1. Cloning DNA Sequences that Encode Eukaryotic Protein
Eukaryotic --- gene contains introns.
--- mRNA does not have introns.
mRNA  cDNA
mRNA --- G cap at 5’ end
(methylated guanine nucleotide
which is joined after transcription)
--- poly(A) tail at 3’ end

2. Isolation of Polyadenylated mRNA

3. Synthesis of cDNA

4. Synthesis of cDNA Klenow fragment RNase H hydrolyzes mRNA.
a product of proteolytic digest of the DNA polymerase I
polymerase activity
3’ exonuclease activity (Figure 4.16)
no 5’ exonuclease activity
RNase H hydrolyzes mRNA.
S1 nuclease removes hairpin loop.

5. Synthesis of cDNA
The final sample contains a mixture of partial and complete ds cDNA.
Much time and effort are spent on identifying clones of a cDNA library with full-length sequences.
Various strategies have been devised to overcome this inconvenience.

6. Cloning of Full-Length cDNA Molecule
Restriction site
5-methyl-dCTP
(Methylation protects cDNA from
cleavage by restriction enzyme.)
Biotin
Streptavidin-coated magnetic bead

7. Cloning of Full-Length cDNA Molecule
: Restriction site
* RNase H removes any RNA that escaped the previous treatment.
* DNA ligase joins DNA segments that were synthesized internally
from bits of mRNA that escaped degradation.

8. Vectors for Cloning Large Pieces of DNA
Vector system
Host cell
Insert capacity (kb)
Plasmid
E. coli
0.1-10
Bacteriophage l
l / E. coli
10-20
Cosmid
35-45
Bacteriophage P1
80-100
BAC
50-300
P1-derived artificial chromosome (PAC)
Yeast artificial chromosome (YAC)
Yeast
100-2,000
Human artificial chromosome
Cultured human cells
>2,000

9. Bacteriophage l 48.5 kb linear DNA Cohesive ends with 5’ 12 nt
cos site
Circularization after infection

10. Life Cycle of Bacteriophage l
Lytic cycle
Cell lysis and release of phage particles
Lysogenic cycle
Prophage state
Integration of DNA into host genome (lysogen)
Induction of lytic cycle by nutrient or environmental stress

11. Bacteriophage l Vector
Insertion or replacement
Plaque formation after up to 25% deletion
Integration-excision (I/E) region
l Vectors
Left arm (20 kb)
Stuffer (14 kb)
Right arm (9 kb)
mcs
Genes for head and tail
Genes for replication and cell lysis

13. Cosmid Vector Cosmid vector l vector vs. cosmid vector
Plasmid with l cos sites
l vector vs. cosmid vector
DNA Size for packaging: 38~52 kb (75-105% of l DNA)
l Phage vectors:
Limitation for the deletion of essential genes
Cosmid vectors:
Accommodate kb DNA in 5 kb cosmid vector

14. Cloning of Genomic DNA into Cosmid Vector

15. High-Capacity Bacterial Vector Systems
Bacteriophage P1-derived artificial chromosomes
100~300 kb of insert DNA
Bacterial artificial chromosome (BAC)
50~300 kb of insert DNA
F-plasmid-based DNA insert-vector construct

16. BAC: Bacterial Artificial Chromosome
Derived from E. coli F (single
copy sex factor) Plasmid
Used to generate genomic
library with average size of
125 kb

17. Genetic Transformation of Prokaryotes
Chemical method: CaCl2 and heat shock
Transformation frequency
10-3 (one transformed cell/1000 cells)
Transformation efficiency :
107 to 108 (transformed colonies / μg of intact plasmid)
Competent cells
cells that are able to take up DNA (plasmid)

18. Genetic Transformation of Prokaryotes
Electroporation
Electric field-mediated membrane permeabilization
Effective way for large plasmids ( > 100kb)
E. coli
electric pulse of 25mF, 2.5 kV, 200 ohms for 4.6 ms
Transformation efficiency : 106 transformants (for ~136 kb) to 109 transformants (for ~3 kb) /mg DNA

20. Conjugation Conjugative function ~ cell-to-cell contact
Mobilizing function ~ DNA transfer
Objective
transfer a plasmid to a cell that is not readily transformed
transfer the plasmid cloning vector from strain B to strain C with the help of strain A

21. Conjugation Strain A contains a conjugative, mobilizing plasmid.
Strain B contains a mobilizing plasmid.
Strain C grows on minimal medium.
Culture in complete growth medium (w/o kanamycin) for conjugation
Selection in minimal medium (with kanamycin) for strain C containing the target plasmid
Occasionally, the targeted recipient cell may receive both types of plasmids. These cells can be excluded by antibiotic selection.