Biomedical Engineering Reference
In-Depth Information
These include the SAGE tagging of Genzyme Molecular Oncology Inc. and the
randomized tagging techniques of Lynx Therapeutics Inc.
a.
Serial analysis of gene expression (SAGE)
is a technique developed by
Genzyme Molecular Oncology Inc
., for profiling the genes present in a popula-
tion of mRNA. By the use of various restriction enzymes, SAGE generates, for
each mRNA, a 10-base tag that usually uniquely identifies a given gene. In the
standard SAGE protocol, the resulting SAGE tags are blunt-end ligated and the
results are sequenced. Such sequencing is faster than sequencing the entire ex-
pressed genes because the tags are much shorter than the actual mRNA they
represent. Once sequencing is complete, one may look up the tag sequences in a
public database to find the corresponding gene. Using the sequence data and the
current UniGene clusters, a computer processing stage determines the genes that
have been expressed. SAGE can be used on any set of expressed genes; it is not
specialized to any particular set. This technology can be adapted for use as addi-
tional information tags appended to the DNA in our database.
b.
Differential expression analysis
is a technique developed by Lynx Thera-
peutics Inc. for finding the difference in gene expression, for example, between
two distinct cell types. The randomized tagging techniques of Lynx Therapeu-
tics Inc. can be adapted to determine the difference between two DNA database
subsets.
c.
Hybrid methods
. One can modify these methods and extend them to ap-
ply to the tagged DNA strands of our database. This requires considerable
changes in the protocols, due to unwanted hybridization that may occur as a
result of combination of synthetic tags with genomic DNA in our database
strands. However, these modified methods can provide further powerful capa-
bilities, for example, the capability for fingerprinting (creating short DNA tags
that are nearly unique IDs for longer DNA strands of the database), identifica-
tion of expressed genes of selected DNA strands, and also the capability for dif-
ferential expression analysis of distinct selected subsets of the Biomolecular
Database.
3.7. Amplification and Reproduction of Biomolecular Databases
Once a Biomolecular Database is created, it is important to be able to accu-
rately replicate it, as it may be consumed during the course of interrogation.
Prudence suggests maintaining each database in an archive, and querying only
daughter databases prepared from the archival forms. Since each database mem-
ber is designed to bear a universal amplification (primer) sequence at the ex-
treme 5'- and 3'-ends, database replication can be performed using PCR.
Because the length of the DNA strands in the database might be quite substan-
tial, including both biological DNA information and many flanking tag se-
quences, the ability to produce full-length amplicons with long templates is
