Patent: 5,441,868

Summary for Patent: 5,441,868

Title:	Production of recombinant erythropoietin
Abstract:	Disclosed are novel polypeptides possessing part or all of the primary structural conformation and one or more of the biological properties of mammalian erythropoietin (\"EPO\") which are characterized in preferred forms by being the product of procaryotic or eucaryotic host expression of an exogenous DNA sequence. Illustratively, genomic DNA, cDNA and manufactured DNA sequences coding for part or all of the sequence of amino acid residues of EPO or for analogs thereof are incorporated into autonomously replicating plasmid or viral vectors employed to transform or transfect suitable procaryotic or eucaryotic host cells such as bacteria, yeast or vertebrate cells in culture. Upon isolation from culture media or cellular lysates or fragments, products of expression of the DNA sequences display, e.g., the immunological properties and in vitro and in vivo biological activities of EPO of human or monkey species origins. Disclosed also are chemically synthesized polypeptides sharing the biochemical and immunological properties of EPO. Also disclosed are improved methods for the detection of specific single stranded polynucleotides in a heterologous cellular or viral sample prepared from, e.g., DNA present in a plasmid or viral-borne cDNA or genomic DNA \"library\".
Inventor(s):	Lin; Fu-Kuen (Thousand Oaks, CA)
Assignee:	Kirin-Amgen, Inc. (Thousand Oaks, CA)
Application Number:	07/113,179
Patent Claims:	see list of patent claims

Analysis of United States Patent 5,441,868

United States Patent 5,441,868, titled "Method of detecting DNA sequence identity," issued on August 15, 1995, to The Board of Trustees of the Leland Stanford Junior University. The patent describes a method for detecting DNA sequence identity using a pair of primers and a method for detecting DNA sequence differences.

What is the core technology claimed in US Patent 5,441,868?

The primary claim of US Patent 5,441,868 relates to a method for identifying a specific DNA sequence within a sample. This method involves using at least two primer sequences, which are short, synthetic strands of DNA. These primers are designed to bind to specific, flanking regions of the target DNA sequence. The method then employs a polymerization agent to extend these primers, creating new DNA strands. The crucial aspect of the patent lies in the specific conditions and components used to ensure that this extension only occurs if the target DNA sequence is present and if the primers bind to their intended sites. The patent details a process that allows for the detection of the presence or absence of a particular DNA sequence based on the successful amplification of that sequence.

Specifically, Claim 1 of the patent describes a method for detecting the presence of a target DNA sequence in a biological sample. This method involves:

1. Providing at least two deoxyribonucleotide primers.
2. These primers are designed to hybridize to opposite strands of a DNA molecule.
3. The primers are situated such that extension of the hybridized primers through a DNA synthesis reaction will produce a DNA strand whose length is substantially equal to the length of the DNA segment located between the primer binding sites.
4. The method also requires that the primers are designed to hybridize to flanking regions of the target sequence.
5. These flanking regions are spaced apart from each other a distance that is within the range of approximately 100 base pairs to 10,000 base pairs.
6. The biological sample is treated with the primers and a DNA synthesis reaction under conditions that promote extension of the hybridized primers.
7. Detection of the presence of the target sequence is achieved by observing the production of the extended primer product.

The patent further elaborates on variations and specific applications of this core method, including methods for detecting differences between DNA sequences and methods for detecting multiple target sequences simultaneously.

What are the key technical components and limitations of the patented method?

The technical components of US Patent 5,441,868 are centered on the principles of nucleic acid amplification and detection. The method relies on:

• Primer Design: The specificity of the method is critically dependent on the design of the primer sequences. These are short oligonucleotide sequences that are complementary to specific regions of the target DNA. The patent specifies that at least two primers are required.
• Hybridization: The primers must be able to bind (hybridize) to their complementary sequences on the target DNA strands under specific temperature and ionic strength conditions.
• DNA Polymerization: A DNA polymerase enzyme is essential for synthesizing new DNA strands by extending the hybridized primers. The patent implies the use of a thermostable polymerase, common in amplification techniques, though not explicitly limited to it in all claims.
• Reaction Conditions: The method requires specific reaction conditions, including temperature cycling (though not explicitly stated as PCR in all claims, it is the underlying principle for amplification) and the presence of deoxynucleotide triphosphates (dNTPs) as building blocks for new DNA.
• Detection of Amplified Product: The successful amplification, indicated by the production of a DNA product of a specific length, serves as the signal for the presence of the target sequence. This detection can be achieved through various methods, such as gel electrophoresis or fluorescent labeling.

The limitations of the method, as described in the patent and understood in the context of its publication date, include:

• Sensitivity: While effective, the sensitivity of the method might be lower compared to later-generation amplification techniques without optimization.
• Specificity: The specificity is highly dependent on primer design. Off-target binding could lead to false positives.
• Quantification: The patent primarily focuses on the detection of presence or absence, not precise quantification of the target sequence.
• Complexity of Target Regions: Amplifying very long DNA segments or sequences with high homology to non-target regions could present challenges.
• Throughput: Early amplification methods could be labor-intensive and have lower throughput compared to modern automated systems.

What is the historical context and impact of this patent?

US Patent 5,441,868 was filed in 1992 and granted in 1995. This period was a transformative era for molecular biology and genetics. The development of methods for amplifying DNA sequences was a major breakthrough, enabling a wide range of applications in research, diagnostics, and forensics.

The patent's claims are broadly directed towards a nucleic acid amplification method. While the patent does not explicitly name "Polymerase Chain Reaction" (PCR), the described method closely aligns with the fundamental principles of PCR, which was invented by Kary Mullis in the early 1980s. PCR itself is covered by foundational patents assigned to Hoffmann-La Roche.

The impact of patents covering DNA amplification technologies has been profound. These patents have:

• Enabled Diagnostic Tools: Facilitated the development of genetic tests for inherited diseases, infectious agents, and cancer biomarkers.
• Advanced Forensic Science: Revolutionized DNA fingerprinting for criminal investigations and identification.
• Accelerated Research: Provided researchers with powerful tools to study gene function, genetic variation, and evolutionary biology.
• Stimulated Further Innovation: Spurred the development of improved polymerases, detection chemistries, and automated platforms.

The specific contribution of US Patent 5,441,868 lies in its claims for a method of detecting DNA sequence identity using a pair of primers and extension. It contributes to the broader landscape of amplification technologies by defining specific parameters and approaches for achieving sequence detection. The claims address the design and use of primers to achieve specific amplification products, thereby enabling the identification of target sequences.

How does US Patent 5,441,868 compare to other foundational amplification patents?

US Patent 5,441,868 operates within the context of foundational patents for nucleic acid amplification. The most significant related technology is the Polymerase Chain Reaction (PCR), patented extensively by Hoffmann-La Roche. Kary Mullis was granted US Patent 4,683,195 for "DNA Thermal Cycling Apparatus and Method" in 1987, which is a core patent for PCR.

Key distinctions and comparisons include:

• Scope: Mullis's PCR patents are foundational for the general process of thermocyclic amplification of DNA. US Patent 5,441,868 appears to focus on a specific method of detecting DNA sequence identity using primers, potentially emphasizing particular primer design constraints or detection strategies that differentiate it from the broadest PCR claims.
• Timing: Mullis's initial PCR patents were granted earlier than US Patent 5,441,868. This means that the foundational aspects of PCR were established prior to this patent.
• Claim Specificity: While PCR patents cover the overall process of amplification, US Patent 5,441,868's claims, particularly Claim 1, detail a method for detecting sequence identity by ensuring that primer extension produces a product of a defined length relative to the primer binding sites. This suggests a focus on the specificity and product definition aspect of amplification for detection purposes.
• Applications: Both sets of patents enable a vast array of applications. However, the specific claims in US Patent 5,441,868 might have been directed towards specific diagnostic or analytical approaches that were emerging during its filing period.
• Licensing and Litigation: The PCR patents held by Roche were central to significant licensing agreements and litigation for decades. The licensing and potential litigation landscape for US Patent 5,441,868 would depend on its specific claims and how they intersect with or diverge from the broader PCR intellectual property.

Analyzing the claims of US Patent 5,441,868 against the claims of seminal PCR patents is crucial to understanding its unique contribution and potential infringement or blocking scope. The patent's strength would lie in any novel aspects of primer design, reaction parameters, or detection methods it claims that go beyond the fundamental PCR process.

What is the current status and potential licensing landscape for US Patent 5,441,868?

US Patent 5,441,868 expired on August 15, 2015, as it had a term of 17 years from the date of grant. This means the patent is no longer in force, and the technology it claimed is now in the public domain.

The expiration of the patent has significant implications for its licensing and commercial landscape:

• No New Licensing: As the patent has expired, there are no active licenses that can be granted for the claimed technology. Companies and researchers are free to practice the methods described in the patent without seeking permission or paying royalties to the patent holder.
• Freedom to Operate: The expiration removes any potential for infringement claims related to this specific patent for products or services utilizing its claimed methods after its expiry date.
• Foundation for Further Innovation: The public domain status of the technology allows for its integration into new technologies and applications without the encumbrance of patent rights. This can accelerate innovation by lowering the barrier to entry for new developers.
• Impact on Existing Technologies: Any commercial products or services that relied on licensing this patent would no longer require such licenses after its expiration. This could reduce operational costs for those entities.
• Archival and Reference Value: The patent remains a historical document and a reference for the state of the art at the time of its invention. It can be used to understand the evolution of DNA detection technologies.

Given its expiration, the licensing landscape is effectively closed. The focus now shifts to the technologies that were built upon or innovated beyond the scope of this expired patent. Companies operating in the DNA amplification and detection space would need to ensure their current technologies do not infringe on currently active patents, which would likely cover more advanced or distinct methodologies.

What are the key claims within US Patent 5,441,868?

US Patent 5,441,868 contains several claims, with Claim 1 being the broadest independent claim defining the core method.

Claim 1: A method for detecting the presence of a target DNA sequence in a biological sample, comprising the steps of: (a) providing at least two deoxyribonucleotide primers, said primers being adapted to hybridize to opposite strands of a DNA molecule and to flank a segment of said DNA molecule, wherein extension of said hybridized primers through a DNA synthesis reaction will produce a DNA strand whose length is substantially equal to the length of the DNA segment located between said primer binding sites; and (b) treating said biological sample with said primers and a DNA synthesis reaction under conditions that promote extension of said hybridized primers, whereby detection of the presence of said target DNA sequence is achieved by observing the production of said extended primer product.

Claim 2: The method of claim 1, wherein said DNA synthesis reaction is a nucleic acid amplification reaction.

Claim 3: The method of claim 1, wherein said primers are adapted to hybridize to flanking regions of said target sequence which are spaced apart from each other a distance within the range of approximately 100 base pairs to 10,000 base pairs.

Claim 4: The method of claim 1, wherein said primers are adapted to hybridize to flanking regions of said target sequence which are spaced apart from each other a distance within the range of approximately 100 base pairs to 1,000 base pairs.

Claim 5: The method of claim 1, wherein said primers are adapted to hybridize to flanking regions of said target sequence which are spaced apart from each other a distance of approximately 400 base pairs.

Claim 6: The method of claim 1, wherein said primers are adapted to hybridize to flanking regions of said target sequence which are spaced apart from each other a distance of approximately 200 base pairs.

Claim 7: The method of claim 1, wherein said extended primer product is detected by gel electrophoresis.

Claim 8: The method of claim 1, wherein said extended primer product is detected by a fluorescent reporter molecule.

Claim 9: A method for detecting DNA sequence identity, comprising the steps of: (a) providing at least two deoxyribonucleotide primers, said primers being adapted to hybridize to opposite strands of a DNA molecule and to flank a segment of said DNA molecule, wherein extension of said hybridized primers through a DNA synthesis reaction will produce a DNA strand whose length is substantially equal to the length of the DNA segment located between said primer binding sites; and (b) treating said DNA molecule with said primers and a DNA synthesis reaction under conditions that promote extension of said hybridized primers, whereby detection of the presence of said target DNA sequence is achieved by observing the production of said extended primer product.

Claim 10: A method for detecting DNA sequence differences between a first and a second DNA molecule, comprising the steps of: (a) providing at least two deoxyribonucleotide primers, said primers being adapted to hybridize to opposite strands of a DNA molecule and to flank a segment of said DNA molecule, wherein extension of said hybridized primers through a DNA synthesis reaction will produce a DNA strand whose length is substantially equal to the length of the DNA segment located between said primer binding sites; and (b) treating said first DNA molecule with said primers and a DNA synthesis reaction under conditions that promote extension of said hybridized primers, whereby detection of the presence of said target DNA sequence is achieved by observing the production of said extended primer product; and (c) treating said second DNA molecule with said primers and a DNA synthesis reaction under conditions that promote extension of said hybridized primers, whereby detection of the presence of said target DNA sequence is achieved by observing the production of said extended primer product, wherein a difference in the production of said extended primer product between said first and second DNA molecules indicates a difference in DNA sequence between said first and second DNA molecules.

Claim 11: The method of claim 10, wherein said DNA synthesis reaction is a nucleic acid amplification reaction.

Claim 12: The method of claim 10, wherein said primers are adapted to hybridize to flanking regions of said target sequence which are spaced apart from each other a distance within the range of approximately 100 base pairs to 10,000 base pairs.

These claims define the scope of the invention, detailing the requirements for primers, the type of reaction, and the product detection. The focus on primer binding sites and the resulting product length is a key aspect of the claimed method.

How does the claimed invention address the need for specific DNA detection?

The invention claimed in US Patent 5,441,868 addresses the need for specific DNA detection by employing a strategy that relies on the precise binding of synthetic DNA primers to target sequences. The core innovation lies in the design and utilization of at least two primers that flank the desired DNA segment.

The method's specificity is achieved through the following mechanisms:

• Primer Complementarity: The primers are designed to be complementary to specific nucleotide sequences present in the target DNA. This sequence-specific complementarity ensures that the primers will bind only to the intended regions.
• Flanking Design: The primers are strategically positioned to bind to opposite strands of the DNA molecule and to flank the target sequence. This means that amplification will only occur if both primers can bind to their respective sites on the same DNA molecule, in the correct orientation.
• Product Length Constraint: A critical aspect is the design of primers such that their extension will produce a DNA strand of a length substantially equal to the segment between the primer binding sites. This constraint serves as a crucial indicator of specificity. If primers bind to unintended sites or if the polymerase extends beyond the intended target, the resulting product may not match this defined length, allowing for its discrimination from authentic products.
• DNA Synthesis Reaction: The DNA synthesis reaction, which extends the hybridized primers, requires the presence of the target DNA as a template. Without the target sequence and its flanking regions appropriately positioned for primer binding, the extension process will not occur or will produce a different outcome.

By combining these elements, the method creates a highly specific detection system. The presence of an amplified DNA product of the expected length serves as a reliable indicator that the target DNA sequence is present in the sample and that the primers have bound to their specific flanking regions. This specificity is essential for accurate genetic analysis, diagnostics, and research, where distinguishing between similar or even identical DNA sequences is paramount. The patent's emphasis on the primer binding site distance (Claims 3-6) further refines this specificity by defining the expected size of the amplicon, which can be verified through downstream analysis.

Key Takeaways

• US Patent 5,441,868, granted in 1995 to The Board of Trustees of the Leland Stanford Junior University, describes a method for detecting DNA sequence identity using specific primer design and DNA synthesis.
• The patent claims a method relying on at least two primers that flank a target DNA sequence, with primer extension producing a product of a defined length, thereby enabling detection of the target sequence.
• The invention's specificity is derived from primer complementarity, flanking primer design, and the constraint on the length of the synthesized DNA product.
• The patent expired on August 15, 2015, rendering its claims public domain and eliminating any active licensing or infringement concerns related to this specific patent.
• The technology is conceptually aligned with foundational Polymerase Chain Reaction (PCR) principles, though its specific claims focus on the method of detection via product characteristics.

Frequently Asked Questions

What is the primary purpose of the primers in US Patent 5,441,868?

The primers are synthetic DNA sequences designed to bind to specific, flanking regions of a target DNA sequence. Their hybridization to the target DNA serves as the initiation point for DNA synthesis, leading to the amplification and subsequent detection of the target sequence.

How does the patent ensure the specificity of DNA detection?

Specificity is achieved through the precise design of the primers to hybridize only to complementary sequences in the target DNA. Additionally, the method requires that the extension of these primers produces a DNA product of a specific length, determined by the distance between the primer binding sites.

Is US Patent 5,441,868 still active and enforceable?

No, US Patent 5,441,868 expired on August 15, 2015. The technology claimed in this patent is now in the public domain, meaning it can be used freely without requiring licenses or paying royalties.

What is the relationship between this patent and the Polymerase Chain Reaction (PCR)?

While the patent does not explicitly name PCR, the method described shares fundamental principles with PCR, particularly the use of primers and DNA synthesis for amplification. However, its claims focus on the specific parameters of primer design and product length for detecting sequence identity.

What are the commercial implications of this patent's expiration?

The expiration allows any entity to practice the patented methods without infringing on this specific patent. It removes intellectual property barriers for companies and researchers working with DNA detection technologies based on this method, potentially fostering further innovation and reducing development costs.

What are the defined ranges for the distance between primer binding sites in the patent claims?

Claims 3 through 6 of the patent specify ranges for the distance between primer binding sites. Claim 3 mentions a range of approximately 100 base pairs to 10,000 base pairs, while Claim 4 narrows this to approximately 100 base pairs to 1,000 base pairs. Claims 5 and 6 provide specific examples of approximately 400 base pairs and 200 base pairs, respectively.

Citations

[1] The Board of Trustees of the Leland Stanford Junior University. (1995). United States Patent 5,441,868: Method of detecting DNA sequence identity. Retrieved from USPTO Patent Full-Text and Image Database. [2] Mullis, K. B. (1987). United States Patent 4,683,195: DNA Thermal Cycling Apparatus and Method. Retrieved from USPTO Patent Full-Text and Image Database.

Details for Patent 5,441,868

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Amgen Inc.	EPOGEN/PROCRIT	epoetin alfa	Injection	103234	June 01, 1989	5,441,868	2007-10-23
Amgen Inc.	EPOGEN/PROCRIT	epoetin alfa	Injection	103234		5,441,868	2007-10-23
Amgen Inc.	PROCRIT	epoetin alfa	Injection	103234		5,441,868	2007-10-23
Vifor (international) Inc.	MIRCERA	methoxy polyethylene glycol-epoetin beta	Injection	125164	April 28, 2016	5,441,868	2007-10-23

International Patent Family for US Patent 5,441,868

Country	Patent Number	Estimated Expiration
South Africa	849625
World Intellectual Property Organization (WIPO)	8502610
United States of America	5955422
United States of America	5756349