Recombinase Polymerase Amplification qPCR Primer Design Portable real-time fluorometer ADVANTAGES AND POTENTIAL APPLICATIONS Advantages

Recombinase Polymerase Amplification qPCR Primer Design Portable real-time fluorometer ADVANTAGES AND POTENTIAL APPLICATIONS Advantages www.phwiki.com

Recombinase Polymerase Amplification qPCR Primer Design Portable real-time fluorometer ADVANTAGES AND POTENTIAL APPLICATIONS Advantages

Dougherty, Julie, Morning Drive On-Air Personality has reference to this Academic Journal, PHwiki organized this Journal Recombinase Polymerase Amplification Radhika Pradhan Aidan Quinn Ziwei Song Class 26-2011 updated Dec. 8, 201 12:45 AM TwistDx Ltd. qPCR Primer Design

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Portable real-time fluorometer ADVANTAGES AND POTENTIAL APPLICATIONS

Advantages From 1 molecule DNA or 10 molecules RNA to detectable levels (billions or trillions) in 5-10 min Low cost in addition to simple reagents means practical applications are enormous Multiplexing allows simultaneous detection of multiple targets Potential applications Therapeutic intervention at the level of pre-mRNA splicing Interfere with improper splicing caused by splice site creation or activation E.g., beta-thalassemia (R. Kole) in which a splice site has been created by a mutation in a hemoglobin gene Use complementary DNA or RNA (antisense) Natural DNA/RNA rapidly degraded: Use modified bases, sugars: PNA, morpholino, 2’ OMe, Normally, DNA-RNA hybrids + endogenous RNase H type activity RNA destruction Modified antisense DNA circumvents this problem (don’t want mRNA destroyed here, want to correct its splicing.) PNA = peptide nucleic acids Class 26-2011 updated Dec. 8, 201 12:45 AM

B. Bias alternative splicing ratios Target the unwanted iso as long as m exon-intron joint. e.g., BCL-2 iso as long as ms, one is pro-apoptotic, one anti-apoptotic. The latter is increased in many cancers Target the anti-apoptotic iso as long as m in cancer cells. e.g., GABA-a-gamma-2 receptor (GABA = gamma amino butyric acid, a neurotransmitter) Long in addition to short as long as ms. Long as long as m associated with mental illness. C. Skip offensive exons e.g., nonsense truncations in dystrophin –> x Nonsense mutation truncates protein Expendable exon (e.g., protein with many repeated domains) Exon must be multiple of 3 in length to maintain reading frame after skipping Antisense-induced skipping Splicing as a target as long as disease therapy Deoxy, or also can add 2’ MOE -O-CH2-CH2-O-CH3 Phosphorothioate deoxyoligonucleotides MOE = methoxyethyl – RNA modification

Morpholino instead of deoxyribose or ribose Modified phosphate RNA modification as long as stabilization Still base pairs OK! ase ase Attached 1 to 4 lysines here PNA = peptide nucleic acid Amide bonds, No ribose B = a nucleic acid base RNA modification Even more extreme in addition to more stable: peptide nucleic acids (PNAs) Base pairs even better than natural nucleic acids (higher melting temperatures) Sazani P, et al. in addition to Kole R. Systemically delivered antisense oligomers upregulate gene expression in mouse tissues Nat Biotechnol. 2002 Dec;20(12):1228-33. EGFP: Enhanced green fluorescent protein = model system Actin promoter, universally expressed. Induced exon skipping yields green fluorescence Mutant globin intron has activated splice sites Antisense “RNA” injected into tail vein, RNA was modified as long as stability Interfere with improper splicing caused by splice site creation or activation

Antisense treatment in cell cultures (ex vivo) from the mouse with the mutant EGFP gene Control oligo (C) (50 nt downstream) was ineffective. Max. effect = 40% No antisense: Dystrophin gene 2400 kb, mRNA = 14 kb, 79 exons: a giant gene Protein maintains muscle cell membrane integrity Mutation: Duchenne’s muscular dystrophy Some cases (~half) are due to stop codons (nonsense) in a repetitious exon (spectrin-like repeat, length = a multiple of 3) Deliver antisense to the ends of exon with the nonsense mutation in mdx mice (model as long as Duchenne’s) to promote the skipping of the nonsense-bearing exon in addition to so avoid truncation of the protein . Use AAV (adeno-associated virus) to deliver the antisense gene Measure: mRNA with skipped exon dystrophin protein muscle histochemistry as long as dystrophin C. Skip offensive exons = 3 X 71 Branch site (consensus = YNYTRAY) protein mRNA 79 Use antisense RNA to target the branch point upstream of the offending exon 23 in addition to the donor splice site downstream of the exon. BP = branch point; SD = splice donor Sequences targeted by antisense

U7 promoter compl. to splice donor site compl. to branch Consensus binding site as long as Sm proteins (to target to pre-mRNA) Double target synergistic (loop) (Kole) ITR = inverted terminal repeat, characteristic of AAV 0 2 4 6 13 weeks Expression of U7 antisense construct Splicing assay (RT-PCR) Dystrophin protein (Western) Endog. U7 U7SmOPT-A.S. RT-PCR Skip exon 23, after 2-4 wks. (slow onset = conclude slow mRNA turnover) normal 0 2 4 6 8 13 weeks transgenic U7 included Top, middle , in addition to bottom Normal Untreated mdx Treated mdx dystrophin dystrophin-associated antigens Muscle immuno-histochemistry

RNAi = RNA interference Short double str in addition to ed RNA molecules trigger the degradation of the complementary sequence in the cell, in addition to can inhibit translation of the targeted mRNA Their introduction into a cell can greatly reduce any protein whose mRNA is targeted. Inhibition is usually incomplete in mammalian cells, but can be considerable (>90%) Thus “gene knockdown” as opposed to knock-out Alternative technologies: Antisense RNA: block translation or splicing Ribozymes: RNAs that cleave other RNAs, sequence specifically siRNA = small inhibitory RNAs shRNA = short hairpin RNAs (both str in addition to s can be coded by one DNA) asRNA = antisense RNA miRNA = microRNAs ncRNA = noncoding RNA Introduction of long DS RNA into mammalian cells will trigger the “interferon response: Cessation of protein synthesis via activation of PKR (protein kinase RNA-activated), in addition to phosphorylation of eIF2 Global degradation of mRNA (without any sequence specificity, RNase L activation) Spread to neighboring cells (induction in addition to secretion of interferon) Most small DS RNAs do not trigger this response(<30 bp) miRNA synthesis in addition to maturation Dougherty, Julie Business For Breakfast - KFNN-AM Morning Drive On-Air Personality www.phwiki.com

mRNA degradation Inhibits translation of an mRNA Generation of siRNA in vitro Chemical synthesis, annealing of 22-mers (bypasses dicing by Dicer) Introduce perfect hairpin RNA into cells, let Dicer make siRNA T7-mediated in vitro transcription of each complementary str in addition to . Anneal to make long DS RNA in addition to transfer to cells. Let Dicer make siRNA in the cell Introduce imperfect hairpin RNA into cells (based on mRNA sequence) in addition to let Dicer make miRNA Also, can use controlled RNase to generate fragments (cheaper) Generation of si RNA in vivo

Got this far Transient nature of the response (~3 days) Limitations of siRNA silencing in mammalian cells Transfection problems (cell type, refractoriness) Can be cell type specific Non-renewable nature of siRNAs ($$) siRNA Incorporation into the RNA-inducing silencing complex (RISC); stability in RISC. Base-pairing with mRNA. Cleavage of mRNA. mRNA Base-pairing with siRNA. The position of the siRNA-binding target region. Secondary in addition to tertiary structures in mRNA. Binding of mRNA-associated proteins. The rate of mRNA translation. The number of polysomes that are associated with translating mRNA. The abundance in addition to half-life of mRNA. The subcellular location of mRNA. Delivery Transfection (lipofection, electroporation, hydrodynamic injection (mouse)) Virus infection (esp. lentivirus (e.g., retrovirus like HIV that can integrate into non-dividing cells) Potential determinants of efficient siRNA-directed gene silencing

R From the label: VEGF = vascular endothelial growth factor Where R is in addition to contains a PEG chain of ~ 450 ethylene glycol units. The chemical name as long as pegaptanib sodium is as follows: RNA, ((2′-deoxy-2′-fluoro)C-Gm-Gm-A-A-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-Am-Gm-(2′-deoxy-2-fluoro)U-Gm-Am-Am-(2′-deoxy-2′-fluoro)U-Gm-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)U-Am-(2′-deoxy-2′-fluoro)U-Am-(2′-deoxy-2′-fluoro)C-Am-(2′-deoxy-2′-fluoro)U-(2′-deoxy-2′-fluoro)C-(2′-deoxy-2′-fluoro)C-Gm-(3’3′)-dT), 5′-ester with ,’-[4,12-dioxo-6-[[[5-(phosphoonoxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[- methoxypoly(oxy-1,2-ethanediyl)], sodium salt. The molecular as long as mula as long as pegaptanib sodium is C294H342F13N107Na28O188P28[C2H4O]n (where n is approximately 900) in addition to the molecular weight is approximately 50 kilodaltons. Macugen is as long as mulated to have an osmolality of 280-360 mOsm/Kg, in addition to a pH of 6–7. Inverted ribo-T 3’-3’ to protect 3’ end Macugen: an RNA aptamer that binds VEGF in addition to is marketed as long as adult macular degeneration (wet type)

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