Biomedical Engineering Reference
In-Depth Information
catenated form the tag. Each of these blocks of distinct address sequences
should exhibit no cross-hybridization under stringent conditions (a Hamming
distance of at least 5), thereby enabling independent encoding and primer exten-
sion and therefore interrogation of up to 20 distinct attributes, each with up to 10
scalar values.
Use of the primer-extension method for logical queries into the Biomolecu-
lar Database is most efficient if performed in solution rather than on a microar-
ray. This creates a challenge in terms of product detection and identification.
The following method enables both to be accomplished. An example is pre-
sented for the fate of one molecule, though it is appreciated that all molecules in
the library are subjected to the same process in parallel. The database member is
a DNA molecule that has been created by the methods described earlier, with a
biological DNA sequence flanked by one or more created tag sequences, which
are to be the templates in a primer-extension reaction. The "bottom" strand is
interrogated in this example. Primers with the following structure (shown in
expanded form below the database element in Figure 4) are created to interro-
gate each tag. Complements to the address sequence in the tag/template are the
same in each primer. Also common to each primer is a "barbed tail" in the form
of a 5'-psoralen group. The irradiation of psoralens with long-wavelength UV is
widely used to cross-crosslink duplex DNA (31,43,64). The message sequence
Figure 4 . Obtaining a bottom strand in single-strand form, hybridized to a cDNA microarray,
via photochemical cross-linkage of psoralen.
must be unique to each variable value, meaning that up to 10 primers are pre-
pared per variable. The primer is also designed to address a unique X base in the
tag/template to be interrogated. The primer-extension reaction is performed us-
ing a dideoxynucleotide terminator complementary to X and bearing a fluores-
cent dye with a unique and readily imaged emission spectrum. The dye color is
specific to the variable, with the same dye/terminator being used for all interro-
gations of that variable. Multiple tags can be interrogated simultaneously be-
cause their dyes are different. The challenge at this stage is to read out the tags
(based on the color(s) of the incorporated fluorescent dye(s)) in the context of
the biological DNA. While the primer is still bound to the template, the psoralen
Search WWH ::




Custom Search