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Chapter: Genetics and Molecular Biology: Lambda Phage Integration and Excision

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DNA Heteroduplexes Prove that Lambda Integrates

Davidson and Sharp directly demonstrated the insertion of lambda DNA into the genome using electron microscopy.

DNA Heteroduplexes Prove that Lambda Integrates

Davidson and Sharp directly demonstrated the insertion of lambda DNA into the genome using electron microscopy. They used a procedure developed to examine sequence homologies between strands from dif-ferent DNA molecules. After denaturation to separate strands of the two input DNA populations, the strands are allowed to anneal. Then the DNA is mixed with a cytochrome C solution to coat the molecules and increase their diameter as well as improve their staining properties. To spread out the long, snarled DNA molecules, a small volume of the DNA plus cytochrome C is layered on top of a buffer. As the protein spreads and forms a monolayer, the DNA molecules are stretched out and can then be picked up on an electron-transparent support and stained. Single- and double-stranded DNA visualized by this procedure are easily distinguished because single-stranded DNA is more flexible and there-fore curlier than double-stranded DNA.

When two homologous DNA strands reanneal, a simple double-stranded molecule results. A heteroduplex between a strand deleted of a stretch of sequence generates a single-stranded “bush” on the other strand, and if the two strands possess a stretch of sequence without homology, a “bubble” is formed. The heteroduplexes formed between an F’-factor containing an integrated lambda genome and lambda DNA showed the structure expected if lambda were to insert itself into the chromosome (see problem 18.4).


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