Management of the 

 Human Genome 

 Program 



Physical Mapping 



Human DNA fragments obtained 

 from rodent somatic cell hybrid 

 background separated on agarose 

 gels. A primer designed to recognize 

 human Alu sequences is used for 

 rapid amplification of regions of 

 fiuman DNA in rodent/fnuman somatic 

 cell hiybrids^ Rodent/human hybrid 

 cells are constructed and used in 

 human genome studies because they 

 contain manageable amounts of 

 human DNA in which genome 

 regions of interest can be manip- 

 ulated and characterized. The TC-65 

 oligonucleotide primer was designed 

 to recognize the human, but not the 

 rodent, Alu sequences and provides 

 specific amplification of human DNA 

 between regions of these ubiquitous 

 Alu sequences when polymerase 

 chain reaction (PCR) methods are 

 used. [Alu sequences are about 

 300 bp long and repeated thousands 

 of times in the human genome.) The 

 specificity of the TC-65 primer is 

 demonstrated in the figure: DNA 

 fragments of total human genome 

 (lane 2) and fragments of different 

 rodent/human hybrid cell lines have 

 been amplified and separated by gel 

 electrophoresis (lanes 3-12). Note 

 the abundance of bands (white) of 

 DNA fragments in lanes 2-12 and the lack of fragments in lanes 13 and 14, where pure rodent genome samples were 

 electrophoresed. No human Alu repeat sequences are found in the rodent genomes, and the rodent Alu equivalent 

 sequences are not amplified; this TC-65 phmer/PCR method is thereby validated. Lane 1 contains standard DNA fragments 

 of known size for determining sizes of DNA fragments in the other lanes. 



This method is useful for rapid comparison of hybrid cell lines' DNA content and overlap and can also be used in 

 preparation of nucleic acid probes from cloned human DNAs — especially for clones in yeast artificial chromosome (YAC) 

 vectors. [Photograph was first published in Proc. Natl. Acad. Sci. USA 86. 6686-6690 (1989). Photograph provided by 

 David L Nelson and C. Thomas Caskey, Baylor College of Medicine.] 



TC-65 



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