Diagnosing Hypolactasia by Genotyping MCM6 Gene SNP C/T-13910
In
human IB3-1 cell lines via Allele Specific PCR
By:
Alec
Aiello, Jon Dokter, Hassan Fadel, and Adam Matynowski
LB 145
Cellular and Molecular Biology
Section
008
Dr.
Doug Luckie
April
22, 2013
Abstract:
Written By: A45273225
Revised By: A45259189
Finalized By: A44174805
The
C/T-13910 mutation on the MCM6 gene is the root cause of the persistence of
lactase-phlorizin hydrolase
(LCT) gene expression (Bulhões et al, 2007). The C/T-13910 mutation involves a single cytosine to
thymine base pair substitution 13,910 base pairs upstream of the LCT gene, in intron 13 (Enattah et al, 2002). The presence of the
mutation causes lactose persistence in up to 97% of adult cases (Bulhões et al, 2007). The purpose of our research was to
successfully develop a cost effective method to diagnose hypolactasia. Allele specific PCR was conducted in order to
properly genotype human IB3-1 cell lines. The PCR results were used to
determine whether samples of DNA were wild type, heterozygous, or homozygous
for the C/T-13910 mutation. Two forward
primers (FWTPrimer/FMTPrimer),
and one reverse primer (Rprimer), were created to
anneal to either mutant or wild-type DNA.
We hypothesized that we could detect the C/T-13910 mutation using PCR
and gel electrophoresis because of a single base pair mismatch on the 3' side
of the forward primers (Bodlaj et al, 2006). Gel
electrophoresis was used to analyze the amplified DNA to identify the presence
of C/T-13910. FWTPrimer and FMTPrimer
were predicted to yield a single band of 1106 base pairs for homozygous genotypes
depending if sample was CC or TT.
Heterozygous DNA was predicted to show a segment of DNA 1106 base pairs
long for both primers, due to a CT genotype (Bulhões et al, 2007). The assay was unsuccessful
due to lack of mutant DNA availability and unsuccessful custom primers (Figure 4).
Mutagenesis was attempted to create mutant DNA but was also unsuccessful (Reikofski & Tao, 1992) Only wild type DNA was used in
the assay and no bands were produced when ran through PCR with both FWTprimer and FMTprimer. A
sociological experiment in which cafeteria menu options were recorded based on
containing lactose was performed to gather information on the limitations
patients of hypolactasia endure daily. A test was
performed, on top of the data collected, to determine whether soy, almond, or
dairy milk was preferred in a blind taste test of 30 individual.
Discussion:
Written
by: A45259189
Revised by: A45273225
Finalized by: A44611726
Allele-specific
polymerase chain reaction (ASPCR) is a fast and cost-efficient assay for
genotyping DNA. Our goal was to discover a method using only ASPCR to
successfully identify the presence or absence of the C/T-13910 mutation on the
MCM6 gene in order to diagnose hypolactasia in
humans. Previous studies have been able to successfully design an assay that
identifies the presence or absence of the C/T-13910 mutation, however such
assays involved time consuming and expensive processes. For example, a study
done in 2007 at the Universidade Federal do Rio
Grande do Sul required the restriction enzyme BsmF1 endonuclease in their PCR assay to identify the C/T-13910
mutation (Bulhoes, 2007). The addition of this
restriction enzyme complicates the assay’s procedure and greatly increases the
cost of each PCR product made, thus making this process an expensive assay (Bulhoes, 2007). Another study performed at Amherst College
in 2012 involved the technique of amplifying the mutation region using PCR, and
then using DNA sequencing to genotype the DNA samples (Emerson, 2012). DNA
sequencing is very time consuming, and expensive (Emerson, 2012). The methods
we performed involved simply one round of PCR and one round of gel
electrophoresis in order to diagnose hypolactasia in
any given DNA sample. With our
experiment, we attempted to prove that it is possible for cheaper and quicker
assay’s to be developed using ASPCR that can diagnose diseases related to
SNP’s, especially those related to hypolactasia.
Our
ASPCR methods involved the use of Yaku-Bonczyk
primers design in attempt to increase primer discrimination against nonspecific
binding (Yaku et
al, 2008). We predicted that the traditional primer design methods would
lead to false-positive tests, because the single base pair mismatch on the 3’
end of the primer would not create enough specificity with the binding site (Järvelä, 2005). This means that our FWT primer could bind
unintentionally with DNA containing the C/T-13910 mutation (Graham et al, 1989). With the Yaku-Bonczyk method, an intentional base pair mismatch near
the 3’ end of the primer for both wild and mutant types would increase
specificity during PCR, thus reducing the likelihood of a false-positive test (Yaku et al, 2008).
We hypothesized that we could detect the C/T-13910 mutation using ASPCR and gel
electrophoresis due to the single base pair mismatch on the 3' side of the
forward primers (Bodlaj et al, 2006). After running PCR with our primers, they were run
through gel electrophoresis to identify the DNA’s genotype based off production
of bands. Before using our designed primers, we performed various control
experiments involved with PCR to confirm that our procedures would be effective
when genotyping our DNA. These control experiments were previously discussed in
the paper. From our methods, multiple trials of PCR and gel electrophoresis
were run in a range of temperatures and times in attempt to acquire our
predicted results. Our methods allowed us to be able to cover a plethora of
variables to narrow down a proper ASPCR assay to diagnose hypolactasia.
After
multiple trials of varied experiments, we ultimately were unable to develop a
successful assay that could diagnose hypolactasia
through identification of the C/T-13910 mutation using ASPCR. In attempt to
produce our own mutant DNA, bands were not produced after our mutagenesis assay
was run, meaning that we failed to create DNA with the C/T-13910 mutation. This
ultimately prevented us from being able to develop an ASPCR assay, because
without mutant DNA, we were unable to use our two designed forward primers to
discriminate against its respective allele specific site. However, we were able
to produce bands from our lambda and control primer assays. This was
significant because it proved that our PCR cocktail reagents and gel
electrophoresis factors could be used for future experimentations.
Additionally, we were successful in extracting human IB3-1 epithelial cells
through the Quiagen genomic extraction kit, producing
a yield of 385ng of DNA. This DNA was used in future experiments to further
develop an ASPCR assay using our designed primers. Although our results did not
correlate with our original predictions, ASPCR remains an attractive method in
diagnosing hypolactasia. ASPCR with gel
electrophoresis is a fast and cost-efficient assay that can be used to diagnose
hypolactasia by identifying the C/T-13910 mutation on
the MCM6 gene due to primer design that can discriminate against a single
nucleotide polymorphism.
Original Predictions
Successful annealing of the FWTprimer
and Rprimer is predicted to show a DNA segment of 984
base pairs in length, demonstrating a homozygous CC genotype, caused by a lack
of the C/T-13910 mutation (Bulhões
et al, 2007). A homozygous TT genotype is expected to show
no electrophoresis band when using FWTprimer, due to
a lack of the presence of the C/T-13910 mutation, which causes the forward
primer to fail to anneal. In contrast,
an electrophoresis band of 984 base pairs in length is expected to be produced
when using FMTprimer with homozygous TT genotypes due
to the presence of the CàT SNP (Bulhões et al, 2007). For
heterozygous CT genotypes, it is expected that both primers will produce faint
984 base pair length amplifications due to the replication of both the mutant
and wild type genotypes.
In
an attempt to gather more information on the lifestyles of individuals
diagnosed with hypolactasia, the group will attempt
to gather data involving all of MSU's cafeterias and calculate the ratio of
lactose-free menu options available for the month of February. We predicted that not all of the food items
available for consumption will be lactose free (Klein et al, 2002).
Ultimate
Findings
Our
first attempts to produce ASPCR using Yaku-Bonczyk
Primer design resulted in no bands being produced. Additional PCR assays were
then ran with varying annealing temperatures above and below the previously
used annealing temperature of 63°C in order to eliminate the possibility of our
primers not annealing due to temperature issues. The additional PCR assays
still resulted in no bands being produced. A new set of primers were then
designed, this time being approved as site specific by the Primer BLAST test.
Despite our newly designed primers being called site specific by the Primer
BLAST test, as well as being ran through PCR with varying annealing
temperatures, our desired bands of length 984 base pairs was still not produced
in our final gel electrophoresis product. Instead, no solid bands were produced
when both the FWTprimer and FMTprimer,
in conjunction with Rprimer, were ran with the same
DNA, leaving only faded bands at the bottom of the gel which suggests the
primers never annealed to the DNA, and the dNTP’s
were unused. To troubleshoot our assay once more, we eliminated the possibility
of the problem residing in our PCR cocktail. To test this, we made three
cocktails: one with FMTprimers, one with FWTprimers, and one with the lambda Rz
primers. All of these cocktails had the same exact reagents except for two
differences, that being each cocktail had their respective forward and reverse
primers and the lambda cocktail had lambda DNA, while the others used our DNA
from genome extraction. The resulting gel product produced a bright band in the
500 base pair length range for the lambda PCR product as expected, however no
bands were produced in the FMTprimer and FWTprimer PCR product (Figure 8). From this gel product,
and the fact that we used a range of different annealing temperatures in
previous PCR’s, we can conclude that our assay’s failure was related to our
primer design.
Initially we were
unable to successfully contact researchers who had previously done work
involving the C/T-13910 mutation in hopes of them sending us some DNA with the
known SNP. As a result, we attempted to create our own mutant DNA through the
process of mutagenesis. Our first attempt of the assay resulted in the
production of a gel electrophoresis product with no bands at the desired 4673
and 4885 base pair length location, but instead produced faint, diffuse bands
near the bottom of the gel that suggests the primers never annealed, possibly
due to the formation of primer dimers (Figure 8). New
primers were then developed, ensuring that they were site specific via the
BLAST Primer test and primer dimer formation would
not occur. Our second attempt of the assay still left us with no bands at the
desired location, leaving us with the possibility that our PCR procedure could
be corrected. Additionally, we produced 10 different variations of each of the
two PCR cocktail’s as stated previously in the methods section, every one
ending in failure of the production of bands. After further research we
discovered that taq polymerase, the enzyme used in
our cocktails for elongation, adds 1000 dNTP’s per
minute time elapsed at 72°C (Schirru et al, 2007). Every
single PCR we produced beforehand was run with an elongation time of two
minutes, leading us to predict that the reason our mutagenesis failed was due
to an insufficient time for elongation (Schirru et
al, 2007).
In
addition to our efforts of producing a successful ASPCR assay to diagnose hypolactasia, a sociological study was conducted in order
to gain a first-hand perspective on living with the hypolactasia
disease. We collected a vast amount of data regarding to the ratio of
lactose-containing foods to lactose-free foods that were present in all of the
cafeterias on Michigan State University’s campus. A total ratio of .84 lactose free meals per
total meals for all eleven cafeterias was calculated. This leads us to conclude that hypolactasia sufferers living on this campus do not
experience a very negative impact to their overall meal choice. Additional sociological
experiment was conducted in which we blind taste tested random individuals with
three different drinks: 2% milk, almond milk and soy milk. The purpose of this
experiment was to determine if people preferred the taste of products that
contained lactose (2% milk) over products that did not contain lactose (almond
milk and soy milk) in order to determine if people with hypolactasia
were required to consume less desirable products. An ANOVA test yielded a
statistically significant p-value for comparison between both almond milk and
2% milk, and almond milk and soy milk (figure), which leads us to reject the
null hypothesis. A non-significant value
was obtained between 2% milk and soy milk, failing to reject the null
hypothesis.
Future
Directions
Although we were
unsuccessful in producing a successful ASPCR assay to diagnose hypolactasia, there are still many additional adjustments
we can make in the future in hopes of achieving our goal. The most important
change we would make is pertaining to primer design, as neither our FWTprimer nor our FMTprimer (in
combination with Rprimer) were able to produce bands
in our gel product. The troubleshooting assumption made for the lack of proper
annealing was the result of a hairpin formation on the designed forward
primers. Our primer design was made to amplify the region of DNA upstream of
our C/T-13910 mutation site, creating an unavoidable area of 3 base pair intraprimer homology on the 3’ end of the forward primers
due to the requirement of having complementary bases upstream and downstream of
the mutation site. In order to avoid this, future experimentation would
comprise of new primers that would amplify a product downstream, rather than
upstream, the mutation site. This would remove the necessity of having intraprimer homology on our forward primers, as the bases
upstream the mutation site does not require intraprimer
homology.
If our newly designed
primers still resulted in improper annealing and did not produce the desired
bands in our PCR product, then we would abandon our Yaku-Bonczyk
primer design and revert to traditional primer design. It is possible that our Yaku-Bonczyk primer design method resulted in the primers
not annealing to the target DNA strand, as there were multiple sites of
non-complimentary base pair binding sites. While it has been demonstrated in
previous studies that the Yaku-Bonczyk primer design
increases discrimination against non-specific binding, other studies have been
able to successfully produce assays using traditional primer design that
diagnose mutations with SNPs (Schirru et al, 2007). The main difference
between the Yaku-Bonczyk primer design and
traditional primer design is that we would remove the intentional base pair
mismatch near the 3’ end of our forward primers, replacing it with the
complimentary base pair to the DNA target region. This would increase the
probability of our primers binding to their respective DNA target region, as
the traditional design method would result in only one base pair mismatch,
instead of two base pair mismatches as expressed in the Yaku-Bonczyk
design method.
An additional obstacle
we encountered with our experimental trials was the inability to
acquire/produce mutant DNA for our assay. We attempted to create our own mutant
DNA by inducing the C/T-13910 mutation into our genomic DNA via the process of
mutagenesis. Ultimately this did not yield our desired first round PCR products
of 4637 and 4885 base pair length DNA containing the C/T-13910 mutation, which
was indicated by no bands being produced from gel electrophoresis. We predict
this failure was likely due to a malfunction of the elongation phase of PCR
because research shows taq polymerase adds 1000 dNTP’s for every minute elapsed, and our elongation phase
was only ran for two minutes (Schirru et al, 2007). Seeing
as our desired PCR products were 4673 and 4885 base pair length, an ample
amount of time would be needed for taq polymerase to
add the dNTP’s to the extending strand. In future
experiments, we would lengthen the elongation phase of PCR to five minutes, as
that would provide sufficient time for taq polymerase
to completely elongate our targeted region of DNA.
Figures and Tables:
Figure 3. Gel electrophoresis of the custom
ASPCR assay along with lambda as gel control. This 1% agarose
gel was run at 95V for 45 minutes before this picture was taken. In wells 1 and
9 there is 9μl of 1kb plus DNA Ladder from Thermo Scientific. In wells 3-5
15μl of FWTPrimer PCR product was loaded. In the
remaining wells 6-8 15μl of FMTPrimer PCR
Product was loaded. Wells 3 and 8 had an annealing temperature of 63°C, wells 4 and 7 had an annealing temperature of 59.9°C,
while wells 5 and 6 had an annealing temperature of 56.5°C. In wells 2
15μl of Lambda PCR product was used as a gel control. From the gel it is easy
to see that the custom PCR assay was unsuccessful because the presence of band
1106 bp long was not visible. The gel however was run
successfully being bands appeared for lambda. The faint bands at the bottom are
most likely primers, which suggests unsuccessful
annealing of the custom ASPCR primers even though Primer BLAST determined the
primers to be template specific.