Unit3-1STUDENTS - Review of Unit 2-3 RNA primer experiment...

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Unformatted text preview: Review of Unit 2-3 RNA primer experiment Eukaryotes-end replication problem DNA damage and mutations Errors during replication Damage from environmental agents, chemical mutagens DNA repair mechanisms Proofreading Mismatch repair Excision repair Unit 3: DNA Cloning and Sequencing Reading: Weaver pp. 51-71, 83-87, 93-104 Outline for Unit 3-1 Recombinant DNA techniques gene cloning Use of restriction endonucleases Cloning vectors Sequencing Studying DNA sequences usually requires the availability of fairly large quantities of the DNA This is facilitated by gene cloning Isolation of a specific DNA fragment of interest (e.g. a gene), and propagating identical copies (clones) of it in a suitable vector. This is achieved using recombinant DNA technology. **Common lab techniques (cloning, polymerase chain reaction, sequencing) are only possible because we learned how bacteria replicate and repair their DNA** Gene Cloning Identification of oriC Have we discussed an example of this? Identification of oriC Nucleases are enzymes that cut DNA Endo nucleases cut DNA at specific sequences within the DNA Exo nucleases cut DNA at its free ends Ligases are enzymes that can join DNA strands Recombinant plasmids The role of restriction endonucleases in cloning Restriction endonucleases recognize and cleave specific DNA sequences How do they distinguish the specific sequence? Access at the major and minor grooves Where do restriction endonucleases come from? Usually isolated from bacteria Named for the bacteria they are isolated from EcoRI isolated from E . co li strain R HindIII isolated from H aemophilus in fluenzae strain R d **The numbering designates the order in which the enzyme was isolated Endonucleases from over 100 bacterial strains have been isolated >150 different specific cleavage sites have been identified Restriction Modification System 1970-Hamilton Smith identifies RMS RMS is used by bacteria to protect themselves from invading pathogens Restriction of invading pathogen is accomplished by degradation of the foreign DNA Degradation of the foreign DNA resulted from endonuclease activity If bacteria produce these enzymes, why isnt the bacterial DNA digested by them? Restriction endonucleases protect bacteria from invasion by foreign DNA (like phage) ( Restrict the host range of the virus) Bacteria use methylation to protect their chromosomal DNA; invading viral DNA is not methylated Many restriction enzymes cannot cleave methylated DNA Methylation of DNA at specific sequences serves to mark self DNA Methylation can prevent restriction endonucleases from cutting Examples Of Restriction Enzymes Most restriction sites are palindromes.Why? Palindromes read the same forwards and backwards Was it a bat I saw?...
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Unit3-1STUDENTS - Review of Unit 2-3 RNA primer experiment...

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