1. Chapter 4 Recombinant DNA Technology

2. Making a Gene Library (Cloned Bank, Gene Bank)
Creating a library
Subdividing genomic DNA into clonable elements and inserting them into host cells
A complete library
Library containing all of the genomic DNA of the source organism
DNA fragmentation by partial digestion
Low concentration of restriction enzyme
Shortened incubation time
Library size
> 3 times the amount of DNA in the genome
4X 106 genome, 1000 bp insert  require 12,000 clones

3. Partial Digestion with a Restriction Endonuclease

4. Effect of increasing the time

5. Determination of Library Size
Genome size: 2.8 X 106 kb
Size of random DNA fragment: 20 kb
f: ratio of the length of the average insert to the size of the entire genome
20 / (2.8 X 106) = 7.14 X 10-6
N : Number of independent recombinants
P: Probability of including any DNA in a random library of N
ln(1-P) ln( )
ln (1-f) ln ( X 10-6)
N = =
= 4.2 X 105

6. Screening Strategies
After a library is created, the clones with the target sequence must be identified.
Sequence-dependent screening
Use DNA hybridization of homologous sequences
Screening of expression library
Screening by immunological Assay
Screening by protein activity

7. Screening by DNA Hybridization
Denaturation
Breaking base pairing by heating of alkali treatment
Renaturation
Annealing of denatured strands by slow cooling
DNA hybridization
Attachment of denatured ss target DNA on membrane (nitrocellulose, nylon)
Annealing with labeled single stand probe (100 to 1000nt)

8. DNA Hybridization Denaturation
Immobilization (e.g. nitrocellulose membrane)
Hybridization with labeled ssDNA probe
Stable binding requires a greater than 80% match within a segment of 50 bases
Autoradiography

10. Possible Sources of Probes
Cloned DNA from a closely related organism (heterologous probe)
Figure 4.15 (production of labeled probe DNA by the random primer method)
Chemical synthesis
based on the probable nucleotide sequence that is deduced from the known amino acid sequence of the target protein.

11. Labeling of DNA Probes Using Random Primers
dNTPs, one of which is labeled with a tag (*)

12. Screening a Library by Colony Hybridization

13. Identifying the Colony Containing the Target gene
Because most libraries are created from partial digestions, a number of colonies may give a positive response to the probe.
 Need to determine which clone contains the complete sequence of the target gene.
By gel electrophoresis
restriction endonuclease mapping
By DNA sequencing

14. Screening by Immunological Assay

15. Detection of Ag-Ab Reaction by Chemiluminescence
HRP (horseradish peroxidase)-Conjugated Secondary antibody
Luminol
HRP
Primary antibody

16. Screening by Protein Activity
e.g. screening by enzyme activity assay

17. Functional (genetic) complementation
Complementation of mutant growth on specific medium