MolbioMT1 - Lecture 6 Telomere: Problem: lagging strand did...

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Lecture 6 Telomere: Problem: lagging strand did not reach to the end of the DNA Goal: extends 3' template strand. Telomere DNA is non protein coding. function: protect the end of the chromosome. rna primer use dna pol to fill in. How to add telomere DNA: 1. Telomerase RNA base pairs to template strand. RNA acs as template. Telomerase Protein: reverse transcriptase (RNA to DNA) from RNA template. complement to TTAGGV (telomere sequence) 2. Telomerase adds nucleotides. 3. RNA template slipping 4. Telomerase fills in gap 5. Slipping, filling Repeated. end of chrom telomere dna now. long template strand (a lot temolmere dna) 6. Made another okazaki frament: primase comes in, fills in, primer removed = gap in temolmere dna Double stranded Breaks: DNA binding Proteins *found at the end of telomeres. it protects telomeres from degraded my nuclease or repaired by the ds break repair pathways Telomerase active in germ and stem cells only =telomeres born with you have for life. every time cells divide telomere get shorter until it breaks off telomere dna=ageing, cancer Techniques of Molecular biology: *what question can we answer with this? 1. DNA Cloning: A method to obtain lots of a specific piece of DNA Why? 1. make DNA libraries 2. Purify a protein 3. Express a protein in a new organism How? 1. Take a piece of DNA (Insert) out into a plamid (vector) 2. Put vector into bacteria. 3. Bacteria will make a bunch of plasmids with the insert DNA.
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1. Insert? 1. Genomic DNA by PCR 2. Use an existing plasmid by PCR or Restriction Digest PCR: make oligos designed to bind to the fragment of interest. =insert. Plasmid: Make oligo that binds Rescription Digest: Use RE( endonuclease tht cut at specfic DNA sequences. 100's of different RE's that cut out at diff sequences. Naturally found in bacteria. 4-8 base pairs, palindromic (read the same way on top and bottom strand when red in 5' to 3' direction). ECR1 and NOT1 makes sticky/staggard ends: 5' or 3' overhang that can base pair to specific complementary DNA SKA1 makes blunt ends: no overhang. no complementary base pairing. ANY two blunt ends can be joined. Why does the bacteria DNA not get cut up?? DNA methylation! methylation will protect the DNA from ECOR1 cutting the DNA What if insert has no good restriction sites?? PCR with specific oligos to add on restriction sites *if multiple restriction sites present, then choose sites that keeps entire insert intact. 2. Plasmid:Vector: circular piece of DNA has components to clone goal: introude vector into bacteria 1. Origin of replication: thats where DNA replication starts. two plasmids from 1. 2. Selection Marker: which bacteria has plasmid Drugs: amplicillian, canamyocin (both prevents bacterial growth) Plates already contain antibiotics, plasmid will contain AMP resistance gene, plated, growth if the plasmid replicated properly! 3. Poly Linker: introduces region with lots of unique (only
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MolbioMT1 - Lecture 6 Telomere: Problem: lagging strand did...

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