 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

3. Introduce into host (transformation)

agar plate with ampicillin 3. Introduce into host (transformation)  Select cells that have plasmid by antibiotic resistance

4. Find the correct clone How do we know which of all these colonies came from a cell that took up a plasmid carrying RPE65?

 Enzyme assay for RPE65 4. Find the correct clone proteins from lysed bacteria trans- retinal HPLC

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

 Ligate to a plasmid vector cDNA cloning +

 Transform into E. coli  Find correct clone cDNA cloning cDNA library Now could we express the protein product??

 Plasmid with transcription and translation signals Expression vector EcoRI expression vector S-D EcoRI S-D EcoRI RPE65 cDNA

 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

Cloning by PCR

 Once amplified, ligate and transform as before + amplified copies of RPE65 gene plasmid vector

 Modified microorganisms:  Insulin, growth hormone, clotting factors, EPO…  HPV vaccine  Ethanol from cellulose  Oil-eating bacteria  Modified plants and animals  BT corn  Roundup-ready soybeans  Golden rice  Modified humans  Gene therapy Genetic engineering