FINDING THE B. lonestari PROBE!!!
Technical Log
Name: HaoQi (Esther) Li
Date: 6/28/06 Wednesday
Work Location: Naval Medical Research Center, Spring Field, MD
Title of Project: Developing an Optimized qPCR for Borrelia lonestari
1. Objective – Why did you hope to accomplish today?
I hoped to carry out the PCR process by myself successfully and start working with qPCR.
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)
Dr. Chen helped me use the scanner to scan my 3 PCR gel results (scanner only connected to his
computer).
Then the rest of the day I did nothing but finding the probe. First I color coded the unique and
semi-unique base pairs of Borrelia lonestari.** (There were two strands of Borrelia lonestari,
AY850064 and AY850063, because they differ by two base pairs and one, AY850063, has an
extra codon.
Key:
Red fill = Unique, i.e. unique
Red box = almost-unique, i.e. share the base pair with 1-10 Borrelia species
Blue box = semi-unique, i.e. share the base pair with 10+ Borrelia species
Then I used the paper cutter and tape to connect the 990 base pairs so that Dr. Ju and I could
easily spot the “unique regions.” Using the program Beacon Designer 4.0, Dr. Ju and I soon
realized that it is easier to input the probe and let the program find the primers instead of vice
versa. Through many tries, we discovered the best probe, which contains 3 unique base pairs, 1
almost-unique base pair, 2 semi-unique base pairs, and most importantly, the probe crosses an
18-base-pair deleted gap (starting at base pair 667) which makes it extremely unique for the
lonestari strands.
Since the probe region only constitutes the loop of the Beacon hairpin, the double strand region
of the hairpin is made from a set of short complementary extra codons, i.e. CGCGAT (sense).
However since it takes over 60˚C to denature the Beacon hairpin, which is much higher than the
low 50's for other parts of the reaction, e.g. primer attachment, Dr. Ju suggested to use only five
base pairs, i.e. CGCGA, so that less energy is required since there is one less hydrogen bond to
break.
Dr. Ju also mentioned that deleting sequences for primers or probes would lower their respective
temperatures, or, adding A's and T's would also lower the temperatures??? I wonder why.
The two primers are on the left and right sides of the probe, with some additional codons in
between each primer and the probe. It is fortunate that the extra codon of AY850063 is in the
additional codon region so that it would not affect the performance of the primers and probe.
The Forward primer has 2 unique base pairs, 2 almost-unique base pairs, and 5 semi-unique base
pairs. The Reverse primer has 1 unique base pair, 1 almost-unique base pair, and 3 semi-unique
base pairs.**
At the top right side the program shows the positions of the forward primer, the probe, and the
reverse primer. The “Status” boxes indicate some valuable information such as the temperature
and the possibility to form crosses.
Beacon Designer 4.0 checked for possible undesired crosses and bindings. The "self dimmer
ΔG" gives the energy for the two primers to bind to each other. The "cross dimmer ΔG" gives
the energy of the primers to bind in other regions of the sequence. The "cross dimmer ΔG" of
the probe gives the possibility for the probe to form unintentional hydrogen bonds (i.e. making a
double loop). Luckily, the program predicts low possibilities for both these unwanted behaviors.
This red circle shows an undesired cross dimer.
Dr. Ju is currently filing a probe order request to the navy headquarters. Hopefully the probe
will arrive next week so I can start qPCR testing.
3. Reflections: It was a very busy day with looking through the thousand-base-pair sequence
and complicated by the internet crashing, but everything worked out!
a. actually accomplished: I found a good probe for B. lonestari using the program Beacon
Designer.
b. concerns: I should really start on my proposal!!!
c. learning (workplace, science, project, yourself): My project is so awesome and I'm doing
REAL SCIENCE!!!   
Question for tomorrow: "Dr. Ju also mentioned that deleting sequences for primers or probes
would lower their respective temperatures, or, adding A's and T's would also lower the
temperatures???"
4. Planning
I hope I can finish the proposal tomorrow so that Dr. Richards can read it over. I plan to print
out this week's logs and let Dr. Richards or Dr. Ju sign them. For the rest of the time I plan to
practice more PCR or qPCR.
signature