isolate a specific gene of interest insert into a plasmid transfer to bacteria grow bacteria to...
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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 cloninghuman
RPE65 gene
plasmid
recombinantDNA
E. colihuman
RPE65 enzyme
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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 cloninghuman
RPE65 gene
plasmid
recombinantDNA
E. colihuman
RPE65 enzyme
ligation
transformation
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Extract from cells Cut into manageable fragments
1. Isolate DNA including YFG
humanDNA
RPE65gene
RPE65gene
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Restriction digest
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
cloning vector(plasmid)
human DNA
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2. Join to plasmid vector (ligation)
AATTC G
GCTTAA
AATTC G
GCTT
AA
“sticky”ends
cloning vector(plasmid)
restrictionfragment
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2. Join to plasmid vector (ligation)
recombinantplasmid
GAATTC
CTTAAG
GAATTC
CTTAAGDNA ligase
what’s missing?what enzyme should we use?
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2. Join to plasmid vector (ligation)
+human DNA fragments
plasmidvector
plasmidlibrary
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3. Introduce into host (transformation)
recombinantDNA
recombinant E. coli
E. coli
+
CaCl2 orelectric shock
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3. Introduce into host (transformation)
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agar platewith ampicillin
3. Introduce into host (transformation) Select cells that have plasmid by antibiotic resistance
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4. Find the correct cloneHow do we know which of all these colonies came from a cell that took up a plasmid carrying RPE65?
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Enzyme assay for RPE654. Find the correct clone
proteinsfrom lysedbacteria
trans-retinal
HPLC
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Why my clones can’t make RPE65 protein: RPE65 gene has introns; bacteria can’t splice Expression signals:
Transcription: bacteria need -10 and -35human gene has TATA, enhancers, etc.
Translation: bacteria need Shine-Dalgarnohuman gene won’t have it
AT
G
TA
G
TATAenhancers
??
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Spliced mRNA → coding sequence with no introns
cDNA cloning: DNA copy of RNA
DNA
mRNAnucleus
cytoplasm
mature RNAAAAAAAAAAAAAAAA
reverse transcriptase
DNA Why does it have to be
DNA?
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Purify mRNA: from what kind of cells?
from where in the cell?
cDNA cloning
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
mRNA
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Add reverse transcriptase to make cDNA
cDNA cloning
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
TTTTTTT
TTTTTTT
TTTTTTT
TTTTTTT
TTTTTTT
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Add reverse transcriptase to make cDNA
cDNA cloning
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Ligate to a plasmid vector
cDNA cloning
+
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Transform into E. coli Find correct clone
cDNA cloning
cDNAlibrary
Now could we express the protein product??
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Plasmid with transcription and translation signals
Expression vector
-10-35
EcoRI
expressionvector
S-D
-10-35
EcoRIS-D
EcoRI
RPE65cDNA
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Enzyme assay for RPE654. Find the correct clone
proteinsfrom lysedbacteria
trans-retinal
HPLC
![Page 23: Isolate a specific gene of interest Insert into a plasmid Transfer to bacteria Grow bacteria to get many copies Express the protein product](https://reader030.vdocument.in/reader030/viewer/2022032612/56649e8a5503460f94b8f13c/html5/thumbnails/23.jpg)
Cloned gene is ready for use!
purify plasmidDNA sequencingexpress proteinetc.
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Cloning by PCR Polymerase chain reaction If DNA sequence is known, amplify specific gene directly
humanDNA
RPE65gene
PCR
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Cloning by PCR• Human DNA• RPE65-specific 20-nt primers• Taq DNA polymerase• dNTPs
5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG3′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCTCC
part of RPE65
heat
5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG TTTGACACCT
5′primer
TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAAC
heat
5′ CCAGGTTGAG TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT
5′
primerCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGA
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Cloning by PCR
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Cloning by PCR Once amplified, ligate and transform as before
+amplified copiesof RPE65 gene
plasmidvector
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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