>>15590497 (lb)

>>15590500 (lb) Supreme Court has ruled that vaccinated people worldwide are products, patented goods, according to US law, no longer human

SUPREME COURT OF THE UNITED STATES

Syllabus

ASSOCIATION FOR MOLECULAR PATHOLOGY ET AL.

v. MYRIAD GENETICS, INC., ET AL.

CERTIORARI TO THE UNITED STATES COURT OF APPEALS FOR THE FEDERAL CIRCUIT

No. 12–398. Argued April 15, 2013—Decided June 13, 2013

Held: A naturally occurring DNA segment is a product of nature and

not patent eligible merely because it has been isolated, but cDNA is

patent eligible because it is not naturally occurring. Pp. 10–18.

(c) cDNA is not a “product of nature,” so it is patent eligible under

§101. cDNA does not present the same obstacles to patentability as naturally occurring, isolated DNA segments. Its creation results in an exons-only molecule, which is not naturally occurring. Its order of the exons may be dictated by nature, but the lab technician unquestionably creates something new when introns are removed from a DNA sequence to make cDNA. Pp. 16–17.

JUSTICE SCALIA, concurring in part and concurring in

the judgment.

I join the judgment of the Court, and all of its opinion

except Part I–A and some portions of the rest of the opinion going into fine details of molecular biology. I am unable to affirm those details on my own knowledge or even

my own belief. It suffices for me to affirm, having studied

the opinions below and the expert briefs presented here,

that the portion of DNA isolated from its natural state

sought to be patented is identical to that portion of the

DNA in its natural state; and that ==complementary DNA

(cDNA) is a synthetic creation not normally present in

nature==.

THOMAS, J., delivered the opinion of the Court, in which ROBERTS, C. J., and KENNEDY, GINSBURG, BREYER, ALITO, SOTOMAYOR, and KAGAN, JJ., joined, and in which SCALIA, J., joined in part. SCALIA, J., filed an opinion concurring in part and concurring in the judgment.

https://www.supremecourt.gov/opinions/12pdf/12-398_1b7d.pdf

>>15590922

>complementary DNA

>(cDNA) is a synthetic creation not normally present in

cDNA is a synthetic creation not normally present in nature

cDNA is not a “product of nature,” so it is patent eligible under§101.

______

In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists want to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to the recipient cell. In molecular biology, cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays and RNA-seq.

cDNA is also produced naturally by retroviruses such as HIV-1, HIV-2, simian immunodeficiency virus, etc.) and then integrated into the host's genome, where it creates a provirus.[1]

The term cDNA is also used, typically in a bioinformatics context, to refer to an mRNA transcript's sequence, expressed as DNA bases (deoxy-GCAT) rather than RNA bases (GCAU).

Synthesis

RNA serves as a template for cDNA synthesis.[2] In cellular life, cDNA is generated by viruses and retrotransposons for integration of RNA into target genomic DNA. In molecular biology, RNA is purified from source material after genomic DNA, proteins and other cellular components are removed. cDNA is then synthesized through in vitro reverse transcription.[3]

RNA Purification

RNA is transcribed from genomic DNA in host cells and is extracted by first lysing cells then purifying RNA utilizing widely-used methods such as phenol-chloroform, silica column, and bead-based RNA extraction methods.[4] Extraction methods vary depending on the source material. For example, extracting RNA from plant tissue requires additional reagents, such as polyvinylpyrrolidone (PVP), to remove phenolic compounds, carbohydrates, and other compounds that will otherwise render RNA unusable.[5] To remove DNA and proteins, enzymes such as DNase and Proteinase K are used for degradation.[6] Importantly, RNA integrity is maintained by inactivating RNases with chaotropic agents such as guanidinium isothiocyanate, sodium dodecyl sulphate (SDS), phenol or chloroform. Total RNA is then separated from other cellular components and precipitated with alcohol. Various commercial kits exist for simple and rapid RNA extractions for specific applications.[7] Additional bead-based methods can be used to isolate specific sub-types of RNA (e.g. mRNA and microRNA) based on size or unique RNA regions.[8][9]

pt 1

>>15590957

Reverse Transcription

First-strand synthesis

Using a reverse transcriptase enzyme and purified RNA templates, one strand of cDNA is produced (first-strand cDNA synthesis). The M-MLV reverse transcriptase from the Moloney murine leukemia virus is commonly used due to its reduced RNase H activity suited for transcription of longer RNAs.[10] The AMV reverse transcriptase from the avian myeloblastosis virus may also be used for RNA templates with strong secondary structures (i.e. high melting temperature).[11] cDNA is commonly generated from mRNA for gene expression analyses such as RT-qPCR and RNA-seq.[12] mRNA is selectively reverse transcribed using oligo-dT primers that are the reverse complement of the poly-adenylated tail on the 3' end of all mRNA. An optimized mixture of oligo-dT and random hexamer primers increases the chance of obtaining full-length cDNA while reducing 5' or 3' bias.[13] Ribosomal RNA may also be depleted to enrich both mRNA and non-poly-adenylated transcripts such as some non-coding RNA.[14]

Second-strand synthesis

The result of first-strand syntheses, RNA-DNA hybrids, can be processed through multiple second-strand synthesis methods or processed directly in downstream assays.[15][16] An early method known as hairpin-primed synthesis relied on hairpin formation on the 3' end of the first-strand cDNA to prime second-strand synthesis. However, priming is random and hairpin hydrolysis leads to loss of information. The Gubler and Hoffman Procedure uses E. Coli RNase H to nick mRNA that is replaced with E. Coli DNA Polymerase I and sealed with E. Coli DNA Ligase. An optimization of this procedure relies on low RNase H activity of M-MLV to nick mRNA with remaining RNA later removed by adding RNase H after DNA Polymerase translation of the second-strand cDNA. This prevents lost sequence information at the 5' end of the mRNA.

pt2

>>15590960

Applications

Complementary DNA is often used in gene cloning or as gene probes or in the creation of a cDNA library. When scientists transfer a gene from one cell into another cell in order to express the new genetic material as a protein in the recipient cell, the cDNA will be added to the recipient (rather than the entire gene), because the DNA for an entire gene may include DNA that does not code for the protein or that interrupts the coding sequence of the protein (e.g., introns). Partial sequences of cDNAs are often obtained as expressed sequence tags.

With amplification of DNA sequences via polymerase chain reaction (PCR) now commonplace, one will typically conduct reverse transcription as an initial step, followed by PCR to obtain an exact sequence of cDNA for intra-cellular expression. This is achieved by designing sequence-specific DNA primers that hybridize to the 5' and 3' ends of a cDNA region coding for a protein. Once amplified, the sequence can be cut at each end with nucleases and inserted into one of many small circular DNA sequences known as expression vectors. Such vectors allow for self-replication, inside the cells, and potentially integration in the host DNA. They typically also contain a strong promoter to drive transcription of the target cDNA into mRNA, which is then translated into protein.

On 13 June 2013, the United States Supreme Court ruled in the case of Association for Molecular Pathology v. Myriad Genetics that while naturally occurring human genes cannot be patented, cDNA is patent eligible because it does not occur naturally.[17]

cDNA is also used to study gene expression via methods such as RNA-seq or RT-qPCR.[18][19][20] For sequencing, RNA must be fragmented due to sequencing platform size limitations. Additionally, second-strand synthesized cDNA must be ligated with adapters that allow cDNA fragments to be PCR amplified and bind to sequencing flow cells. Gene-specific analysis methods commonly use microarrays and RT-qPCR to quantify cDNA levels via fluorometric and other methods.

pt3

>>15590964

Viruses and retrotransposons

Some viruses also use cDNA to turn their viral RNA into mRNA (viral RNA → cDNA → mRNA). The mRNA is used to make viral proteins to take over the host cell.

An example of this first step from viral RNA to cDNA can be seen in the HIV cycle of infection. Here, the host cell membrane becomes attached to the virus’ lipid envelope which allows the viral capsid with two copies of viral genome RNA to enter the host. The cDNA copy is then made through reverse transcription of the viral RNA, a process facilitated by the chaperone CypA and a viral capsid associated reverse transcriptase.[21]

cDNA is also generated by retrotransposons in eukaryotic genomes. Retrotransposons are mobile genetic elements that move themselves within, and sometimes between, genomes via RNA intermediates. This mechanism is shared with viruses with the exclusion of the generation of infectious particles.[22][23]

pt4

A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which constitute some portion of the transcriptome of the organism and are stored as a "library". cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. Similarly, tissue-specific cDNA libraries can be produced. In eukaryotic cells the mature mRNA is already spliced, hence the cDNA produced lacks introns and can be readily expressed in a bacterial cell. While information in cDNA libraries is a powerful and useful tool since gene products are easily identified, the libraries lack information about enhancers, introns, and other regulatory elements found in a genomic DNA library.

cDNA Library Construction

cDNA is created from a mature mRNA from a eukaryotic cell with the use of reverse transcriptase. In eukaryotes, a poly-(A) tail (consisting of a long sequence of adenine nucleotides) distinguishes mRNA from tRNA and rRNA and can therefore be used as a primer site for reverse transcription. This has the problem that not all transcripts, such as those for the histone, encode a poly-A tail.

mRNA extraction

Firstly, the mRNA is obtained and purified from the rest of the RNAs. Several methods exist for purifying RNA such as trizol extraction and column purification. Column purification is done by using oligomeric dT nucleotide coated resins where only the mRNA having the poly-A tail will bind. The rest of the RNAs are eluted out. The mRNA is eluted by using eluting buffer and some heat to separate the mRNA strands from oligo-dT.

cDNA construction

Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymidine nucleotides) is tagged as a complementary primer which binds to the poly-A tail providing a free 3'-OH end that can be extended by reverse transcriptase to create the complementary DNA strand. The mRNA is removed by using an RNAse enzyme, leaving single-stranded cDNA (sscDNA). The sscDNA is converted into double-stranded DNA with the help of DNA polymerase. However, for DNA polymerase to synthesize a complementary strand, a free 3'-OH end is needed. This is provided by the sscDNA itself by coiling on itself at the 3' end, generating a hairpin loop. The polymerase extends the 3'-OH end, and later the loop at 3' end is opened by the scissoring action of S1 nuclease. Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids.

The cloned bacteria are then selected, commonly through the use of antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library.

cDNA Library uses

cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library. cDNA libraries are used to express eukaryotic genes in prokaryotes. Prokaryotes do not have introns in their DNA and therefore do not possess any enzymes that can cut it out during transcription process. cDNA does not have introns and therefore can be expressed in prokaryotic cells. cDNA libraries are most useful in reverse genetics where the additional genomic information is of less use. Additionally, cDNA libraries are frequently used in functional cloning to identify genes based on the encoded protein's function. When studying eukaryotic DNA, expression libraries are constructed using complementary DNA (cDNA) to help ensure the insert is truly a gene.[1]

cDNA Library vs. Genomic DNA Library

cDNA library lacks the non-coding and regulatory elements found in genomic DNA. Genomic DNA libraries provide more detailed information about the organism, but are more resource-intensive to generate and keep.

Cloning of cDNA

cDNA molecules can be cloned by using restriction site linkers. Linkers are short, double stranded pieces of DNA (oligodeoxyribonucleotide) about 8 to 12 nucleotide pairs long that include a restriction endonuclease cleavage site e.g. BamHI. Both the cDNA and the linker have blunt ends which can be ligated together using a high concentration of T4 DNA ligase. Then sticky ends are produced in the cDNA molecule by cleaving the cDNA ends (which now have linkers with an incorporated site) with the appropriate endonuclease. A cloning vector (plasmid) is then also cleaved with the appropriate endonuclease. Following "sticky end" ligation of the insert into the vector the resulting recombinant DNA molecule is transferred into E. coli host cell for cloning.

See also

Functional cloning

>>15590997

https://en.wikipedia.org/wiki/CDNA_library

>>15591024

yes, so you can be infected by an mrna derived 'virus' or vaccine to turn you into a cdna host/carrier, thus a patentable nonhuman product. And a chimera, and a machine.