Johns Hopkins researchers have announced the
development of a Web-based automated computer program that
they say greatly simplifies the time-consuming and
error-prone process of manually designing artificial pieces
of DNA.
The program, called GeneDesign, guides the design of
blueprints for DNA segments to the exacting specifications
required for studying gene function and genetically
engineering cells. The blueprints are then used by
companies or other investigators to synthesize the gene.
A report on the program appears in the April issue of
Genome Research and online in mid-February. The
publicly accessible Web site can be found at
slam.bs.jhmi.edu/gd.
GeneDesign automates the process of determining which
base pairs — the building blocks of DNA —
should be linked together in a particular order to make a
gene, according to senior author Jef Boeke, professor of
molecular biology and
genetics and director of the High Throughput Biology
Center at the School of Medicine. A gene codes for a
specific protein, and the order of the hundreds or
thousands of base pairs making up that gene determines the
order of the amino acid building blocks making up that
protein.
"GeneDesign not only guides the user in designing the
gene but also automatically diagnoses design flaws in the
sequence of bases making up the gene," Boeke said.
Simplifying creation of so-called designer or
artificial genes is important because slight changes in the
choice of base pairs making up specific parts of the gene
can have significant effects on how the gene works and how
efficiently it can be inserted into cells. "In the past,"
Boeke said, "researchers had to use many different programs
to address all the requirements of the separate steps of
synthetic gene design."
The researchers have so far used GeneDesign to make a
variety of synthetic sequences, including a Ty1 element, a
mobile piece of genetic material found in yeast cells. Ty1
elements can move from one yeast cell and "jump" into a
specific spot in one of a second yeast's chromosomes. This
jumping movement can cause mutations or bring in additional
genetic material to the yeast.
GeneDesign consists of six modules that can be used
individually or in series to automate the tasks required to
design and manipulate synthetic DNA sequences. The program
allows the user to start with either the sequence of the
amino acid making up the protein or the bases making up the
gene that codes for that protein. Then the user moves
through a series of steps that guide the design of the gene
and vector that will carry the gene into the cell. Users
can follow the main "Design a Gene" path or use the modules
individually as needed. Vectors are mobile pieces of DNA
that are used to carry artificial genes into cells.
A major advantage to GeneDesign is the ability to
choose specific codons that work especially well in
specific organisms, Boeke said. A codon is a trio of bases
in a gene that codes for a specific amino acid building
block. Most amino acids are represented by more than one
codon. For example, the codons GCU, GCC, GCA and GCG can
each code for the amino acid alanine.
Human, bacterial and yeast cells often differ in the
codon they prefer to use for a particular amino acid.
"GeneDesign automatically chooses the best codon to use
depending on whether the gene is supposed to work in a
human cell, a bacterium or a yeast cell," Boeke said. "When
you're working with hundreds of codons, that's a
significant help."
The program also simplifies the design of genes that
will make proteins with desired, specific modifications
— for example, changes that make them work more
efficiently.
Another advantage of GeneDesign is ease of creating
restriction sites, places along the DNA where the gene can
be cut, said Sarah M. Richardson, a doctoral candidate in
the Department of Genetic Medicine and first author of the
paper. Scientists use molecular scissors called restriction
enzymes to make these cuts, which allow them to do the
cutting and pasting needed to put artificial genes into
vectors.
"GeneDesign guides the choice of the series of base
pairs where the restriction enzymes cut the DNA,"
Richardson said. "That lets investigators use different
restriction enzymes to make cuts exactly where they want
to."
If the same restriction site sequence occurred
throughout the gene, the specific restriction enzyme that
recognizes that site would make multiple cuts, according to
Richardson. "That would make it impossible to do the
precise cutting and pasting needed to make and use
artificial genes," she said.
However, even a successfully designed gene would not
benefit researchers if there were only one copy of it. "To
make use of artificial genes, we need to make millions of
copies of them for experiments using a process called
polymerase chain reaction," Boeke said. "By putting
restriction sites into specific spots along the gene, we
can cut it into bite-sized pieces that are easily
duplicated millions of times. So the ability to cut and
paste genes back together again is critical for designing
genes to the right specifications, rapidly replicating them
and putting them into vectors to genetically engineer
cells."
The other authors of the paper are Sarah J. Wheelan
and Robert M. Yarrington, both of the High Throughput
Biology Center.
This work was supported by National Institutes of
Health grants, including a Research Roadmap grant.