Appeal 2007-3395
Application 10/260,733
cDNA sequences which are complementary to that target element
(Bao at col. 6, ll. 51-55).
C. Kuukasjärvi
[28] According to Kuukasjärvi, a major limitation in analyzing genetic
changes in the early stages of tumorigenesis is that the higher number
of normal cells in a tissue sample often mask genetic alterations in
premalignant and small malignant tumors (Kuukasjärvi at 94).
[29] While standard CGH7 allows genome-wide screening for DNA
sequence copy number abnormalities, it is said to require 0.5 to 1 µg
of genomic DNA, corresponding to roughly 50,000 to 100,000 diploid
cells from the tumor sample (Kuukasjärvi, ¶ bridging 94-95).
[30] Microdissected tumor samples are said to be too small to provide
enough DNA for standard CGH and, therefore, the DNA must be
amplified using PCR with degenerate probes (DOP-PCR) prior to
CGH analysis (Kuukasjärvi at 95).
[31] Kuukasjärvi describes an improved DOP-PCR method which includes
incorporating fluorescent labeled nucleotides directly into the PCR
reaction (Kuukasjärvi at 95).
[32] To test the sensitivity of DOP-PCR CGH, Kuukasjärvi made serial
dilutions of a known concentration of DNA extracted from the MCF-7
breast cancer cell line and used the dilutions as starting material in
DOP-PCR (Kuukasjärvi at 97).
[33] According to Kuukasjärvi, DOP-PCR could amplify template DNA
from concentrations as low as 25 pg DNA (Kuukasjärvi at 97).
7 Standard CGH uses chromosomes as the target elements for hybridization
(see e.g., Kuukasjärvi at 96 and Figure 3).
11
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