Gene Sequencing programs and probes, PCR practice 3, qPCR 1
Technical Log
Name: HaoQi (Esther) Li
Date: 6/27/06 Tuesday
Work Location: Naval Medical Research Center, Spring Field, MD
Title of Project: Developing an Optimized qPCR for Borrelia lonestari
toa: 8:00am tod: 4:30pm
1. Objective – Why did you hope to accomplish today?
I hoped to carry out the PCR process by myself successfully for the second time, start
working on qPCR, analyze the ClustalW sequence alignments, and start working on my
proposal.
2. Events – a. summarize the schedule of events. Procedures performed in detail,
results, analysis, with tables, charts, diagrams, and scanned sketches
b. summarize communications, emails (attached), conversations, pertinent material
read (annotated bibliography)+ reactions, browser used (search identifier,
annotation)
Gene Sequencing
Dr. Ju and I looked at the first ClustalW sequence alignment and realized that the colors
were not set properly. All adenines were marked red instead of the differences. Thus I
have to set the colors to mark only the differences.
Dr. Ju explained that after that is done, I have to find unique sequences of Borrelia
lonestari (AY850064 and AY850063) then we will send those sequences for a biotech
company to synthesize. So basically I think my project is not extremely complicated! ;)
When I failed to change the colors of ClustalW (the best I got was an non-exportable java
window) I asked Lois from Blair Magnet to show me how to use GeneDoc. It worked
wonderfully.
And then I used the realignment tool from ClustalW then copied and pasted the results as
a .msf file into GeneDoc to detect the differences as “Borrelia sequences
GeneDocClustalW.msf”. The program behaved strangely in the middle of printing and
resulted in “Borrelia sequences GeneDocClustalWb.msf” with some misalignment, but I
managed to print out all of the original “Borrelia sequences GeneDocClustalW.msf”.
Dr. Ju taught me the two types of probes, the TaqMan and the Beacon. It is not very
specific so the probe has to be complimentary to a really unique strand with 3+ codon
differences to work well. However, the TaqMan gives a stronger signal. The ideal length
for TaqMan is 30-50 basepairs.
(TaqMan is an analogy to PacMan.
http://www.vetscite.org/issue1/tools/leute_2_0800.htm )
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Pierce/realtimepcr.htm
So in this downloaded picture, the green ball is the "F", which stands for "FAM Reporter"
and it fluoresces when it is broken from the rest of the probe. The red ball is called "Q"
which stands for "quencher". The quencher we will use will be Black Hole quencher 1
(Bhq1), which can work with FAM. There is also Bhq0 and Bhq2 which work with other
reporters and absorbs different light waves. (In the experiment with B. recurrentis
described in the 1997 article, ROX or TET was the other probe so Bhq1 was not used.)
The second probe is called a Beacon and it is less specific because of its hairpin structure.
Since the sequence has to match exactly because of the high energies required to break
the hairpin structure, even one unique codon can be made into a probe with beacon. Thus
the ideal length is shorter than TaqMan with only 25-30 base pairs. Contrary to TaqMan,
beacon lights up when the hairpin structure is stretched out on the template DNA, and is
removed from the template without being broken when the DNA polymerase comes
along.
Dr. Ju told me that Beacon is mainly used for its specific bindings despite that the signal
is not as strong as TaqMan. Then I suggested that since there are different florescent
colors of the reporter, and then if we could have different probes mixed into a template
and still use TaqMan. So the target template would have the greatest mixture of colors.
PCR
Then from 9am to 10:10am, I performed PCR all by myself and put them in the PCR
machine. Everything went smoothly.
After lunch, we looked at the results from the PCR Practice 3; however my three lanes
(on the opposite end of the standard) failed to show up, perhaps because I failed to load
enough DNA samples.
Record Book
Joey showed the group (Sindhu, Rebecca, Wei, me) how to write in the Record Book. I
learned that each entry needs to include:
 Title
 Date every page
 Explanation
 Protocol
o At least 1 protocol of each type
o Anything that is out of norm or could be a source of error
 Results
 Summary (if necessary)
Before lunch I entered more Borrelia sequences into GeneDoc. After lunch I asked Dr.
Li and she said that it is not possible for ClustalW to distinguish the different sequences,
thus I can only use GeneDoc.
qPCR
After the gel electrophoresis was set running, I looked at the results of yesterday’s qPCR
run.
The threshold 30 was set arbitrarily. The orange line goes over the threshold earlier (at
cycle 26.27) because it has a higher concentration of original DNA present, 105copies/1l,
whereas another site only has 103copies/1l and crossed the threshold later at 33.90
cycles of amplification. The control of water stayed 0 because there was no DNA to be
amplified.
The bumps before the logarithmic shaped fluorescence curve show the unstableness of
the fluorescence [as Joey described the phenomenon as an “ADHD” child].
3. Reflections
a. actually accomplished: I did a PCR by myself and printed out the sequences from
GeneDoc.
b. concerns: No conrcerns.
c. learning (workplace, science, project, yourself): I learned about the probes and how
to write in the record book.
4. Planning
I hope to learn more about the probes, i.e. how FAM and Black Hole actually perform
their tasks. I also would like to organize the sequences and find a unique places for B.
lonestari. I also would like to perform qPCR by myself tomorrow and perfect my PCR
skills.
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