Download DNA cloning

853
MCB 3020, Spring 2005
Chapter 31:
Genetic Engineering and
Biotechnology I
Genetic Engineering I
I. Genetic Engineering
II. Cloning vectors
854
I. Genetic Engineering
i. Some uses
ii. Restriction enzymes
iii. DNA cloning
iv. DNA library
855
Genetic Engineering
DNA manipulation using molecular
biology techniques
Typical procedures
• DNA cloning
• identification of genes of interest
• expression of genes to make a
desired product
856
i. Some uses of genetic engineering
857
• Industrial or biotechnology products
– (eg. alkaliphilic proteases, Taq polymerase)
• Medical products
– (eg. insulin, hepatitis B vaccines, gene therapy)
• Agriculture and Environment
– (eg. plant resistant to pesticides, insects, disease)
• Basic research
ii. Restriction enzymes
a. natural role
b. recognition sequence
c. cut sites
d. modification enzymes
858
ii. Restriction enzymes
859
Enzymes that break double-stranded
DNA at specific sequences.
a. Natural role
Used to protect bacteria from
viruses (by cutting viral DNA)
Bacterial DNA is protected by
modification enzymes.
TB
b. Recognition sequence
DNA sequence where cutting occurs
860
EcoRI recognition sequence
cut sites
GAATTC
CTTAAG
Palindromic recognition sequence
TB
After cutting
G
CTTAA
861
sticky ends
AATTC
G
double stranded break
TB
DraI recognition sequence
862
TTTAAA
AAATTT
DraI
TTT
AAA
AAA
TTT
After cutting: blunt ends
TB
c. Modification enzymes
863
• covalently modify DNA, often by
methylation
• prevent cutting by the corresponding
restriction enzyme
• recognize the same site on DNA as
the corresponding restriction enzyme
• protect bacteria from their own
restriction enzymes
Typical modification enzyme
EcoRI methylase
CH3
864
methylation
GAATTC
CTTAAG
CH3
After modification, the EcoRI restriction enzyme
will NOT recognize the methylated DNA. TB
iii.DNA cloning
Isolation and insertion of a DNA
fragment (insert) into a vector.
ori
+
Cloning vector
a small independently replicating
genetic element into which
genes can be recombined
865
Basic steps of DNA cloning
866
1. Isolate source DNA
2. Digest source DNA and vector
using restriction enzymes
3. Ligate the source DNA to the vector
4. Introduce DNA into a host
5. Identify the clone of interest
TB
source DNA
cloning vector
1. Isolate
867
2. Digest DNA and vector
3. Ligate
cloned DNA
(insert)
vector
One to many clones representing the source DNA
4. Introduce DNA into host
host cell
TB
5. Identify clone of interest
1. Isolate source DNA
x
868
gene of interest
x
eg. BamHI
2. Digest source DNA
and vector using
restriction enzymes
x
source DNA
3. Ligate source DNA into vectors.
ori
restriction site
+
source
DNA
x
cloning
vector
+
869
cloned
DNA
(insert)
4. Introduce DNA into a host.
+
DNA library
E. coli
Plate on agar
870
5. Identify the clone of interest
871
Plate on selective medium to find
colonies with cloned DNA
E. coli with cloned DNA
Identify colonies with gene of
interest
iv. DNA library
A large number of clones representing
the entire genome of an organism.
872
The source DNA for DNA libraries is
typically the genomic DNA.
x
Introduce
into host
cells; plate
II. Cloning vectors
A. important features
B. examples
restriction site
ApR
ori
873
A. Important features
874
1. means of replication (ori)
2. unique restriction sites (single cut)
3. selectable markers
restriction
4. gene inactivation marker
ApR
ApR
ori
= ampicillin resistance gene
TcR = tetracycline resistance gene
site
TcR
Selectable marker
In genetic engineering, a gene whose
product can be used to select the cells
that carry the plasmid of interest
Gene inactivation marker
In genetic engineering, a gene that is
disrupted (inactivated) when a second
gene of interest is cloned into the plasmid
or DNA
875
4a. Selectable markers and gene inactivation876
ApR
ori
BamH1 Uncut vector allows cells to grow on
TcR
ampicillin (Ap) and tetracycline (Tc).
transform
E. coli
+ ampicillin
replica
plating
+ ampicillin
+ tetracycline
What happens when foreign DNA
is inserted into the BamH1 site?
• the TcR (tetracyline resistance) gene is inactivated
Selectable markers and gene inactivation877
ApR
ori
BamH1
TcR
ApR
ApR
BamH1
digest
TcR
+
TcR
insert
ApR
When foreign DNA is inserted,
• TcR gene is inactivated
• cells will grow on Ap, but NOT tetracycline
In this gene inactivation system, what
happens when E. coli is transformed with a
mixture of vector and [vector with insert]?
+ ampicillin
replica
plating
878
+ ampicillin
+ tetracycline
Cells containing the cloned DNA (insert), are
Ap-resistant (ApR) but Tc-sensitive (TcS ).
4b. Another gene inactivation marker is the 879
lacZ gene (codes for beta-galactosidase)
ApR
BamH1
beta-galactosidase
cleaves X-gal and
produces a blue color
lacZ
ori
O
Cl
O
X-gal
(CLEAR)
N
O
Br
HO
beta-galactosidase
Cl
N
Br
OH
BLUE
product
880
Colonies containing vector WITHOUT
an insert are blue.
ApR
ori
BamH1
lacZ
+ ampicillin
+ X-gal
(We don't want these.)
When foreign DNA is inserted,
it inactivates lacZ
881
• beta-galactosidase is not made
• X-gal is not cleaved
• colonies with insert are white, NOT blue
insert
X-gal
(CLEAR)
X
X
(LacZ-)
+ ampicillin
+ X-gal
B. Examples of cloning vectors
1. Plasmids
2. Phage
3. Cosmids
4. YACs
882
883
1. plasmid vector (holds ~10 kb)
vector
BamHI
R
source DNA
Ap
pBR322
TcR
BamHI sites
ori
ApR = ampicillin resistance gene
TcR = tetracycline resistance gene
ori = origin of DNA replication
BamHI = unique restriction site
BamHI digestion
TcR
884
source DNA
ligation
cloned
DNA
mixture of [vector with cloned DNA], and vector
TB
2. Phage vector (holds about 20 kb) 885
a. Phage lambda (l)
l
dsDNA
1/3 of genome
non-essential for lytic growth
(can replace this section
with foreign DNA)
TB
886
e.g. of phage vector: Charon 4A
(genetically altered l derivative)
EcoRI sites
cos site
lacZ gene
1. restriction
2. ligation
cloned DNA
TB
3. Package the cloned DNA into
capsids in vitro.
887
4. Infect host cells and plate to
obtain plaques
lawn of E. coli cells
plaques
(regions of dead cells caused by lytic phage)
clear plaques (LacZ-)
888
blue plaques (LacZ+)
5. Isolate DNA from clear plaques.
(Blue plaques do NOT have insert.
TB
We don't want these.)
b. Phage M13 vectors
• Phage M13: a ssDNA virus that
has a dsDNA replicative form
• Used to produce ssDNA for
DNA sequencing and sitedirected mutagenesis
• Double-stranded replicative
form is used for cloning
889
3. Cosmid (holds up to 45 kb)
Plasmids with cos (cohesive end) sites
for in vitro packaging into l capsids.
1. clone DNA fragments
2. linearize
3.
package
in
vitro
cos
890
plasmid
4. Yeast Artificial chromosomes (YACs)891
(holds up to 800 kb)
Features of YACS:
ori
telomeres
centromere
cloning site
selectable marker
200-800 kb inserts
(Human genome ~ 3 x 109 bp or 3 x 106 kb)
Comparison of clone sizes
Plasmids up to ~10 kb
Charon phage up to ~ 20 kb
Cosmids up to ~ 45 kb
YACS up to ~800 kb
892
C. Hosts for cloning vectors
893
Escherichia coli
Bacillus subtilis
Saccharomyces cerevisiae (yeast)
mammalian cells
Study objectives
894
1. Name three procedures typically used in genetic engineering.
2. What are some uses of genetic engineering? Know the examples presented.
3. What are restriction and modification enzymes? What is their natural role?
Describe the general features of the recognition site of restriction enzymes.
You do NOT need to memorize the sequences of the recognition sites.
4. What is DNA cloning? What is a cloning vector?
5. Understand in detail the basic steps involved in cloning DNA.
6. What is a DNA library? What is the typical source DNA for a library?
7. Know the important features of a cloning vector and their roles in cloning.
8. Describe how antibiotic resistance genes and the beta-galactosidase gene
can be used to determine if foreign DNA has been inserted into a vector.
9. Understand why the following are important for cloning vectors: selectable
markers, gene inactivation, means of replication, unique restriction sites.
10. How the following are used in DNA cloning: plasmid vectors (example,
pBR322) phage vectors (examples, Charon 4A and M13) cosmids, and YACs.
11. Compare and contrast the different DNA cloning vectors. What features are
specific to each cloning vector?
12. Know that specific host cells facilitate cloning. Know the examples presented.
895
MCB 3020 Spring 2005
Chapter 31:
Genetic Engineering and
Biotechnology II
Last time:
896
I. Genetic Engineering
II. Cloning vectors
Today:
III. Identifying clones of interest
IV. Expression vectors
V. Polymerase chain reaction (PCR)
VI. Cloning and expression of
mammalian genes in bacteria
VII. Applications of genetic engineering
III. Identifying clones of interest
A. antibodies
B. DNA and RNA probes
C. complementation
897
A. antibodies (immunoglobulins)
soluble immune system proteins
that bind specific antigens*
898
This antigen is a protein.
(*Antigens are "nonself" (foreign) molecules that interact with
components of the immune system.)
TB
Using antibodies to identify clones
1. Purify protein of interest (protein X).
899
X
2. Prepare antibody ( ) that specifically
binds to protein X.
3. If a DNA clone expresses protein X,
it includes the gene for protein X
4. Use antibody to test clones
for production of protein X.
TB
Using antibodies to identify clones of
interest
900
DNA Library
transform E. coli
transformant
colonies
1. replica plate cells to filter paper
transformant cells on filter paper
TB
2. lyse cells
901
3. bind the antibody
4. detect the antibody
contains a DNA clone
expressing the protein of interest
TB
B. DNA and RNA probes
902
Probe: labeled DNA or RNA that
can bind a particular DNA by
complementary base paring.
32P
(Probes can be short single-stranded
oligonucleotides with a radioactive or
fluorescent label attached)
Uses of DNA probes
903
1. Detect DNA with a sequence
related to a DNA of known sequence.
2. Detect genes that encode proteins
of partially known sequence.
TB
904
transformant cells on filter paper
1. lyse cells
2. denature DNA
3. bind and detect probe
contains a clone with sequences
complementary to the probe TB
C. Complementation: How could genes of 905
interest be identified by complementation?
mutation
X
human DNA library
E. coli coenzyme B12 mutant
(can't make coenzyme B12)
Restoration of the wildtype phenotype by a second DNA molecule TB
IV. Expression vectors
906
A. Factors affecting protein expression
B. Typical expression vector
PO
Vectors used to
produce large
amounts of
protein.
gene for
regulatory
protein
ori
ApR
Expression vectors
907
Vectors used for the production of
proteins.
usually used to get a high
level of gene expression
TB
908
A. Factors affecting protein expression
1. Gene copy number
2. Promoter strength and regulation
3. Translation initiation
4. Codon usage
5. Protein and mRNA stability
TB
B. Typical expression vector
909
P O unique
restriction site
lacI gene
(encodes
repressor
protein) ori
P = promoter
O = operator
gene of
interest
selectable
marker
TB
910
Some repressor proteins mediate gene induction.
Lactose ( ) induces the expression of lac genes
or whatever genes follow the lac promoter.
CAP
site
+
normal lac operon
P O
lacZ
lacY
lacA
genetically engineered gene
P O
gene of interest
protein of interest
V. Polymerase chain reaction (PCR)
Process for producing large
amounts of DNA from a small
amount of template DNA.
A. applications
B. reaction components
C. procedure
911
A. PCR applications
912
amplification of small amounts
of DNA for
gene cloning
mutagenesis
amplification of related sequences
TB
B. PCR reaction components
913
4
(~10
template
molecules)
thermostable DNA polymerase
(Taq or Pfu polymerase)
17
(10
2 DNA primers
molecules)
the 4 deoxynucleotides
buffer
TB
The 2 DNA primers bind on opposite 914
strands of DNA
primers
template
Heat to separate strands
Cool to anneal to primers
5'
5'
Primer #1
Primer #2
TB
C. procedure
915
1. denature template DNA
DNA
polymerase
template
primers
denature at 94°C
TB
2 anneal primers
916
anneal at ~ 50ºC
primers bind by complementary base pairing
TB
3. extend with DNA polymerase
917
extend at 72ºC
4. repeat steps 1-3, ~ 35 times (35 cycles)
TB
second cycle
918
denature
TB
second cycle
919
anneal
TB
second cycle
920
extend
TB
35 cycles
template
921
final product
primers are incorporated into product
TB
Amount of product from 1 molecule
922
= (number of templates) x 2 (number of cycles)
= (1) x
35
2
= 3.4 x
10
10
molecules
34,000,000,000
TB
923
VI. Cloning and expression of
mammalian genes in bacteria
A. Problems
introns
large genomes
posttranslational modifications
(like glycosylation, attaching a sugar)
TB
One solution to the intron problem:
cDNA ("complementary DNA")
B. cDNA
924
mRNA
reverse
transcriptase
AAAA...
TTTT...
primer
AAAA...
TTTT...
alkali (removes mRNA)
TB
925
DNA polymerase
specific nuclease
cDNA
clone
TB
VII. Applications of genetic engineering
926
A. General uses
B. Mammalian proteins
C. Vaccines
D. Plants
E. Gene transfer to plants by bacteria
TB
A. General uses
Microbial fermentations
(eg. antibiotic production)
Vaccines
Mammalian proteins
Transgenic plants and animals
Environmental biotechnology
Gene therapy
927
TB
B. Mammalian proteins
Insulin
alpha-interferon
clotting factors
928
C. Vaccines
Hepatitis B
TB
D. Genetic engineering in plants
Herbicide resistance
Insect resistance
Disease resistance
Improved product quality
Production of pharmaceuticals
929
TB
E. Gene transfer to plants by bacteria930
cloned DNA
R
Kan
transfer
sequences
plasmid used for gene transfer
KanR = kanamycin resistance
TB
931
D-Ti
Plant cell genome
Agrobacterium
tumefaciens
Provides genes
needed for DNA
Transfer
regeneration
transgenic plant
TB
Study objectives
932
1. Understand the details of how antibodies, nucleic acid probes and
complementation are used to identify particular clones.
2. Know the main factors that affect protein expression from expression
vectors.
3. Understand the polymerase chain reaction, its uses, and the details
of the procedure presented in class.
4. Understand how cDNA is made and how it solves some of the problems
of cloning eukaryotic genes.
5. What are some of the applications of genetic engineering?
6. Understand how Agrobacterium can be used to transfer genes to plants.