Lecture 17

Lecture 17 - LS3 MOLECULAR BIOLOGY LECTURE 16 Overview...

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LS3 MOLECULAR BIOLOGY LECTURE 16 Overview Dr. Randolph Wall Reading: Watson et al. 654-660
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From Lodish et al (2004) Molecular Cell Biology. Ligation of Restriction Fragments with Complementary Sticky Ends via T4 Ligase - used for inserting DNA into a vector to make recombinant DNA Insert DNA
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Reverse Transcriptase (RT) allows the conversion of mRNA into cDNA oligo-dT hybridized to polyA as primer for cDNA synthesis -> First strand synthesis Initial product is DNA-mRNA duplex Degrade mRNA -> single-stranded cDNA
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cDNA library Attach restriction site linkers to blunt-end cDNA, digest linkers & ligate sticky ends into plasmid vector, transform E. coli Second DNA strand synthesized using random short oligonucleotide primers, DNA polymerase & dNTPs
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DNA Cloning in a Plasmid Vector -> Amplification of cloned DNA Fragment re
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Assembling a Genomic library in Phage lambda Cleave genomic DNA to ~25kb fragments by partial digestion with Sau3A1 ( v GATC) Remove replaceable central region of λ phage genome by BamHI cleavage (G v GATCC) Ligate λ arms to insert genomic DNA (i.e., ~25kb insert DNA fragments isolated from partial Sau3A1 cleavage) Package recombinant λ◊ P q+ ÷ℜ °¼ ª * in vitro Infect E. coli -> plaques for screening
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PCR Reactions PRIMERS: Short synthetic oligonucleotides (15-20 nt long) complementary to the known DNA sequences flanking the specific region to be amplified POLYMERASE: Taq polymerase, isolated from thermophilic bacteria Thermus aquaticus, used because of its resistance to heat denaturation REACTIONS: Cycles of DNA synthesis repeated many times in vitro to amplify a specific region from as little as one copy of DNA Cycle #1 Cycle #2 // Cycle #25 DNA denaturation, 95°C DNA denaturation, 95° C DNA denaturation, 95°C primer annealing, ~50°C primer annealing, ~50°C primer annealing, ~50°C primer elongation,72°C primer elongation,72°C primer elongation,72°C EXAMPLE:
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The Polymerase Chain Reaction (PCR) is used to amplify DNA of known sequence. The PCR cycle repeated 25-30X -> ~1 billion copies of amplified DNA
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Influenza Virus Two viral glycoproteins in spikes on virion envelope (H & N) determine infectivity of flu viruses & are major antigens for anti- flu virus antibodies Activities of H and N Hemagglutinin (H, 15 types): trimeric protein spikes, bind to sialic acid-containing sugars on host cell membranes -> viral entry into infected cell - H binding determines flu virus host specificity Neuraminidase (N, 9 tyes): clips sialic acid on cell membranes of infected cells and new flu virus particles budding from infected cells, thereby releasing newly replicated flu viruses able to efficiently infect other host cells
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PCR: Reconstruction of the 1918 Flu Virus RNA Flu virus genome composed of 8 RNA strands -> 11 viral proteins RT-PCR conducted on flu RNA preserved in tissues from fatal cases of 1918 flu (in 1997) -Four formalin-preserved parafin-embedded lung biopsy tissue samples from 1918 kept in
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Lecture 17 - LS3 MOLECULAR BIOLOGY LECTURE 16 Overview...

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