1. steps
• Isolation
• Insertion of target DNA into vector
• Cloning vectors
• Isolation and identification of recombinant
genes
• Expression of cloned genes
5. Step 3-Splice (or ligate) DNA into some kind of cloning vector to
create a recombinant DNA molecule
Six different types of cloning vectors:
1. Plasmid cloning vector
1. Phage cloning vector
2. Cosmid cloning vector
3. Shuttle vectors
4. Yeast artificial chromosome (YAC)
5. Bacterial artificial chromosome (BAC)
6. Fosmid cloning vector
6. Cloning Vector Types
• For different sizes of DNA:
– plasmids: up to 5 kb
– phage lambda (λ) vectors: up to 50 kb
– BAC (bacterial artificial chromosome): 300 kb
– YAC (yeast artificial chromosome): 2000 kb
• Expression vectors: make RNA and protein
from the inserted DNA
– shuttle vectors: can grow in two different species
7. Plasmid Cloning Vectors:
Bacterial plasmids, capable of replicating autonomously.
Plasmid vectors used in cloning.
Feeatures (e.g., E. coli plasmid vectors):
1. Origin sequence (ori) required for replication.
2. Selectable trait that enables E. coli that carry the plasmid to
be separated from E. coli
3. Unique restriction site
4. Simple marker allows to distinguish plasmids that contain
inserts (e.g., lacZ+ gene)
11. Some features of pUC19 plasmid vector:
1. High copy number in E. coli, ~100 copies/cell, provides high yield.
2. Selectable marker is ampR
.
3. Cluster of several different restriction sites
4. Add X-gal to medium; turns blue in presence of -galactosidase.
5. Plaque growth: blue = no inserted DNA and white = inserted DNA.
6. Plasmids are transformed into E. coli by chemical incubation or
electroporation (electrical shock disrupts the cell membrane).
7. Good for <10kb; Cloned inserts >10 kb typically are unstable.
14. Phage cloning vectors:
1. Engineered version of bacteriophage (infects E. coli).
2. Central region of the chromosome (linear) is cut with a
restriction enzyme and digested DNA is inserted.
3. DNA is packaged in phage heads to form virus particles.
4. Phages with both ends of the chormosome and a 37-52 kb
insert replicate by infecting E. coli.
5. Phages replicate using E. coli and the lytic cycle (see Fig. 3.13).
6. Produces large quantities of 37-52 kb cloned DNA.
7. Like plasmid vectors, large number of restriction sites available;
phage cloning vectors useful for larger DNA fragments than
pUC19 plasmid vectors.
16. Cosmid cloning vectors:
1. Features of both plasmid and phage cloning vectors.
2. Do not occur naturally; circular.
3. Origin (ori) sequence for E. coli.
4. Selectable marker, e.g. ampR
.
5. Restriction sites.
6. Phage cos site permits packaging into phages and
introduction to E. coli cells.
7. Useful for 37-52 kb.
17. Shuttle vectors:
1. Capable of replicating in two or more types of hosts..
2. Replicate autonomously, or integrate into the host genome and
replicate when the host replicates.
3. Commonly used for transporting genes from one organism to
another (i.e., transforming animal and plant cells).
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Example:
*Insert firefly luciferase gene
into plasmid and transform
Agrobacterium.
*Grow Agrobacterium in large
quantities and infect tobacco
plant.
18. Yeast Artificial Chromosomes (YACs):
Vectors that enable artificial chromosomes to be created and cloned
into yeast.
Features:
1. Yeast telomere at each end.
2. Yeast centromere sequence.
3. Selectable marker (amino acid dependence, etc.) on each arm.
4. Autonomously replicating sequence (ARS) for replication.
5. Restriction sites (for DNA ligation).
6. Useful for cloning very large DNA fragments up to 500 kb; useful
for very large DNA fragments.
19. Bacterial Artificial Chromosomes (BACs):
Vectors that enable artificial chromosomes to be created and cloned
into E. coli.
Features:
1. Useful for cloning up to 200 kb, but can be handled like regular
bacterial plasmid vectors.
2. Useful for sequencing large stretches of chromosomal DNA;
frequently used in genome sequencing projects.
3. Like other vectors, BACs contain:
1. Origin (ori) sequence derived from an E. coli plasmid called
the F factor.
2. Multiple cloning sites (restriction sites).
3. Selectable markers (antibiotic resistance).
20. Fosmid:
1. Based on the E. coli bacterial F-plasmid.
2. Can insert 40 kb fragment of DNA.
3. Low copy number in the host (e.g., 1 fosmid).
4. Fosmids offer higher stability than comparable high copy number
cosmids. Contain other features similar to plasmids/cosmids
such as origin sequence and polylinker.
21. Isolation and identification of
recombinant genes
• Colony hybridization (replica method)
• Hybrid release translation method
• Immunochemical detection
22. Replca plate method
Clones on agar plates
Replca on nitro cellulose membrane
Lyse cells and denature DNA
with alakli and fix by baking
Immobilised DNA
Colony with complementary DNA
Mark with radio active
ink
Incubate 32p a 650
c wash ,
expose to xray
23. Hybrid released translation
method
Double stranded cDNA
Single standed cDNA
mRNA of desired protein
mRNA- Cdna hybrid
Double stranded DNA
denaturation
Single stranded DNA
Used for isolation of rdna gene
As probe
24. Immunochemical detection
method
• Fixed cell lysate with primary Ab on
polyvinyl sheet
• Lysed ag
• Replca
• Exposed to secondary ab
• Autoradiography color change
25. Expression of cloned genes
• Post translational modifications
– Folding
– Secretion
– Modifications
