Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-04
2002-06-11
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C702S019000, C702S020000, C536S022100
Reexamination Certificate
active
06403314
ABSTRACT:
TECHNICAL FIELD
The present invention relates to computational methodologies for designing hybridization assays, polymerase chain reaction amplifications, and anti-sense drugs and, in particular, to a computational method and system for predicting the hybridization potential of a probe molecule/target molecule pair.
BACKGROUND OF THE INVENTION
The present invention relates to computationally predicting the stability of non-covalent binding between a probe molecule and a target molecule. The current application will specifically address hybridization of deoxyribonucleic acid (“DNA”) polymers, although the techniques and methodologies described in the current application may be applied to DNA and ribonucleic acid (“RNA”) hybridization, RNA/RNA hybridization, hybridization of various synthetic polymers, and hybridization of other types of polymer molecules.
DNA molecules are linear polymers, synthesized from only four different types of subunit molecules: (1) deoxy-adenosine, abbreviated “A,” a purine nucleoside; (2) deoxy-thymidine, abbreviated “T,” a pyrimidine nucleoside; (3) deoxy-cytosine, abbreviated “C,” a pyrimidine nucleoside; and (4) deoxy-guanosine, abbreviated “G,” a purine nucleoside.
FIG. 1
illustrates a short DNA polymer 100, called an oligomer, composed of the following subunits: (1) deoxy-adenosine 102; (2) deoxy-thymidine 104; (3) deoxy-cytosine 106; and (4) deoxy-guanosine 108. When phosphorylated, subunits of the DNA molecule are called nucleotides, and are linked together through phosphodiester bonds 110-115 to form the DNA polymer. A linear DNA, such as the oligomer shown in
FIG. 1
, molecule has a 5′ end 118 and a 3′ end 120. A DNA polymer can be chemically characterized by writing, in sequence from the 5′ end to the 3′ end, the single letter abbreviations for the nucleotide subunits that together compose the DNA polymer. For example, the oligomer 100 shown in
FIG. 1
can be chemically represented as “ATCG.” A nucleotide comprises a purine or pyrimidine base (e.g. adenine 122 of the deoxy-adenylate nucleotide 102), a deoxy-ribose sugar (e.g. ribose 124 of the deoxy-adenylate nucleotide 102), and a phosphate group (e.g. phosphate 126) that links the nucleotide to the next nucleotide in the DNA polymer. RNA polymers are similar to DNA polymers, except that 2′-hydrogens, such as 2′-hydrogens 126, are replaced with hydroxyl groups and the pyrimidine uridine replaces the pyrimidine thymine, where the 5′-methyl group of thymine is replaced by a hydrogen in uridine.
The DNA polymers that contain the organizational information for living organisms occur in the nuclei of cells in pairs, forming double-stranded DNA helices. One polymer of the pair is laid out in a 5′ to 3′ direction, and the other polymer of the pair is laid out in a 3′ to 5′ direction. The two DNA polymers in a double-stranded DNA helix are therefore described as being anti-parallel. The two DNA polymers, or strands, within a double-stranded DNA helix are bound to each other through attractive forces including hydrophobic interactions between stacked purine and pyrimidine bases and hydrogen bonding between purine and pyrimidine bases, the attractive forces emphasized by conformational constraints of DNA polymers. Because of a number of chemical and topographic constraints, double-stranded DNA helices are most stable when deoxy-adenylate subunits of one strand hydrogen bond to deoxy-thymidylate subunits of the other strand, and deoxy-guanylate subunits of one strand hydrogen bond to a deoxy-cytidylate subunits of the other strand.
FIGS. 2A-B
illustrate the hydrogen bonding between purine/pyrimidine base pairs of two anti-parallel DNA strands.
FIG. 2A
shows hydrogen bonding between an adenine and a thymine, and
FIG. 2B
shows hydrogen bonding between a guanine and a cytosine. Note that there are two hydrogen bonds 202 and 203 in the adenine/thymine base pair, and three hydrogen bonds 204-206 in the guanine/cytosine base pair, as a result of which GC base pairs contribute greater thermodynamic stability to DNA duplexes than AT base pairs. AT and GC base pairs, illustrated in
FIGS. 2A-B
, are known as Watson-Crick (“WC”) base pairs.
Two DNA strands linked together by hydrogen bonds form the familiar helix structure of a double-stranded DNA helix.
FIG. 3
illustrates a short section of a DNA double helix 300 comprising a first strand 302 and a second, anti-parallel strand 304. The ribbon-like strands in
FIG. 3
represent the deoxyribose and phosphate backbones of the two anti-parallel strands, with hydrogen-bonded purine and pyrimidine base pairs, such as base pair 306, interconnecting the two strands. Deoxy-guanylate subunits in one strand are generally paired with deoxy-cytidylate subunits in the other strand, and deoxy-thymidylate subunits in one strand are generally paired with deoxy-adenylate subunits in the other strand. However, non-WC base pairings may occur within double-stranded DNA. Generally, purine/pyrimidine non-WC base pairings contribute little to the thermodynamic stability of a DNA duplex, but generally do not destabilize a duplex otherwise stabilized by WC base pairs. Such base pairs are referred to below as “non-WC” base pairs. However, purine/purine base pairs may destabilize DNA duplexes, as may, to a lesser extent, pyrimidine/pyrimidine base pairs. Such base pairings are referred to below as “anti-WC” base pairs.
Double-stranded DNA may be denatured, or converted into single-stranded DNA, by changing the ionic strength of the solution containing the double-stranded DNA or by raising the temperature of the solution. Single-stranded DNA polymers may be renatured, or converted back into DNA duplexes, by reversing the denaturing conditions, for example by lowering the temperature of the solution containing the single-stranded DNA polymers. During the renaturing process, complementary bases of anti-parallel strands form WC base pairs in a cooperative fashion, leading to regions of DNA duplex. However, many different types of associations between and within DNA polymers may occur that may lead to many different types of mismatching between single strands of DNA. In general, the longer the regions of consecutive WC base pairing between two single strands of DNA, the greater the stability of hybridization of the two polymers under renaturing conditions.
The ability to denature and re-nature double-stranded DNA has led to development of many extremely powerful and discriminating assay technologies for identifying the presence of single-stranded or double-stranded DNA of particular base sequences or containing particular sub-sequences within complex mixtures of different DNA polymers and other bio-polymers and chemical substances. These methodologies include the polymerase chain reaction (“PCR”), molecular-array-based hybridization assays, fluorescent in situ hybridization (“FISH”), and anti-sense nucleic acid bio-polymers that may be used as therapeutic agents or research tools to block expression of particular genes within an organism.
FIG. 4
illustrates probe/target hybridization that underlies hybridization assays. A probe 402 is synthesized to contain a short single-stranded DNA of a particular sequence 404 attached to a second chemical entity 406 represented in
FIG. 4
as an unfilled disk. The nature of the second chemical entity 406 varies depending on the technique in which the probe is employed. The probe is brought into contact with a solution of single-stranded DNA polymers 408-412 having different sequences. The solution is then modified to become a renaturing environment, for example by changing the ionic strength of the solution or lowering the temperature of the solution, to allow for hybridization of the single-stranded-DNA portion of the probe molecules 404 with single-stranded DNA molecules having complementary sequences 410. Thus, a probe molecule, in figurative terms, fishes out a single-stranded DNA polymer having a complementary or near-complementary sequence from a complex solution of DNA
Lange Daniel H.
Sampas Nicholas M.
Wolber Paul K.
Yakhini Zohar H.
Agilent Technologie,s Inc.
Fredman Jeffrey
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