Download Recombinant DNA and gene cloning To use an unique feature(s) of

Recombinant DNA and gene cloning
To use an unique feature(s) of your gene/gene product to isolate the DNA fragment
containing your gene from a library of DNA fragments.
Difficulty in isolating genes (needle in a long line of connected needles):
A gene is a small part of a large DNA (0.01% of an average chromosome);
DNA pieces all have same physical-chemical characteristic;
Unique feature of a gene/gene product (preconditions):
partial sequence of the gene (based on protein sequence)
activity (enzyme activity)
genetic defect (temperature/chemical sensitivity)
Strategy: 1) break up the DNA; 2) separated each fragement into a unique locations
(library); 3) screen your gene out from the library.
Tools
1) restriction endonuclease (restriction enzymes, recognize and make a stagger cut at a
symmetric DNA sequence of 4-8 bp)
2) DNA Ligase
3) bacterial plasmid (vectors): 1) ori, 2) antibiotic resistant marker gene, 3) cloning sites
that can be cut with a unique restriction enzyme
4) bacteria host
5) a genomic DNA library: a large collection of host strains, each contain a distinct
piece of DNA fragments on the plasmid vector. (The size of the collection is so big
that every gene of genome can be found in the library.)
Construction of genomic library
1) make random genomic DNA fragments to a certain size (> larger than the average
gene size using partial digestion with a restriction enzyme, so that at least some of the
fragments contain the intact gene)
2) Link the DNA fragments to the precutted DNA vector by ligase (recombinant)
3) Introduce the recombinant DNAs into bacterial (‘transformation’ by heatshock or
electroporation)
4) bacterial harboring recombinant plasmid grow on antibiotic solid media as single
colony/strain (DNA in the cells are identical “clone”)
5) the library- Sufficient large number of different bacterial colonies
Screening and isolation of your gene:
Hybridization screening:
1) grow mixture of bacterial strains on solid media at a density each strain grow as a
single colony
2) blot the colonies on nylon membrane, cell lysed and DNA denatured
3)
4)
5)
6)
use radioisotope labeled DNA piece (probe) to hybridize the membranes
identify the colony that light up on Xray film—your clone
go back to the original plate to recover the colony of bacterial
grow up large amount of the candidate bacterial cells and isolate the recombinant
plasmid DNA
7) sequence your gene.