine with cytosine. The sequence of bases in one strand of DNA is thus complementary to the sequence on the other strand.
A DNA chain has polarity: one end of the chain has a free 5′-OH (or phosphate) group (termed “the 5′ end”) and the other a free 3′-OH (or phosphate) group (“the 3′ end”). By convention, the nucleotide sequence is written or read left-to-right in the direction from the 5′ end to the 3′ end. The two strands of a DNA double helix have opposite polarities. Thus the 5′ end of one strand pairs with the 3′ end of the other strand and the complementarity of the two strands is revealed by comparing one strand read in the 5′ to 3′ direction with the other strand read in the 3′ to 5′ direction.
Genetic information is encoded in the particular sequence (order of occurrence) of nucleotides along a DNA molecule and DNA sequencing is the process of determining that order in a particular DNA molecule.
2.2. Enzymes used DNA Sequencing
Two classes of enzyme activity which have been employed in certain methods used to sequence DNA are DNA polymerase and exonuclease activity.
A DNA polymerase is an enzyme that has the ability to catalytically synthesize new strands of DNA in vitro. The DNA polymerase carries out this synthesis by moving along a preexisting single DNA strand (“the template”) and creating a new strand, complementary to the preexisting strand, by incorporating single nucleotides one at a time into the new strand following the base-pairing rule described above.
In contrast to polymerase activity, exonuclease activity refers to the ability of an enzyme (an exonuclease) to cleave off a nucleotide at the end of a DNA strand. Enzymes are known which can cleave successive nucleotides in the single DNA strand of a single-chain DNA molecule, working from the 5′ end of the strand to the 3′ end; such enzymes are termed single-stranded 5′ to 3′ exonucleases. Other enzymes are known which perform this operation in the opposite direction (single-stranded 3′ to 5′ exonucleases). There also exist enzymes which can cleave successive nucleotides from the end of a single strand of a double-stranded DNA molecule. These enzymes are termed double-stranded 5′ to 3′ or 3′ to 5′ exonucleases, depending on the direction in which they proceed along the strand. Exonucleases are also characterized as being distributive or processive in their action. Distributive exonucleases dissociate from the DNA following each internucleotide bond cleavage, whereas processive exonucleases will hydrolyze many internucleotide bonds without dissociating from the DNA.
2.3. Sequencing of DNA
Approaches to DNA sequencing have varied widely. Use of these enzymes or other chemical methods, as described below, has made it possible to sequence small portions of the human genome. Despite these successes, most of the human genome remains unexplored. Of the 3,000,000,000 base pairs in the human genome, only about 20 million base pairs have been sequened (GenBank® Release 74—December 1992).
2.3.1. Sequencing Ladder Methods
Many techniques for sequencing DNA have involved generating fragments of labeled DNA, the lengths of which are sequence-dependent, and separating the fragments according to their lengths by electric field-induced migration in a gel, so as to be able to discern the DNA sequence from the appearance of the separated fragments. Such a pattern of sequence-dependent fragment lengths is known as a sequencing ladder. The fragments can be generated by either: (a) cleaving the DNA in a base-specific manner (see FIG.
4
), or (b) synthesizing a copy of the DNA wherein the synthesized strand terminates in a base-specific manner (see FIG.
5
).
The Maxam-Gilbert technique for sequencing (Maxam and Gilbert, 1977, Proc. Natl. Acad. Sci. USA 74:560, which is incorporated herein by reference) involves the specific chemical cleavage of DNA. According to this technique, four samples of the same labeled DNA are each subjected to a different chemical reaction to effect preferential cleavage of the DNA molecule at one or two nucleotides of a specific base identity. By adjusting the conditions to obtain only partial cleavage, DNA fragments are thus generated in each sample whose lengths are dependent upon the position within the DNA base sequence of the nucleotide(s) which are subject to such cleavage. Thus, after partial cleavage is performed, each sample contains DNA fragments of different lengths each of which ends with the same one or two of the four nucleotides. In particular, in one sample each fragment ends with a C, in another sample each fragment ends with a C or a T, in a third sample each ends with a G, and in a fourth sample each ends with an A or a G. The fragments so generated are then separated from one another by electric field-induced migration in a polyacrylamide gel. The four individual sets of fragments produced by cleavage using chemical reactions of different specificity are run side-by-side, in separate lanes of the gel. The DNA fragments are then visualized, and sequence is determined by the observing the position in the gel of the generated fragments.
FIG. 4
schematically depicts the visualization of DNA fragments that are generated by cleaving the labelled DNA having the sequence 5′-AAGTACT-3′-label. The fragments from the four
Alexander Lyle A.
Pennie & Edmonds LLP
Praelux Incorporated
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