Chapter 4 Recombinant DNA Technology

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

Partial Digestion with a Restriction Endonuclease

Effect of increasing the time

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(1 - 0.95) ln (1-f) ln (1 - 7.14 X 10-6) N = = = 4.2 X 105

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

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)

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

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.

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

Screening a Library by Colony Hybridization

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

Screening by Immunological Assay

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

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

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