2. Index
1. Gene library
2. Types of gene library
3. Difference between genomic and cDNA library
4. Requirments of genomic library
5. partial RE digestion
6. Screening
3.
4. Gene library
•Large collection of DNA fragments cloned from a specific organism, tissue, organ
or cell type. Collectively, the clones may represent the complete genomic sequence
of the organism.
5. Types of gene library
1. Genomic library: Library produced by cloning random fragments of a genome
6. Types of gene library
1. Genomic library: Library produced by cloning random fragments of a genome
2. cDNA library: A library of complementary DNA sequences formed from
transcribed mRNA
7.
8.
9.
10. Requirements of genomic libraries
1. Size of the library: Must contain a certain number of recombinant to ensure the
presence of a particular sequence
2. Genomic DNA: Fragmented randomly by physical shearing or restriction
enzyme digestion to give a size range appropriate for the chosen vector.
Sometimes, combination of restriction enzymes used to partially digest the DNA
3. Vectors: Plasmid, bacteriophage, cosmid etc. are used. Choice of vector depends
on the genome size. Ligated by T4 DNA ligase
11. Partial RE digestion of DNA
•Digestion with RE for different times/different concentration
•Larger the size of the fragments, the less number of clones required
12. Screening
•The process of searching through a library or other collection of recombinant
transformants in the attempt to identify a clone containing a recombinant vector
having at least a portion of the gene recognized by the probe.
•Screening may include:
1. Phenotypic screening: The protein product changes color of the colony
13. Screening
•The process of searching through a library or other collection of recombinant
transformants in the attempt to identify a clone containing a recombinant vector
having at least a portion of the gene recognized by the probe.
•Screening may include:
1. Phenotypic screening: The protein product changes color of the colony
2. Antibiotic resistance: Gene encodes resistance to antibiotic
3. Immunoscreening: Using antibody (Ab) that can recognize the protein
produced by the gene
16. Screening by functional complementation
•Requires a strain unable to produce the desired product/show desired function
•Cloned DNA will contain the gene for the product
•Selection for restoration of lost product
17.
18. Cdc mutant = Cell division cycle mutant
Ura3 = an enzyme that catalyzes one reaction in the synthesis of pyrimidine ribonucleotides (a component of RNA)
