ECE 416 Homework 6 solutions
Due: Wed, April 24th
OFFICE HOURS: APR 23, 430PM
“Next-Gen. Sequencing, Raman Spectroscopy, and Quantum
Dots”
I. Next-generation Sequencing Techniques
Q1: Assume that there are 3 billion base pairs in a human
DNA sequence, and that you have 50 copies of identical full
DNA molecules. Next, assume that you will use enzymes to
break the full DNA molecule into sections that can be
anywhere between 80 and 120 base pairs in length. As a
result, the location of enzyme chopping (called enzyme
cleavage) is not necessarily the same on all 1000 DNA
copies, but that on average, the base pair length is 100. The
chip has 8 channels, and each channel is 75mm long, 1 mm
wide, where both the top and bottom are active (usable)
surfaces.
You wish to distribute the cleaved DNA fragments onto an
Illumina microfluidic chip. Assume that each fragment must
attach to a distinct location on the chip, and not overlap with
any of its neighbors.
a) Using information presented in class (or in the
Voelkerding supplemental reading from the Next
Generation Sequencing lecture):
i) Determine the size of an Illumina "polony" and
estimate the number of polonies per square
millimeter.
ii) How much total surface area would be needed to
immobilize all your DNA fragments?
iii) If a single Illumina microfluidic chip contains 8 flow
channels (75 mm long, 1 mm wide), how many chips will
be needed for this? (assume that polonies will grow on
both the upper an lower surfaces of the microfluidic
channels).
b) The laser used in the Illumina imaging system is capable of
delivering 100 mW of total power. The laser illumination is
broadened into a line that is 1 micron wide and 25mm long,
so it can be scanned down the length of the microfluidic chip
(illuminating all 8 flow channels at once). Consider detection
of the "G"-nucleotides, in which each DNA fragment is
labeled with only one Cy5 molecule, and that the laser has a
wavelength of 633 nm. Assume that the quantum efficiency
(or quantum yield) for Cy5 is 28%, the fluorescent lifetime is
2 nanoseconds, and that the fluorescence emission is
gathered by a lens with a focal length of 2.9 mm and a
numerical aperture (NA) of 0.85.
i) How many photons/sec from the laser reach one polony
area in a region illuminated by the laser line?
ii) Assuming a laser line scan rate of 1 cm/sec, how many
photons are emitted by a single fluorescent dye
molecule? (consider how many times a single
fluorophore would be excited and relax in the course of
the scan line passing over the molecule, and factor in
the quantum efficiency). How many photons are emitted
by one polony?
II.
Raman Spectroscopy
Q2: Describe the difference between the two types of
scattering discussed in class, specifically discussing
changes in energy of the particles and commonalty of the
process. Why do different molecules produce different
Raman signatures?
Relative Raman Intensity
(a.u.)
Q3: From the Raman spectrum given below, what is the
actual scattered wavelength of the two most prominent
peaks (1030 and 1570 𝒄𝒎−𝟏 )? Assume that a laser with a
wavelength of 785 nm is used to excite the Raman spectrum.
250
200
150
100
50
0
-50
500
1000
1500
-1
Raman Shift (cm )
III.
Quantum Dots
Semiconductor quantum dots (QDs) have the potential to enable
the development of hybrid inorganic-bioreceptor sensing
materials. The following question highlights the use of QD–protein
assemblies as chemical sensors – in this case for detection of
maltose, a specific type of sugar molecule. E. coli bacteria have
been engineered to produce a maltose-binding protein (MBP) that
can be covalently attached to a QD. The sensors are selfassembled in solution in a controllable manner. The broad
emission spectrum of semiconductor QDs makes them an ideal
candidate for FRET based reactions. The chain of events for
maltose detection are shown in the diagram below, in which 2
FRET photon transfers are used (From the QD to Cy3, and then
from Cy3 to Cy3.5). The quantum dot is excited by a λ=400 nm
laser.
Q4: What is the emission wavelength if there is no Maltose
binding protein (MBP)?
Q5: What is the emission wavelength if no maltose is
introduced into the reaction?
Emission
Absorption
Q6: As you increase the concentration of maltose, describe
what you would see on a spectrometer.
Q7: If MBP were not tagged with Cy3, how would your
observation on the spectrometer change?
Q8: Is this an efficient way to do this study? Explain your
answer in terms of spectral overlap.