Recombinant DNA3_08

Recombinant DNA3_08 - Recombinant DNA 3 Today's Outline How...

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Recombinant DNA 3 Today’s Outline How to Clone a Gene, Continued Ligating Fragments into Plasmids T4 DNA Ligase CIP Blue-White Screening PCR DNA Sequencing Libraries Genomic -uses restriction enzymes cDNA RT-PCR
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To clone DNA fragments into plasmid vectors: Can use restriction enzymes Requires a ligation reaction to seal or ligate the DNA ends (T4 DNA ligase) The desired result is a recombinant DNA molecule: the vector now contains an inserted piece of DNA (or insert) Fig. 12-03, p.378
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Ligation Reactions Catalyzed by T4 DNA Ligase Requires ATP Ligates two DNA ends (new phosphodiester bond) Requires 5’Ps Fig. 12-04, p.379
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Mechanism of DNA Ligase Requires ATP or NAD+ Lys residue in Ligase is adenylated Transfer of AMP to the 5’P Attack on AMP by 3’OH Phosphodiester bond formation; release of AMP
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Restriction Enzyme Digestion with a Single Enzyme Results in Compatible, Cohesive Ends that Can Ligate or Re-anneal: AGTGCGTGCCC GAATTC GATAT TCACGCACGGG CTTAAG CTATA +EcoRI digestion
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Restriction Enzyme Digestion with a Single Enzyme Results in Compatible, Cohesive Ends that Can Ligate or Re-anneal: AGTGCGTGCCC G 3’ 5’ AATTC GATAT TCACGCACGGG CTTAA 5’ 3’ G CTATA GAATTC CTTAAG GAATTC CTTAAG Any other fragment with EcoRI ends can also be ligated: G 3’ CTTAA 5’ 5’ AATTC 3’ G
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This note was uploaded on 03/31/2008 for the course BCHM 4115 taught by Professor Emgregory during the Spring '08 term at Virginia Tech.

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Recombinant DNA3_08 - Recombinant DNA 3 Today's Outline How...

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