isolate a specific gene of interest insert into a plasmid transfer to bacteria grow bacteria to...

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

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

Extract from cells Cut into manageable fragments

1. Isolate DNA including YFG

humanDNA

RPE65gene

RPE65gene

Restriction digest

GAATTCCTTAAG

GAATTCCTTAAG

GAATTCCTTAAG

cloning vector(plasmid)

human DNA

2. Join to plasmid vector (ligation)

AATTC G

GCTTAA

AATTC G

GCTT

AA

“sticky”ends

cloning vector(plasmid)

restrictionfragment

2. Join to plasmid vector (ligation)

recombinantplasmid

GAATTC

CTTAAG

GAATTC

CTTAAGDNA ligase

what’s missing?what enzyme should we use?

2. Join to plasmid vector (ligation)

+human DNA fragments

plasmidvector

plasmidlibrary

3. Introduce into host (transformation)

recombinantDNA

recombinant E. coli

E. coli

+

CaCl2 orelectric shock

3. Introduce into host (transformation)

agar platewith ampicillin

3. Introduce into host (transformation) Select cells that have plasmid by antibiotic resistance

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

Enzyme assay for RPE654. Find the correct clone

proteinsfrom lysedbacteria

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 -35human gene has TATA, enhancers, etc.

Translation: bacteria need Shine-Dalgarnohuman gene won’t have it

AT

G

TA

G

TATAenhancers

??

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?

Purify mRNA: from what kind of cells?

from where in the cell?

cDNA cloning

AAAAAAAAAA

AAAAAAAAAA

AAAAAAAAAA

AAAAAAAAAA

AAAAAAAAAA

mRNA

Add reverse transcriptase to make cDNA

cDNA cloning

AAAAAAAAAA

AAAAAAAAAA

AAAAAAAAAA

AAAAAAAAAA

AAAAAAAAAA

TTTTTTT

TTTTTTT

TTTTTTT

TTTTTTT

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

cDNAlibrary

Now could we express the protein product??

Plasmid with transcription and translation signals

Expression vector

-10-35

EcoRI

expressionvector

S-D

-10-35

EcoRIS-D

EcoRI

RPE65cDNA

Enzyme assay for RPE654. Find the correct clone

proteinsfrom lysedbacteria

trans-retinal

HPLC

Cloned gene is ready for use!

purify plasmidDNA sequencingexpress proteinetc.

Cloning by PCR Polymerase chain reaction If DNA sequence is known, amplify specific gene directly

humanDNA

RPE65gene

PCR

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

Cloning by PCR

Cloning by PCR Once amplified, ligate and transform as before

+amplified copiesof RPE65 gene

plasmidvector

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