Genotypic Amplification of the G310S Mutation of the TBX1
Gene Using Allele Specific PCR on DNA from IB3 Cell Lines
Mitch Huber, Caitlin Bouchey, Jake Pristas, and Susannah
Lyddon
Abstract
The G310S mutation on the TBX1 gene can
result in DiGeorge Syndrome (DGS) (Yagi, 2003), which causes multiple
biological abnormalities. (Wilson et al., 1993). Oligonucleotide primers
were designed to anneal to either DNA containing the G310S mutation or wild
type DNA, using the Yaku-Bonczyk method for primer design. The purpose was to
use allele specific polymerase chain reaction to determine the presence of the
G310S mutation in either mutant or wild type DNA using designed primers. It was
hypothesized that a double base pair mismatch in the forward mutant and forward
wild type primers would be sufficient to discriminate between mutant and wild
type DNA for the G310S mutation because the two primers are specific to their
respective DNA strands (Xu et al., 2011). Gel electrophoresis was
conducted on the prepared DNA cocktail and a band length of 1005 base pairs
will be observed due to the primers binding 1005 base pairs apart. It is
predicted that the double base pair mismatch will be sufficient to discriminate
between wild type and mutant DNA for the G310S mutation because the double base
pair mismatch decreases the frequency of false positives (Yaku et al.,
2008). These tests are scientifically significant because they can help
researchers efficiently identify the G310S mutation on the TBX1 gene when the
22q11.2 deletion (the predominant cause of DGS) is not present in a DGS
patient. PCR was conducted on Lambda virus genomic DNA and primers specific to
its nucleotide sequence, and the expected bands of approximately 500 base pairs
were observed on a gel. The team investigated the sociological effects of DGS
by creating a test to mirror the learning disabilities faced by DGS patients.
Participants watched an educational video and subsequently took a quiz on it.
Participants were split into four conditions: Control, vision impaired
(watching the video in a dark room while wearing sunglasses), hearing impaired
(watching the video while wearing noise-cancelling headphones), and total
cognitive disability (no video, questions only). Impaired participants were
found to give significantly fewer correct answers than control group
participants.
Figure
A)
B)
Figure 3. a). Ultraviolet image of an agarose gel containing
GeneRuler 1kb Plus DNA Ladder and DNA from CF IB3 cells b). and associated
analysis 1μL of purified IB3 DNA was placed into a cocktail along with
10μL of PCR buffer, 1μL of a forward primer, 1μL of a reverse
primer 2μL of TAQ polymerase, 1μL of free nucleotide bases, and 34μL
of nuclease-free water. The primer sets consisted of published forward and
reverse (labeled A in the above image), custom wild type forward and reverse
(labeled B), and custom mutant forward and reverse (labeled C). All cocktails
for each primer set were run through 30 cycles of PCR with 30 seconds for
denaturation (95¼C), 30 seconds for annealing (In the above figure: I = 40¼C,
II = 45¼C, III = 50¼C), and 1 minute for elongation (72¼C). GeneRuler 1kb Plus
DNA ladders (labeled L) were interspersed throughout the gel to make band
length comparison easier. One well was unused (labeled U), and serves as a
legend for ladder band lengths. Smearing occurred in the majority of the wells
due to nonspecific annealing brought on by low temperatures. However, specific
faint bands were found in wells IIIA and IIIB. The band lengths were calculated
to be 1112bp and 1533bp respectively (Image B). These are well above the
expected lengths for both the published primers and the custom wild type
primers.
Discussion
Experimentation
DGS normally stems from a deletion on
the 22q11.2 region on chromosome 22 (Merscher et al., 2001). DGS is a
major consequence of this deletion. Studies have been performed to find a gene
within the deletion syndrome that could result in the many abnormalities of
DGS. The TBX1 gene has been linked to the syndrome due to its location on
the gene and how it is responsible for development in the head and the pharynx
(Jerome and Papaioannou, 2001). A mutation in the TBX1 gene was found to
also result in DGS. This mutation is a single nucleotide polymorphism
(SNIP) at the 928th
base pair of the gene and changes a guanine to an adenine. The amino acid
sequence at the 310th
amino acid is then changed from a glycine to a serine (G310S), resulting in
inactive and dysfunctional proteins (Yagi et al., 2003). The
query for this experiment was whether or not DNA amplification using polymerase
chain reaction (PCR) could be used to detect the G310S mutation. It was
hypothesized that using a 15 base pair primer that was designed to match the
wild-type allele with one mismatched base and a reverse primer would be able to
correctly identify if a DGS patient without the deletion on the 22nd chromosome does not
have the G310S mutation on the TBX1 gene. The wild-type primer would only bind
to the wild-type DNA strand, proving to researchers that the G310S mutation is
absent. It was also hypothesized that using a 15 base pair primer that was
designed to match the mutated allele with 2 mismatched bases and a reverse
primer would be able to correctly identify if a DGS patient without the
deletion on the 22nd
chromosome has the G310S mutation on the TBX1 gene because the mutant primer
would only bind to the mutant DNA strand (Yaku et al., 2008). The Yaku-
Bonczyk method was utilized in primer design to decrease the frequency of false
positives found in testing. Instead of a traditional single base pair mismatch,
in which only the 3Õ most base of the wild type primer is non- complementary to
the mutant DNA, the Yaku- Bonczyk method also mismatches the third most 3Õ
base. Wild type primers have been found to anneal to mutant DNA, as the single
base pair mismatch is sometimes simply skipped over. This additional mismatch
decreases the frequency of non-complementary annealing found with conventional
primer design by creating a more pronounced sequence of non-complementary bases
(Yaku et al., 2008). An objection to the Yaku Bonczyk method is that it
can be viewed as resulting in false negative results. (Yaku et al.,
2008). However, the team would rather sacrifice many products with a high
chance of obtaining a false positive to obtain fewer results with a very low
window of falsification. To gain a more comprehensive understanding of DGS and
the diseaseÕs phenotype, a DIY experiment was designed. Researchers showed a
video from the Khan Academy covering Medieval and Byzantine Art in coordination
with a series of questions after viewing the video to a number of participants.
One group was the control, the video was shown without mimicked visual or
auditory defects. The second group mimicked the visual disabilities present in
DGS patients. They watched the video with the lights off and were wearing
sunglasses. The third group mimicked the auditory defects and watched while
wearing noise-cancelling headphones playing music at a low volume. A fourth
group did not watch the video at all, only the questions were answered to
mimick a learning disability in the way that it is hard for those individuals
to learn without assistance. Researchers gained a better understanding of the
everyday hindrances of DGS through the DIY experiment. The researchers relayed
that understanding in a sociological video journal outlining the experiment.
Predictions
Three different primers were designed
for the experiment in order to discriminate whether or not a patient had the
G310S mutation in the TBX1 gene. Two forward primers, FprimerWT and
FprimerM, were created so that they would bind to two different DNA sequences.
The FprimerWT would only bind to DNA where the mutation is not present
(wild-type DNA). The FprimerM would only bind to DNA where the mutation
is present (mutant DNA). Both forward primers had a mismatch base 3 base
pairs in that would not match either the mutant or wild-type DNA.
Changing the 3rd
base pair increases the specificity of the primers so that a false positive is
much less likely to take place (Yaku et al., 2008). The third primer was
a reverse primer, 991 base pairs away from the forward primers, which would
stop at the point of amplification. If the primers result in a positive
test, then the band length should be 1005 base pairs long. If the primers
result in a negative test, then there should not be a band because the primer
will not anneal to the DNA sequence since there are two mismatched bases.
For a homozygous wild-type DNA sequence, the gel should show a band that
is 1005 base
pairs long when using the FprimerWT and no band when using FprimerM. For
a homozygous mutant DNA sequence, the gel should show a band that is 1005 base
pairs long when using the FprimerM and no band when using FprimerWT (Wittwer
et al., 1993).
Ultimate Findings
Distinct PCR bands were not amplified
during the experiment. In order to determine the best conditions for DNA
amplification, many different trials were performed adjusting concentration of
Taq polymerase, concentration of PCR buffer, primer annealing temperatures, and
concentration of DNA. The best results were obtained using a 2X concentration
of Taq polymerase, a 2X concentration of PCR buffer and a temperature of 50¡C.
The best results were obtained with a lower temperature that resulted in smears
on the gel. This is a result of non-specific annealing; primers bound to
nonspecific sites on the template DNA inside the cocktail (Rychlik et al., 1990).
As a result of the DIY experiment, it
was concluded through the video showing that it is much harder to learn and
comprehend when faced with visual and auditory defects. After analyzing the
number of correct answers that subjects answered after viewing the video, it
was gathered that a higher rate of correct answers were obtained when
participants could hear and see the video as opposed to participants who
mimicked the visual and auditory defects present in many DGS patients. The p
value obtained after an analysis was less than .0001 after a one-way ANOVA test
was done, meaning that the results of the four conditions are significantly
different. The Tukey HSD test gave p values that were all less than .01 when
comparing the means from each condition to each other except for the comparison
of the mean of the control condition and the mean of the vision impaired
condition. That comparison was shown to be not significant.
Errors
When performing the experiment, there
were a few occurrences that may have affected the outcome. The primers did not
bind to the DNA with which they were mixed in the PCR cocktail, resulting in no
amplification of the target sequence. This could have been the result of the
annealing temperatures of the primers not being met when put through the PCR.
The annealing temperatures were altered in an attempt to have the primers bind.
The cycling times for PCR were adjusted to investigate their effects.
Concentration of Taq polymerase, PCR buffer, and DNA were also experimented
with to investigate their effect on obtaining an amplified target sequence.
Future Research
The next step in the teamÕs research is
to obtain mutant DNA in an effort to amplify the target sequence. With access
to mutant DNA, many new approaches would be possible. The optimal cocktail
recipe to obtain an amplified sequence could be found by changing the
concentration of individual components. Subsequently, the optimal cocktail
could be used as a diagnostics test for patients to determine if the G310S
mutation is the cause of their DGS. Other research projects can be devised from
the basis of the teamÕs research. Primers that are derived from the ones in
this experiment could be created and tested. Different mutations in the
TBX1 gene that are known to result in phenotypes similar to DGS could be
tested. Other primers could be designed for that mutation and tested in
patients as well. The research team could then look at the other genes
that are deleted in the Chr22q deletion syndrome and possibly find mutations in
those genes that could lead to phenotypes present in DGS or Chr22q deletion
syndrome. Primers that detect the entire 22q11.2 deletion could be designed.
Designing all of theses different primers for all of the known mutations that
result in DGS can be used to determine the underlying cause in specific
patients. This data can show how relevant each mutation is in DGS.
In retrospect of the sociological facet
of this experiment, the research team could attempt to live with a resulting
deformity of the disease. This would provide an in depth personal experience of
the struggles with which DGS patients cope. The team could go to a hospital and
stay for the duration of an average surgery and then mirror the medical
correction process for this deformity by simulating the recovery from a surgery
common to patients afflicted by DGS. Surveying DGS patients on their feelings
regarding the disease could also reveal societal aspects of DGS.
DIY Video Link
http://www.youtube.com/watch?v=-DqUI0HYkPU&feature=youtu.be