Genome Project, the international research program to map and sequence
all the human genes, is an immense scientific effort that has made
demands on biological research techniques and led to new biological
tools. Among the many developments resulting from genomic research
is one by a Livermore team that can find minute changes in the DNA
of individual cells and thereby significantly improve the detection
of cancer and other diseases.
new technique, called in situ rolling circle amplification (IRCA),
is a fast and inexpensive method to precisely locate a damaged or
abnormal gene that indicates the presence of or tendency toward
a particular disease. IRCA can find a single cell containing an
abnormal gene from among thousands of cells, making the technique
ideal for tissue biopsies. The process is so sensitive that it can
detect a mutation in a single DNA base, the smallest unit of genetic
information. (An average human cell contains about 6.5 billion DNA
The technique involves locating
the gene of interest in intact cellsin which fluorescent molecules
have been incorporatedand massively amplifying (duplicating)
critical sections of DNA so a fluorescent signal can be detected.
No other method is available that can detect a single DNA or RNA
base change within a cell or tissue.
IRCA is a way of putting
our newly gained knowledge of the human genome to beneficial use,
says team leader Allen Christian, a molecular biologist and chemical
engineer. The discovery, he says, moves the research findings of
Human Genome Project from the laboratory to the clinic. The
genome project has given us the sequences of all the human genes.
Our job as scientists is to apply that knowledge. We know that certain
DNA sequence variations signal a diseased state, so we can use that
information to diagnose disease at the earliest stages, when treatment
The Livermore research
team, which worked for about a year on the process, included biologist
Jim Tucker and technicians Melissa Pattee and Christina Attix. (Christian
and Tucker were part of a Livermore team that won an R&D 100
Award last year for gene recovery microdissection, a process that
identifies expressed genesthat is, genes that have been turned
onfrom a specific chromosome region.)
The Livermore breakthrough
is a significant extension of rolling circle amplification (RCA),
which was developed at Yale University in the mid-1990s. RCA is
limited to identifying DNA that has been extracted from a cell.
In contrast, the Livermore technique works inside cells, thereby
preserving the chemical environment of the cell and its neighbors.
In addition, IRCA provides answers in a couple of hours, compared
to a wait of several days required with tests using traditional
methods. The technique can also be used to detect and measure messenger
RNA in single cells, something that could not be done previously.
(Genes produce proteins with the aid of messenger RNA.)
|The team that developed the
in situ rolling circle amplification technique. From left, Allen
Christian, Melissa Pattee, Jim Tucker, and Cristina Attix.
Synthetic Probes Start the Process
The technique starts with the laboratory production
of linear probes (or stretches) of DNA encompassing about 100 bases.
The DNA has been treated to remove one of its double strands. The
probes are applied to target DNA under study, which also have been
made single-stranded. Because DNA prefers to be double-stranded,
the probe will seek out a target whose DNA sequence is a counterpart
to that of its own.
In the cell nucleus, the
linear probes two ends attach through hydrogen bonds to the
target DNA and in the process wrap around that DNA to form a circle.
An enzyme called a ligase then padlocks the circular
probe onto the target DNA by forming chemical bonds that are much
firmer than hydrogen bonds. In this way, the probe is prevented
from detaching from the target.
Typically, two probes are
applied to the target DNA:
a normal sequence probe and a mutant sequence probe. The normal
probe can only attach to the normal DNA sequence, and the mutant
probe can only attach to a mutant sequence.
Each probe contains a DNA
sequence that allows a short piece of DNA, called a primer, to initiate
a reaction that quickly and repeatedly replicates the probe. The
duplicating reaction is catalyzed by a polymerase enzyme, which
rolls out hundreds or thousands of linear copies of the circular
probes DNA. The single strands of targeted DNA make it easier
for the polymerase to make copies of the probe.
Every time the probe is replicated,
a binding site for a fluorescently labeled molecule (or beacon)
is created. Within
a few seconds, enough beacon sites are created to allow the fluorescent
signal to be seen under a microscope and permit
a normal base to be distinguished from an abnormal base. Were
in essence sending up signal balloons that help us to detect the
products of the polymerase reaction, says Christian.
The match between a normal
probe and target produces a green signal after amplification, whereas
a match between a mutant probe and target produces a red signal.
If the probe and the target do not match, there is no signal because
the ligase will not work.
The in situ rolling circle
amplification technique reveals a mutated tp53 gene in a human
lymphoblastoid cell by coloring it red, whereas a normal gene
is colored green.
has numerous medical applications, especially in the diagnosis and
treatment of diseases that have genetic markers. Christian expects
that one of the techniques first routine clinical uses will
be as a fast, inexpensive assay for the presence of mutations that
are relevant to a particular cancer. IRCA will allow physicians
and medical researchers to identify and localize genes that are
known to be or strongly suspected of being responsible for causing
certain cancers, says Christian. The technique will also help
physicians to choose and customize cancer therapies for their patients
and monitor the effectiveness of therapies.
Christian notes that many
cancer diagnostic procedures currently in use involve the same dyes
and stains that were first discovered more than 100 years ago. These
dyes and stains cannot detect the subtle genetic changes that are
believed to occur when tissue first becomes cancerous.
On a different application,
IRCA could be used to identify
the strain of bacteria infecting a person. This capability would
enable physicians to select the most effective antibiotics. Similar
approaches may work for detecting viruses ranging from the common
cold to hepatitis, herpes, and HIV.
IRCA also has applications
in agriculture, toxicology, pharmacology, and environmental science.
The technique will assist scientists in rapidly identifying which
strains of a plant, tree, or vegetable have desired genetic characteristics.
In this way, IRCA has the potential to improve the quantity and
quality of food. In basic cell research, the advance could help
to determine the genetic composition of bacterial, plant, and human
cells. For pharmacology, the technique will provide an important
tool to test promising new drugs and measure cells responses
technique may also have an important role to play in fighting bioterrorism.
Portable detectors using IRCA may offer advantages over units using
the polymerase chain reaction technology to amplify short stretches
of DNA or RNA and thereby identify a potential bioagent.
reports that the research team has been flooded with calls and e-mails
from researchers across the country since the procedure was first
described in a paper published in Proceedings of the National Academy
of Sciences in 2001. The Laboratory is currently negotiating with
companies to license the process.
IRCA stands to improve human health and advance a large number of
Key Words: cancer
detection, in situ rolling circle amplification (IRCA), R&D
For further information contact Allen Christian (925) 424-5909 (firstname.lastname@example.org).