Finding a single piece of DNA that carried a specific sequence was hopeless. For organisms with large genomes, however, this procedure generated a smear of DNA because of the millions of fragments. The pieces would migrate at different rates, depending on size. They could then separate the resulting pieces by loading the collection onto an agarose gel and applying an electric current. Scientists knew that they could chop up DNA using restriction enzymes, proteins that cut DNA at particular sequences. In the mid-1970s, Ed Southern (at the Medical Research Council Mammalian Genome Unit in Edinburgh) wanted to develop a method that would pinpoint a particular gene amidst the more than a billion building blocks - or basepairs - that compose the frog Xenopus laevis genome. This situation severely restricted efforts to define genetic differences that characterize species, individuals, and specific cell types, thus hampering the study of subjects as diverse as evolution and the physiological characteristics of distinct tissues. Until the mid-1970s, the ability to locate most genes or sequences of interest on the chromosomes of complex organisms was nearly impossible. Technology has always defined the strength of genetic analysis. Its ability to establish family relationships as well as individual identity has helped solve crimes, settle paternity and immigration disputes, establish the bases of inherited diseases, enhance transplantation biology, save endangered species, establish human origins and migrations, and advance countless other beneficial endeavors. Using this technology, Alec Jeffreys devised 'genetic fingerprinting', a way to distinguish every person from every other person, except an identical twin. Suddenly scientists could study genetic variation in detail and decipher gene structures. By inventing a method for detecting specific DNA sequences amidst the huge genomes of complex organisms, Edwin Southern infused genetic analysis with tremendous power. Biotechniques 5, 638-650.The 2005 Albert Lasker Award for Clinical Medical Research honors two scientists who revolutionized human genetics and forensic diagnostics. Research applications of transgenic mice. For an example of copy standards in Southern blots, refer to Camper SA. Remember to reserve one lane for genomic DNA only with no spike. Thus, to prepare a 1 copy standard: add 1.83 pg of transgene DNA to 2 microgram tail DNAįor use as a transgene PCR standard, use 200 ng of the spiked tail DNA as a substrate in a 25 ul PCR reaction as described: genotyping transgenic mice.įor use in Southern blot analysis, digest the tail DNA as you would for Southern analysis, and add the transgene insert DNA (not the entire plasmid) just before you load your gel. Transgenic mice will be hemizygous for the transgene, not homozygousġ.83pg transgene should be added to 2 ug of genomic DNA Mass of transgene DNA = 1.83 picograms per 1 ug genomic DNA Mass of transgene DNA = (5,480 bp cloned DNA) X (1 µg genomic DNA) or Mass of transgene DNA = 5,480 bp cloned DNA orġ micrograms genomic DNA 3 X 10 9 bp genomic DNA Mass of transgene DNA = N bp transgene DNAġ microgram genomic DNA 3 X 10 9 bp genomic DNAĮxample: for a 5,480 bp transgene insert or plasmid Since the transgenic founder mice are hemizygous: Assumption: the Haploid content of a mammalian genome is 3 X 10 9 bpĪssumption: you have 2 micrograms of tail DNA available
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