Detection
of the Single Base Mutation, C1824T, in the LMNA Gene, in Human Cells Using Yaku and PCR Methods.
By:Chuck Ternes, Stephen Manning, Jenna Serino, Victoria Thomas
LB 145 Cell and Molecular Biology
Lab
Instructors: Natalie Palumbo and Zachary Gaudette
Abstract
The
genetic disease Hutchinson Gilford Progeria Syndrome
(HGPS) is most commonly caused by the single base pair mutation C1824T on the
608th codon of the LMNA gene (Gordon et al,2003).
The experimental techniques of polymerase chain reaction, gel electrophoresis,
genomic DNA extraction, site directed primer
mutagenesis and yaku primer design were used in order
to determine if the DNA from human epithelial cells could be accurately
identified either as wild or mutant for the C1824T mutation. We
hypothesized that experimental yaku primers will
target the single base pair mutation with the specificity needed to distinguish
wild and mutant genotypes that vary by only one basepair.
The intentional misconfiguration at position 3 and
variable matching base pairs on position 1 of the detection primers will allow taq to replicate the DNA contingent upon the complementary
matching at position 1 of the primers with the DNA (Yaku
et al., 2008). The wild type primer will only bind to and
replicate only wild type DNA, whereas mutant primers will only bind to and
replicate mutant DNA. Experimentally designed primers amplified bands of
approximately 440 bp in length with non-specific
distinction between wild and mutant DNA sequence. The sociological implications
of HGPS were examined though disability replication of stiff joints, and were
found to have a negative correlation on all aspects of our surveyed health
categories including physical and psychological health but not a statistical correlation
(p>0.05) . This research is profound for understanding ways of accurately
identifying the presence of subtle genetic mutations.
Figure 4:
a) Gel electrophoresis replication of combination primer and DNA cocktails for
detection of wild and mutant DNA sequences. Mastermix
cocktails were utilized in creating the PCR reaction mixtures. Thermo cycling
conditions were identical for all PCR cockatils.
The cycling including 35 cycles of 30,30,45 sec at 94,57, and 72°C respectively
with 5 min initial denaturation at 94°C and final
extension at 72°C.Wells that are labeled DL indicate the use of 1 kb plus DNA
ladder. Wells 10 and 11 contain successful mutagenesis primer PCR amplification of bands at 472 bp. Wells 2 and 3 show successful amplification of a 438 bp band using MM as predicted, well 5 contained MW which
also produced a band but not as expected. Wells 6 and 7 contained PCR cocktails
with WM which amplified bands of approximately 440 bp
but not as predicted. Again, with respect to figure 3, Wells 8 and 9 contained
WW which was unsuccessful in amplification of a 438 bp
band, contrary to our predicted observation and previous success seen in figure
2. The results were ambiguous for all combinations of primer and DNA. b)
Confirmation of band lengths as target PCR products was done using a semi-log
plot constructed from the 1 Kb plus invitrogen DNA
ladder in well 7 labeled DL. The measured band length of all highlighted bands
corresponded to 6.00 cm which was approximately 449 bp,
providing an extremely close approximation of theorized band lengths for PCR
products.
Discussion
Hutchinson-Gilford Progeria Syndrome (HGPS), a rare, genetic condition that
affects one in 8 million human births, is a fatal disease that is characterized
by premature aging (Snigula, 1977). This disease is
caused by the de novo point mutation, C1824T, on the 608th codon of the LMNA gene. This mutation creates a cryptic
splice that leads to an expression of a truncated abnormal Lamin A protein which causes the cells
nucleus to become unstable and die prematurely (Paradisi
et al, 2005).
PCR has been used in the past to
determine genetic disorders such as Progeria;
however, we are interested in seeing if a PCR assay can be designed to detect a
single-base pair substitution accurately (Scaffidi et
al, 2006). We hypothesized that the
experimental Yaku primers would provide the
specificity needed to use PCR to target the single base mutation that causes
HGPS because of an intentional mismatch on the 3rd base in from the 3' end of the
2 reverse primers (Yaku
et al, 2008) (Figure 5). This hypothesis was tested
using the experimental techniques of gel electrophoresis, genome extraction,
primer mutagenesis, and yaku primer design.
Control primers were adapted from a
previous study of an alternative Progeria mutation (Fukuchi
et al, 2004). They were used to
verify that our PCR techniques were viable and that we were running the gel
electrophoresis correctly. Bands
were produced at 394 bp, which were expected from the
literature (Figure 1). The
successful replication of the controls primers reaffirmed our protocol.
The F1 and W1R primers, along with the purified wild-type
DNA, were run through PCR at temperatures of 57 and 58 degrees Celsius. They
successfully annealed at both temperatures, each producing bands in the gel at
the expected 438 bp band length with respect to the
ladder (Figure 2). The presence of the bands in the wild-type DNA indicates the
absence of the C1824T mutation, and supports our hypothesis.
Mutagenesis was utilized to produce
amplified DNA that contained the C1824T mutation that causes HGPS in order to
produce bands for our M1R primer. Initially, a gel was run and a band was
produced unexpectedly for the W1R primer and designed mutant DNA, while the
bands that were expected to show up for the W1R primer and wild-type DNA did
not appear, even though they had previously been shown to (Figure 3). In
determining these errors, we ran a final PCR and gel with each combination of
primer and DNA, which included the M1R primer with mutant and wild-type DNA as
well as the W1R primer with mutant and wild-type DNA (Figure 4). We did get
faint bands for the M1R and mutant DNA, but then we also got bands for M1R and
wild-type DNA as well as W1R and mutant DNA. While we expected our W1R and
wild-type DNA to produce bands, once again they did not. This is particularly
unusual due to the fact that we have had previous, definite success with the
W1R and wild-type DNA (Figure 2). While these final results do not support our
hypothesis that we could provide the specificity to determine the presence or
absence of the mutation, they do not explicitly refute it either. Alterations
can be made to the Yaku primer design to feasibly
increase the efficiency of the intentional mismatch, including designing
entirely new primers, which our research team is giving thought to (Yaku et al., 2008).
Along with the PCR tests, we also
tried to understand the sociological and psychological effects and social
stigma that comes with having Hutchinson-Gilford Progeria
Syndrome. We conducted a sociological experiment in which each member of
the group simulated living the life of someone with Progeria.
Our focus was on how one of the limitations of Progeria,
joint stiffness, could be replicated by wearing restrictive, elastic wrap
bandages on our knees and elbows. We hypothesized that the hindrances of joint
movement on group members would cause a decrease in the capability to take care
of oneself and carry out daily routines as well as a decrease in attitude,
while increasing pain/discomfort, correlating to an overall decrease in
self-esteem (Barnes, 2009). A limitation to our experiment was that it was
designed only for a short period of time whereas the conditions of Progeria will last a lifetime. While our hypothesis was
supported by the trends of increased perception of disability over time, each
person had an entirely unique experience (Figure 6). This led us to conclude
that disability can truly be just a perception. A longer experiment may have shown
more of significance between life with the bands and life without them, or in
terms of this experiment, life with Progeria versus
life as a healthy individual. However, our conclusions indicate that the longer
we wore the bands, the more we may have accepted our limitations and ceased to
be considered “disabled”.
Future
Directions:
The experimental
testing of both genome purified and mutant DNA, synthesized from site directed
primer mutagenesis, yielded results that were somewhat ambiguous in terms of
conclusive findings. The results of our final PCR reactions and gel
electrophoresis of the products allowed us to conclude that our experimental
primers were non-specific in distinguishing the DNA sequence of wild and mutant
DNA. We noted that the use of site directed primer mutagenesis to synthesis our
own DNA template for testing may have led to the production of DNA that did not
simulate the DNA of a patient with progeria. If we
were to progress in this research further we would want to use our primer design
tested in this study on DNA from an actual progeria
patient. Although we were unsuccessful in attempts to acquire mutant DNA
samples for this investigation, we would ultimately need to include such
testing before we can conclude the capacities of our primer specificity.
Secondarily we cannot confidently refute our hypothesis
statement that yaku primer design will give the
necessary specificity needed to distinguish between the single base pair
difference in wild and mutant DNA sequences. Our results did not reflect
defective methodology, nor did it access all variations of experimental design,
specifically yaku primer design. We would design new
experimental primers still using the yaku primer
approach but with a modified intentional mismatch combination on the third base
pair in from the 3’ end of the reverse primer. This might include using a purine – purine mismatch for the
reverse primers. Specifically we might try a G-G or a G-A mismatch that would
more effectively push the primer and DNA strand apart as suggested in the work
of Yaku et al. In the event of successful distinction
between mutant and wild genotypes we would want to do blind sample testing to
confirm validity of the prospective “screening” application of our primers.
Sociological
Experiment video: http://www.youtube.com/watch?v=u9eeTj6vmoI