Dealing with DNA on a Large Scale 
Maynard V. Olson, Ph.D. — Investigator 
Dr. Olson is also Professor of Genetics at the Washington University School of Medicine. He was trained as 
a chemist, receiving his B.S. degree from the California Institute of Technology and his Ph.D. degree from 
Stanford University. After five years on the faculty at Dartmouth College, he moved to the University of 
Washington and changed fields from chemistry to genetics. He has served on the National Research 
Council Committee on the Mapping and Sequencing of the Human Genome and presently serves on the 
NIH Program Advisory Committee of the National Center for Human Genome Research. 
MOST human cells contain 6 billion base 
pairs (bp) of DNA. Embedded therein are 
an unknown number of genes, perhaps 100,000, 
that direct the biochemical events in the cells. At 
present, well over 95 percent of this DNA re- 
mains unexplored. 
Geneticists have developed powerful methods 
with which to study DNA in small packets. Gene- 
splicing techniques, DNA sequencing, and meth- 
ods of reintroducing altered DNA molecules into 
cells allow the detailed structural and functional 
analysis of DNA molecules containing up to tens 
of thousands of base pairs. Our laboratory seeks 
to extend these approaches to encompass mole- 
cules ranging up to millions of base pairs in size. 
In the short run, this research should allow the 
analysis of larger functional units of DNA — for 
example, large human genes, clusters of coregu- 
lated genes, and such structures as centromeres 
and telomeres, which govern the behavior of hu- 
man chromosomes during the cell division cycle. 
In the long run these methods should allow the 
systematic analysis of the whole human genome 
— the entire complement of DNA sequences — 
thereby creating tools, such as detailed maps, that 
would be of permanent value in biology. 
The heart of our approach is a new method of 
cloning large DNA molecules from any organism. 
DNA cloning, which was the root technology of 
the recombinant-DNA revolution of the 1970s, 
depends on splicing t-ecfor sequences onto other 
DNA molecules to create a new replicon — that is, 
a DNA molecule that will replicate inside a host 
cell, allowing large numbers of identical copies 
to be produced in cell culture. The replicons in 
the new cloning system are yeast artificial chro- 
mosomes (YACs). DNA sequences in the YAC 
vectors impart the properties of a true yeast chro- 
mosome to the foreign DNAs to which they are 
spliced. Once introduced into host yeast cells, 
the YACs replicate at each cell cycle during the 
growth of the host and segregate faithfully into 
the two progeny cells. 
One advantage of YACs over previous cloning 
systems is that there is no absolute upper size 
limit. At present, we can prepare large collec- 
tions of YAC clones, each containing a different 
segment of human DNA, averaging hundreds of 
thousands of base pairs in size, a 1 0-fold improve- 
ment over the capacities of previous cloning sys- 
tems. Another advantage is that the methods of 
packaging, maintaining, and replicating DNA are 
more similar in yeast than in bacteria to the analo- 
gous methods in cells of higher organisms. Con- 
sequently, it will likely be possible to propagate 
more segments of human DNA in yeast cells than 
in bacterial hosts. 
The first phase of this project has involved es- 
tablishing basic "library" technology for the YAC 
system. We have prepared more than 80,000 YAC 
clones containing segments of human DNA that 
average 250,000 bp in size. A typical segment of 
human DNA is represented six to seven times in 
this library on YACs of independent origin. We 
have also developed efficient screening methods, 
based on the polymerase chain reaction (PCR), 
which allow YACs containing panicular seg- 
ments of the human genome to be identified in 
the library. Screening can be carried out even if 
one's only prior knowledge of the segment is the 
sequence of a mere 100-200 bp of the DNA. 
The YAC library has yielded clones containing 
more than 200 different human genes. These 
clones are being employed in many collaborating 
laboratories for such purposes as aiding in the 
search for genes involved in specific human dis- 
eases. Examples include the successful search 
during the past year for the gene that is mutated 
in neurofibromatosis, carried out in part in the 
laboratory of Francis Collins (HHMI, University 
of Michigan). Andrew Feinberg (HHMI, Univer- 
sity of Michigan) has also used YACs to character- 
ize a large region of chromosome 1 1 that is impli- 
cated in the etiology of a childhood cancer, 
Wilms' tumor, as well as several developmental 
defects. YACs are also playing a key role in the 
worldwide effort to identify the gene that is mu- 
tated in Huntington's chorea, a fatal, adult-onset 
neurological disease. 
Current research is focused on improving the 
power of the YAC system for the functional and 
structural analysis of human DNA. Functional 
studies depend on the transfer of YAC clones 
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