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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