every cell in the human body contains the same set of genes. But
not all of the genes are used, or expressed, by those cells. For
example, some processes that are particular to cells in the liver
are completely unused in brain cells. Ever since genomic research
began, scientists have been searching the tangle of DNA for the
expressed genes, the ones that really matter.
If one thinks of the nucleus
of a cell as a library, then the chromosomes in the cell are bookshelves
and the genes are the books on each shelf. Almost every cell in
an organism contains the same libraries and the same sets of books.
The books represent all of the information (the DNA) that every
cell in the body needs so that it can grow and carry out its various
functions. Two challenges complicate the process of locating our
genes: Not all of the genes are expressed in any one tissue, and
less than 10 percent of our DNA is actually used to make genes.
Only occasional passages in the librarys written material
A team at Livermore led
by molecular biologist Allen Christian has developed Gene Recovery
Microdissection (GRM), a process that can weed out the unexpressed
genetic material from a piece of DNA. With GRM, scientists can
isolate all of the genes in a chromosomal region that are being
used by a specific tissue at any point in time. GRM can be used
for any plant or animal species. A variant of this method can
also be used to clone all of the DNA of any organism, including
bacteria, even those that cannot be cultured.
Its not always
necessary to sequence the entire genome of a species to locate
its gene, says Christian. With GRM, we can focus on
particular regions of a genome that are of interest.
Twice Does the Trick
The product of gene expression
is messenger RNA (ribonucleic acid), or mRNA. Typically, before
work begins to isolate expressed genes, the mRNA molecules are
converted into more stable complementary DNA molecules called
cDNA, which has exactly the same sequence as the mRNA but is easier
to handle in the laboratory. Then the cDNA is combined on a microscope
slide with chromosomes. The cDNA molecules hybridize to the chromosome
regions corresponding to the genes of which their parent mRNA
is a product. Using tiny glass needles and microdissection, scientists
can isolate regions of the chromosomes of interest and, with them,
the hybridized cDNA molecules. Finally, amplification by polymerase
chain reaction (PCR) is used to produce many copies of the molecules
in preparation for DNA sequencing.
The basic technique of
using microdissection to isolate genes has existed for about five
years. But no commercially available gene libraries have been
generated because of inefficiencies in the hybridization and subsequent
PCR amplification processes. Because genes are typically represented
only once in a chromosome, a maximum of one cDNA molecule will
be present for each expressed gene following microdissection.
Successful hybridization, dissection, and PCR amplification of
a single molecule is virtually impossible. Gene libraries made
with this procedure are too incomplete to be useful.
Livermores GRM process
overcomes this inefficiency by combining cytogenetics and genomics
with chromosome microdissection. CRM increases both the number
of targets available for cDNA hybridization and the total number
of cDNA molecules in each region following hybridization.
The trick is to perform
PCR amplification in situ, on the slide rather than in a tube,
which is the conventional means. And it occurs twice. First, prior
to hybridization, random-primed PCR of the chromosomes on the
slide produces many copies of the target DNA, significantly improving
the chances of cDNA hybridization. Second, following the hybridization,
another PCR amplification using primers specific for the ends
of the cDNA molecules increases the numbers of bound cDNA molecules.
Instead of isolating just one cDNA molecule per expressed gene
in a region, the GRM process recovers hundreds or even thousands
of cDNA molecules. This simple step makes possible the production
of highly useful chromosome-region-specific libraries.
GRM has other advantages.
While cells generally contain only one or two copies of a gene,
some genes make thousands of copies of mRNA and others make only
a few copies. Finding mRNA molecules with a low number of copies
amid the noise of the more numerous gene products
can be difficult with conventional methods of making cDNA libraries.
But the hybridization step in GRM results in a balanced library
in which mRNA molecules with high and low numbers of copies are
Several companies offer
processes that provide partial information about gene expression
and genomic location. But
no other single technique identifies both known and unknown expressed
genes and determines the part of the genome that regulates their
expression. GRM makes possible in one process what multiple processes
could previously handle only in part, and it does so cost effectively.
Current estimates are that the costs associated with GRM will
be substantially less than those of traditional methods. The process
is also significantly faster.
amplified by Gene Recovery Microdissection. Amplification
is by polymerase chain reaction and produces many copies of
stable complementary DNA molecules in preparation for sequencing.
GRM was invented
to allow researchers to identify cancer genes in chromosomal regions
for which no genomic information existed. Initially, these were
regions for which scientists had good evidence of their importance
in rat mammary cancer but almost no other knowledge. To identify
the genes expressed in these regions, researchers needed a quick,
simple, inexpensive, and reliable method of identifying and characterizing
both new and previously known genes in chromosomes.
GRM focuses on data that
current genomic sequencing efforts do not provide, namely, information
concerning the expression of genes in specific regions of abnormal
cells, such as those found in cancerous tissue. We are using
GRM to learn which genes are expressed in certain parts of chromosomes
in cancer cells, says Christian. We can then compare
our data with data from the Human Genome Project and learn how
these particular cancer cells differ from normal cells.
will be used to generate chromosome-specific
and chromosome-region-specific libraries of genes that are expressed
for any tissue, normal or diseased, of any organism that can have
its chromosomes spread on a microscope slide. Once these libraries
have been produced, they can easily be placed on microarrays and
made available to other investigators for more detailed analyses,
including gene expression studies. GRM can thus be used to create
a systematic approach to identifying genes expressed in virtually
every species of interest to humans. This capability opens the
door to sequencing many plant and animal species that might otherwise
be ignored because of the prohibitive cost of genomic analysis.
Agriculture, environmental sciences, and veterinary medicine will
GRM technology provides
the preliminary step toward a full genomic analysis of an organism,
allowing time and money to be saved during the full analysis.
This invention will enable scientists to identify genes that are
expressed after exposure to drugs, environmental chemicals, or
radiation. Toxicologists can study the reactions of cells and
organisms to chemical and radiation exposure, furthering basic
understanding of the molecular mechanisms involved in responses
to adverse environments. Similarly, the pharmaceutical industry
will be able to decipher biological responses to drugs.
Gene Recovery Microdissection (GRM), genomic research, R&D
information contact Allen Christian (925) 424-5909 (firstname.lastname@example.org).