BIOL 2030 Lecture Notes - Lecture 20: Hybridization Probe, Complementary Dna, David Baltimore
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Cloning Genes and mRNA Transcripts
March 14, 2016
Identification of specific DNA (gene) sequences by:
• Complementation
• Hybridization
Genome = all genetic information for an organism
• Restriction enzymes are used to cut DNA into fragments which are then spliced into a
complementary restriction site in a plasmid that contains antibiotic resistance
• Recombinant plasmids are used to transform an antibiotic sensitive strain under
conditions that ensure each bacterial cell receives no more than one plasmid
• The cells are grown on antibiotic containing solid nutrient medium; only those with
plasmid survive
Cloning by Complementation: requires a mutant strain of the bacterium or other host organism
• Host E. Coli with mutant in His-biosynthetic genes; these cells cannot synthesize
histidine and hence, cannot grow on minimal medium
• A plasmid carrying a copy of His+ biosynthetic gene is introduced to E. Coli: the cells
can now synthesize histidine and grow on a minimal medium
• Steps:
o Purify total DNA from wild type strain of E. Coli (genomic DNA w His+)
o Cleave E. Coli with restriction endonuclease
o Construct a genomic library with E. Coli DNA in a suitable cloning vector
o Transform a mutant strain that lacks ability to synthesize His
o Plate is transformed by bacterial mutant cells on minimal medium
o Colonies of cells that grow on plates are selected as they contain cloned histidine
biosynthetic gene
• Limitation: need mutant strain with complementary normal gene in biosynthetic
pathways; genes that code for structural proteins are harder to complement
Cloning by Hybridization
• Requires hybridization probe that is complementary (or partially complementary) to the
targeted cloned DNA sequence
• Bacterial colonizies are transferred to a nitrocelluloae filter
• Filter with bacteria are lysed and the DNA is denatured (using NaOH to break bonds and
bacterial cells)
• Radioactively labeled nucleic acid probe in added
• Some of the radioactive nucleic acid probe molecules become hybridized to the DNA of
some colonies
• Creates exposed x-ray film
Complexity of Eukaryotic Genome
• Intergenic region
• Introns
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