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

Isolation of Polyadenylated mRNA

Synthesis of cDNA

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.

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.

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

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.

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) 100-300 Yeast artificial chromosome (YAC) Yeast 100-2,000 Human artificial chromosome Cultured human cells >2,000

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

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

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

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 33-47 kb DNA in 5 kb cosmid vector

Cloning of Genomic DNA into Cosmid Vector

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

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

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)

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

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

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.