Biomedical Engineering Reference
In-Depth Information
Table 4.2 Comparison of single channel conductance from the GP-10 connector and
a
-hemolysin
Conductance (nS/pore)
a
at 0.5 M NaCl at 1 M KCl
Connector 3.6 10.2 1.57
0.16 4.84
0.15
a
-HL 1.5 [
49
] 1.8 0.31
0.05
b
0.94
0.01
c
Ratio (Connector/
a
-HL) 2.4 5.7 5.1 5.1
a
The data for connector conductance at 0.5 M NaCl, and 1 M KCl were obtained from a total of
38 and 36 insertions, respectively. The data for
a
-HL conductance at both 0.5 M NaCl and 1 M
KCl were from a total of four insertions, respectively
b
Pore
diameter (nm)
Cross-section
area (nm
2
)
Proteins
Conductance of
a
-HL at 1 M NaCl has been reported to be 0.68 nS/pore [
59
]
c
Conductance of
a
-HL at 1 M KCl has been reported to be 0.80 nS/pore [
60
] or 1.0 nS/pore [
29
]
4.5 Translocation of Double-Stranded DNA
4.5.1 Translocation of Double-Stranded DNA Through Phi29
Connector Channels
The principle of nucleic acid translocation through nanopores was demonstrated
almost 15 years ago using
-hemolysin channels. The concept is based on the
classic 'coulter-counter' whereby the passage of non-electrolytes through a pore
against an electrochemical potential gradient physically blocks the ion flow, which
can be detected. In presence of linear dsDNA of varying lengths (12-5,000 bp),
numerous transient current blockade events, (each representing the translocation
of a single DNA molecule) were observed (Fig.
4.7b
). When circular plasmid
DNA (Cx43) was added, no such current blockade events were observed, presum-
ably due to the supercoiled nature of plasmid DNA. However, upon digestion of
circular plasmid DNA with DNaseI, a burst of DNA translocation events were
observed, demonstrating that the phi29 channel is only capable of translocating
linear dsDNA (Fig.
4.7c, d
).
Occasionally, in presence of multiple pores, blockade events with plateaus at
one or more discrete current levels were observed, indicating the simultaneous
translocation through multiple pores. The rate of current blockade events was
observed to increase with increasing concentration of DNA as well as by increasing
the applied voltage, as expected.
a
4.5.2 Characterization of DNA Current Blockade Events
One of the parameters in individual pore blockade events is the depth of current
blocks and is expressed as current blockade percentage, calculated as follows:
the size of current blockade resulting from the DNA translocation through
Search WWH ::
Custom Search