Presentation on theme: " Isolate a specific gene of interest Insert into a plasmid Transfer to bacteria Grow bacteria to get many copies Express the protein product "— Presentation transcript:
Isolate a specific gene of interest Insert into a plasmid Transfer to bacteria Grow bacteria to get many copies Express the protein product Why? Sequence the gene Study the enzyme Understand regulation Genetic screening Gene therapy …etc. Gene cloning human RPE65 gene plasmid recombinant DNA E. coli human RPE65 enzyme
1) Isolate DNA including YFG 2) Join to plasmid vector (ligation) 3) Introduce into host (transformation) 4) Find correct clone 5) Express the protein product Steps in gene cloning human RPE65 gene plasmid recombinant DNA E. coli human RPE65 enzyme ligation transformation
Extract from cells Cut into manageable fragments 1. Isolate DNA including YFG human DNA RPE65 gene RPE65 gene
Restriction digest GAATTC CTTAAG GAATTC CTTAAG GAATTC CTTAAG cloning vector (plasmid) human DNA
2. Join to plasmid vector (ligation) AATTC G CTTAA AATTC G CTTAA “sticky” ends cloning vector (plasmid) restriction fragment
2. Join to plasmid vector (ligation) recombinant plasmid GAATTC CTTAAG GAATTC CTTAAG DNA ligase what’s missing? what enzyme should we use?
2. Join to plasmid vector (ligation) + human DNA fragments plasmid vector plasmid library
3. Introduce into host (transformation) recombinant DNA recombinant E. coli E. coli + CaCl 2 or electric shock
Why my clones can’t make RPE65 protein: RPE65 gene has introns; bacteria can’t splice Expression signals: Transcription:bacteria need -10 and -35 human gene has TATA, enhancers, etc. Translation:bacteria need Shine-Dalgarno human gene won’t have it ATG TAG TATAenhancers ? ?
Spliced mRNA → coding sequence with no introns cDNA cloning: DNA copy of RNA DNA mRNA nucleus cytoplasm mature RNA AAAAAAAAAAAAAAA reverse transcriptase DNA Why does it have to be DNA?
Purify mRNA: from what kind of cells? from where in the cell? cDNA cloning AAAAAAAAAA mRNA
Add reverse transcriptase to make cDNA cDNA cloning AAAAAAAAAA TTTTTTT
Add reverse transcriptase to make cDNA cDNA cloning
Enzyme assay for RPE65 4. Find the correct clone proteins from lysed bacteria trans- retinal HPLC
Cloned gene is ready for use! purify plasmid DNA sequencing express protein etc.
Cloning by PCR Polymerase chain reaction If DNA sequence is known, amplify specific gene directly human DNA RPE65 gene PCR
Cloning by PCR Human DNA RPE65-specific 20-nt primers Taq DNA polymerase dNTPs 5 ′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG 3 ′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCTCC part of RPE65 heat 5 ′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG TTTGACACCT 5 ′ primer TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAAC heat 5 ′ CCAGGTTGAG TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT 5 ′ primer CATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGA