The purpose of this project was to colour code the entire length
of chromosomal 8, and to test the effect of radiation on cells.
The findings show that through multiple tests, it is possible to
M-Band the entire chromosome. Primarily, the expectations were very
low, simply because there were so many errors to consider, and hurdles
to cross. The procedure that was originally designed, however, worked
remarkably well.
The
first step was the harvesting of cells. High quality, elongated
chromosomes had to be created to achieve a refined barcode for chromosome
8. Horse serum and acetic acid were added in order to accomplish
this. In the future other substances could be used to improve the
quality of the chromosomes. Some potential substances include, caluculin
A (which prevents chromosomes from condensing) and fresh KCl. Figures
8 and 9 are examples of metaphase spreads. Figure 8 was one of the
first ones that was done. It is less spread, and the chromosomes
seem small and too close together. This may be due to a variety
of reasons such as humidity in the air and improper timing. An important
aspect of the metaphase spread is the process of making slides.
When making slides, it is important to let the cells run down the
slide in order to have the cells evenly distributed. The more slides
I made, the better they turned out. Figure 9 is a slide that was
made in the later part of the project and is much more desirable
for FISHing.
The
second part of the experiment, PCR (polymerase chain reaction) worked
well. Figure 10 depicts a PCR in which all the probes are the same
length. The ladders on the sides are standards through which the
lengths can be approximated. With the help of the ladders, it can
be seen that the probes are copied properly. On the other hand,
Figure 11 shows a PCR that didn’t quite work. There are no
bands in some of the wells. This suggests that the probe was not
properly amplified during PCR. Mistakes like this can make the FISH
process worthless, which is why gel electrophoresis is performed
as a check. This way, time and money are not wasted in performing
FISH with samples that have no probe.
Throughout
the experiment, I was faced with many different challenges. Most
of these problems were addressed through experimentation. One of
the problems was in figuring out the optimal length for each segment
of colour in the chromosome 8 barcode. Less than 5 million base
pairs of the same colour was too little to observe using the microscope,
but more than 15 million was just too imprecise, and did not allow
enough bands to be produced along the chromosome. Therefore, segments
of 5, 10, or 15 million base pairs were used depending on the brightness
of the fluors for that section, and the position of the segment
along the chromosome.
Another
concern was the limited number of fluors that worked. Figure 12
depicts the wavelengths of colour that were used in the M-Banding.
Of the 8 fluors, Alexa 532, Alexa 488, and Cy-5 did not result in
detectable signals. This may have been due to fluorescence quenching,
which results when electrons in the outer orbital from several fluor
molecules are too close to each other. These “crowded”
electrons do not get excited by the UV light, and the colour appears
dimmer as a result. More work in adjusting fluor concentration and
probe position may result in the detection of these three fluors.
The fluors that were used and worked were: TMR (orange), Alexa 594
(red), FITC (green), DEAC (aqua), and Alexa 633 (far red). These
fluors were used in patterns to M-Band the entire chromosome.
M-
banding is a process that is relatively cost efficient because the
fluors that are involved are used in small quantities. The one difficulty
of M-banding is that it is much more time consuming than whole chromosomal
FISH since many different probes have to be mixed together instead
of using just one probe for the whole length. An advantage is that
the barcode of chromosome 8 is reproducible, and therefore of great
value, because it can be used to detect any inversions in cells.
Figures
13 and 14 are of chromosome 8, with fragments that are painted.
Some of the fluors were not as bright as others. In Figure 14, it
can be seen that the red fluor is much brighter than the green.
This problem was resolved by painting more megabases of the chromosome
with the dimmer colour, and fewer megabases with the brighter fluor.
Finally, once all the fluors were tested, a final FISH was done
to produce a complete coloured barcode for the chromosome. Figure
16 is a depiction of a preliminary coloured barcode. The colours
that are used are shown in Figure 15. In the future, the barcode
can be further refined to have a wide range of colours, improving
the accuracy of radiation testing.
Once
the barcode was established, it was used to observe the effects
of radiation. Figures 17, 18, and 19 depict the results. It can
be seen that radiation can cause an incredible amount of mutations,
if cells are exposed to the rays. Using M-Band, the amount of radiation
(dose) that is safe for human cells can be calculated.