Download L27- Cloning

What causes LCA2 blindness?
light
change [Na+]
send signal on
optic nerve
trans-retinal
cis-retinal
RPE65
LCA2 blindness:
light
change [Na+]
send signal on
optic nerve
trans-retinal
cis-retinal
trans-retinal
Is LCA2 allele dominant or recessive?
chromosome 1
normal
allele
transcription
translation
RPE65
RPE65 gene
no functional
protein
LCA2
allele
What are the genotypes?
What is the probability of another LCA2 child?
Rr
Rr
R = normal RPE65
r = LCA2
rr
1 in 4 chance
next child will
be blind by age 20
How could we prevent or cure this disease?
Rr
Rr
R = normal RPE65
r = LCA2
rr
1 in 4 chance
next child will
be blind by age 20
What would we need to have
in order to do gene therapy?
Where can we find the RPE65 gene?
Joe
human
cell
human
DNA
OK, but now what?
RPE65
gene
Gene cloning

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.
human
RPE65 enzyme
human
RPE65 gene
plasmid
recombinant
DNA
E. coli
Steps in gene cloning
human
RPE65 gene
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
ligation
plasmid
recombinant
DNA
transformation
human
RPE65 enzyme
E. coli
1. Isolate DNA including YFG

Extract from cells

Cut into manageable fragments
RPE65
gene
human
DNA
RPE65
gene
Restriction digest
GAATTC
CTTAAG
GAATTC
CTTAAG
cloning vector
(plasmid)
GAATTC
CTTAAG
human DNA
2. Join to plasmid vector (ligation)
AATTC
G
G
CTTAA
restriction
fragment
“sticky”
ends
cloning vector
(plasmid)
2. Join to plasmid vector (ligation)
DNA ligase
recombinant
plasmid
what’s missing?
what enzyme should we use?
2. Join to plasmid vector (ligation)
plasmid
vector
+
human DNA fragments
plasmid
library
3. Introduce into host (transformation)
recombinant
DNA
+
E. coli
CaCl2 or
electric shock
recombinant E. coli
3. Introduce into host (transformation)
3. Introduce into host (transformation)

Select cells that have plasmid by antibiotic resistance
agar plate
with ampicillin
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?
4. Find the correct clone

Enzyme assay for RPE65
transretinal
HPLC
This won’t work.
Why not?
proteins
from lysed
bacteria
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

enhancers
TATA
TAG
Why my clones can’t make RPE65 protein:
cDNA cloning: DNA copy of RNA

Spliced mRNA → coding sequence with no introns
DNA
nucleus
mRNA
cytoplasm
AAAAAAAAAAAAAAA
mature RNA
reverse transcriptase
DNA
Why does it
have to be
DNA?
cDNA cloning

Purify mRNA: from what kind of cells?
from where in the cell?
AAAAAAAAAA
AAAAAAAAAA
AAAAAAAAAA
mRNA
AAAAAAAAAA
AAAAAAAAAA
cDNA cloning

Add reverse transcriptase to make cDNA
AAAAAAAAAA
TTTTTTT
AAAAAAAAAA
TTTTTTT
AAAAAAAAAA
TTTTTTT
AAAAAAAAAA
TTTTTTT
AAAAAAAAAA
TTTTTTT
cDNA cloning

Add reverse transcriptase to make cDNA
cDNA cloning

Ligate to a plasmid vector
+
cDNA cloning

Transform into E. coli

Find correct clone
cDNA
library
Now could we express the protein product??
Expression vector

Plasmid with transcription and translation signals
-35
-10
-35
S-D
EcoRI
-10
S-D
EcoRI
RPE65
cDNA
expression
vector
EcoRI
4. Find the correct clone

Enzyme assay for RPE65
transretinal
HPLC
proteins
from lysed
bacteria
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
RPE65
gene
human
DNA
Cloning by PCR
• Human DNA
• RPE65-specific 20-nt primers
• Taq DNA polymerase
• dNTPs
part of RPE65
5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG
3′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCTCC
heat
5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG
TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT 5′
heat
primer
5′
CCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGA
TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT 5′
primer
Cloning by PCR
Cloning by PCR

Once amplified, ligate and transform as before
+
amplified copies
of RPE65 gene
plasmid
vector
Genetic engineering
Genetic engineering

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
Recombinant DNA
technology:
Unlimited possibilities
Many questions