PCR detection of the N314D mutation on the GALT gene for Galactosemia Type I in S9 human epithelial cells

By: Nick Campbell, Allison Chan, Alexus Cox, and Davis Thomas

“Team Spider-man”

LB 145 Cell and Molecular Biology

Michigan State University

11/23/11

Abstract

Galactosemia Type I is an autosomal recessive disease caused by the N314D point mutation on the galactose-1-phosphate uridylyltransferase (GALT) gene. The N314D mutation occurs with an adenine-guanine transition on the 968^th nucleotide on the GALT gene (Elsas et al, 1995). We hypothesized that using our primers designed to amplify segments of DNA on the GALT gene under specific temperatures, controlled cocktail ingredients, and experimentally designed durations of PCR cycles, we will be able to accurately diagnose the presence or absence of the N314D mutation (Elsas et al, 1995). We also hypothesized that the primers designed with the Yaku method will anneal more specifically because the intentional mismatch creates less of a chance of false diagnoses (Yaku et al, 2008). The significance of this research and purpose was to verify that PCR is an efficient and accurate way in diagnosing Galactosemia Type I with our precise primers and annealing temperatures determined. Our research was significant as additional evidence that the Yaku primers produce less false positives in our comparison to non-Yaku primers. Identification of unsuccessful and unexpected amplified DNA was analyzed under agarose gel electrophoresis by comparing the band lengths of the amplified DNA against the 1Kb+ ladder. Our results refuted our hypothesis by portraying the two mutant primers’ ability to bind and amplify DNA on wild-type DNA while wild-type primers were unable to amplify DNA on wild type DNA. Four weeks were also taken to study four subjects’ transitions to a strict no lactose diet, as a patient of Galactosemia would adhere to. Changes in cholesterol were analyzed. We predicted that our HDL levels to increase and LDL levels to decrease because it has been verified through previous research that the increase of lactose in one’s diet is directly related to high cholesterol levels because of the high amounts of saturated fats in dairy products containing lactose (Sears, 1978). Results showed that 72.4% of the foods available for a combo exchange meal plan were unable to be consumed because of their lactate content. Our paired-sample t-test yielded a p-value > 0.05 which verified that any change in HDL/LDL levels cannot be linked to the absence of lactose/galactose in diet.

Discussion

Experiment Summary

Galactosemia occurs in approximately one in 50,000 people and if not diagnosed at birth, life-threatening complications emerge within a few days including brain damage, enlarged liver, and kidney damage (Sockwell and Glen, 1959). Patients with this disease cannot digest food containing the sugar galactose, due to the mutation of the GALT gene on chromosome nine, causing the enzyme that would normally break down the galactose to be ineffective (Elsas et al, 1994). The N314D mutation specific to Galactosemia Type I involves a single base pair substitution at the 968^th base pair from adenine to guanine (Elsas et al, 1994). There has been diagnostic research in which PCR has been used to amplify the N314D mutation, but there has been little evidence showing that PCR has been utilized strictly for determining presence of the Galactosemia disease in the human genome (Elsas et al, 1994; Schweitzer, 1995). Our goal was to show not only that PCR is a reliable diagnostic tool for infants needing to be tested for Galactosemia Type I, but to also compare the different degrees of success between using complete base pair match primers and intentional mismatch primers, known as Yaku primers, on the GALT gene (Yaku et al, 2008). We hypothesized that using our primers designed to amplify segments of DNA on the GALT gene under specific temperatures, controlled cocktail ingredients, and experimentally designed durations of PCR cycles, we will be able to accurately diagnose the presence of the N314D mutation (Elsas et al, 1995). We also hypothesized that the primers designed with the Yaku method will anneal more specifically because of the intentional mismatch creating less chance of false diagnoses (Yaku et al, 2008).

Along with primer design, we conducted a psychological and nutritional experiment; four weeks were used to analyze the effects of a strict galactose restricted diet. For 30 days, we were utilized as four human subjects that once digested galactose multiple times per day to go without any milk products. We hypothesized that our LDL cholesterol levels would decrease and our HDL cholesterol levels increase because lowering lactose intake will decrease the amount of saturated fat consumed, which is linked to cholesterol levels (Sears 1978).

Primer Design

We designed two Yaku forward primers, YWF and YMF and two non-Yaku, forward primers, NWF, and NMF. These primers were designed to bind specifically to the base pair 34,649,442; the base pair location of the point mutation (Kent et al, 2002). The YWF and YMF primer were designed with a method to limit the amount of false positives by adding an intentional mismatch on the primer that would bind to base pair 34,649,440; two base pairs before the N314D mutation on base pair 34,649,440 (Yaku et al, 2008). The intentional mismatch is 3 base pairs from the 3’ end of the primer. This would prevent the YWF primer from annealing improperly if the N314D mutation is present. Our goal was to find the most optimal way one could attempt to amplify the DNA of the N314D mutation, with our designed primers, to successfully diagnose somebody that may or may not have Galactosemia Type I.

Genomic Purification

From the genome preparation of one million human epithelial cells we initially predicted that the yield from the purified samples would be between 3-8 μg DNA because this is the typical DNA yield obtained when using the Generation Capture Column Kit. After executing the protocol there was 800 ng DNA that were purified. By analyzing wild type DNA (S9 cells epithelial cells), without Galactosemia, PCR and specific primers and the length of the amplified DNA were analyzed through gel electrophoresis to attempt to verify the absence of the N314D mutation.

Controls

Previously successful PCR primers AF and AR were used as a control to ensure that our assay was adequate enough to successfully achieve DNA amplification using PCR (Elsas et al, 1994). These primers were predicted to amplify DNA to create a band length of 934 base pairs long because of previous PCR amplification success (Elsas et al, 1994). Through PCR and gel electrophoresis a small band length of less than 100 base pairs appeared. Smaller bands may be due to mispriming, where primers may have additional complementary regions with the DNA template (Don et al, 1991).

Furthermore, our second control for this experiment included the PCR and gel electrophoresis analysis of the Escherichia coli pathogen. Cells from Escherichia coli were used in a PCR cocktail in order to amplify a 521 bp long segment of DNA. This control validated the amounts and concentrations of the ingredients in our PCR cocktail. We confirmed that 5.0µl of 1X ThermoPol buffer from NEB, 0.2µl of 1 U/ul Taq polymerase, 2.0µl of 2uM forward primer, 2.0µl of 2uM reverse primer, 1.0µl 200mM dNTPs, 1.0µl 50mM MgCl₂ and 37.0µl nuclease-free water were all adequate enough in order to amplify DNA successfully. It also allowed us to become familiar with the process of experimenting with different annealing temperatures in order to find the most ideal temperature.

PCR Analysis

We predicted that from the genomic DNA, PCR would amplify the N314D mutation of the GALT gene located on chromosome 9 because this is where the A-to-G transition of nucleotide 968 takes place (Lin et al, 1994). This will transpire due to the selected primers, reaction cocktail, specific PCR temperatures and times, and repeated cycles. When we analyzed NWF and YWF primers they appeared to have a band length less than 100 base pairs. This could happen when the primers are too similar with each other and interactions can occur between them (Brownie et al, 1997). As an additional control for determining the possible primer dimer we removed the DNA template but the primers did not anneal to each other. This confirmed that the low band length was not due to the primers annealing to one another.

We predicted that primer YMF and NMF would anneal to the mutant type template because our primers are designed to anneal to only one complementary strand (Schochetman et al, 1988). Through PCR and gel electrophoresis we analyzed that the YMF and NMF annealed to the wild-type DNA template with a consistent 500 base pair band length. The consistency of the mutant primers annealing to the wild-type DNA may be that the mutant forward primers are annealing to an alternative place on the gene, called mispriming (Chou et al, 1992). Our hypothesis was supported through analysis of 500 base pair band length that the Yaku designed primers were more specific than the non-Yaku designed primers. The non-Yaku primers showed a greater amount of non-specific binding resulting in less specificity.

For all the primers including control primers, YWF, NWF, YMF, and NMF the predicted band length amplification was unsuccessful but bands were acquired on gels for each primer. However, they ranged from 500 bp to less than 100 bp. Primers range around 20 base pairs with this the likelihood of a particular region of the genome being complementary to 12 of the 20 base pairs is about 180 places in the genome, which the primers could match up and anneal to (Cha and Thilly, 1993). Errors in these cocktails, which caused the unsuccessful amplification, may be due to adding the Taq polymerase before running PCR. This could be solved by running the hot start procedure.

Physical, Psychological, and Emotional Aspects from Galactosemia

During the 30 days experiment of adhering to the strict Galactosemia diet, all dairy products, which contain lactose, were eliminated from our diets. Lactose is a disaccharide that is composed of two monosaccharides, glucose and galactose. Therefore we excluded all lactose because galactose is a component. Our transition into this diet was analyzed on changes in HDL, LDL, and total cholesterol levels. We predicted that with the decrease of galactose in our diet cholesterol levels would also decrease because high intakes of galactose products have been linked to high levels of saturated fat, and as a result increase LDL levels (Sears 1978). Our obtained p-value>0.05 refuted our hypothesis. This could be due to not being on the diet long enough or the unawareness of galactose in our food.

Also, we collected statistical data that verified how difficult it is being an MSU student with Galactosemia. The food, available at a local store, Sparty’s, was assessed and the percentage of food unable to be consumed at Sparty’s for the average MSU student’s combo exchange meal plan was determined. We found that 27.6% of the merchandise available for a combo exchange meal plan excluded lactose and 72.4% of the merchandise contained lactose, therefore could not be consume. This demonstrated the difficulty we experienced finding appropriate food to fit our lifestyle.

Future Direction

YWF and NWF primers were designed to anneal to the wild-type DNA template with a 1,038 bp band length. Through PCR and gel electrophoresis a small band length of less than 100 base pairs was amplified. A control cocktail was made without the DNA and confirmed that the low band length was not due to primer dimer. However, another frequently encountered problem in PCR amplification is the appearance of smaller bands due to mispriming, where primers may have additional complementary regions with the DNA template (Don et al, 1991).

YMF and NMF primers annealed to the wild-type DNA template, but were designed to anneal to the mutant-type DNA template. The band length appeared to be about 500 base pairs and was expected that no band would amplify. This could also be an amplification of the primer pairs annealing to non-target sequences on the DNA template (Chou et al, 1992). To troubleshoot for mispriming the hot start procedure would be used, which would increase the specificity and precision of PCR amplification reducing non-target annealing (Chou et al, 1992). With this there should be no band length with YMF and NMF primers and wild-type DNA template.

For the control primers, YWF, NWF, YMF, and YWF the hot start procedure would be used. The hot start procedure is when after the 5 minute pre-denaturation step at 94°C, taq polymerase and MgCl₂ are added (Hoover and Lubkowski, 2002). Using the hot start procedure would decrease the chance of mispriming. Along with running hot starts, the annealing temperatures will be varied in order increase PCR specificity.

Also, our experiment could be improved by obtaining mutant DNA samples for Galactosemia. The NMF and YMF could be run in PCR with the mutant DNA and a 1,042 base pair should appear. If the predicted band length of 1.042 base pairs appeared when using the YMF and NMF with the mutant DNA, this would be used as a control to confirm that when the mutant primers were ran with wild-type DNA, mispriming had occurred.

The nutritional aspect of our experiment could be improved by maintaining the galactose-free diet for a longer period of time. Also, the psychological aspect of the experiment could be improved by conducting a survey using ourselves as the subjects. We would be able to analyze psychological effects we went through while be on the diet.

Figure 5. Unsuccessful PCR amplification of N314D mutation using NMW and YMF primers under varying conditions. Lanes 1 and 6 contain 1Kb+, 0.1µg/µL ladder. Lanes 2, 3, 4, and 5 contained mutant primers on wild-type DNA. Lanes 2 and 3 had NMF and lanes 3 and 4 had YMF. Thermocycling conditions included 5 minute denaturation at 94°C, 30 cycles of annealing of 30 seconds at 50°C and 45 seconds at 72°C, and final elongation at 72°C for 7 minutes for lanes 2, 3, 4, and 5. Bands appeared at lanes 2-5 at 510 base pairs.1% TBE (Tris-base EDTA) buffer on agarose gel was used to run the bands at 110V. Annealing temperature was held constant at 50°C and concentration of DNA in the wells was held constant as well. Mutant primers were not supposed to portray bands on wild-type DNA. We concluded that mispriming may have occurred. In order to conclude this, we would have to run the designed mutant primers on mutated DNA.

View our Physical, Psychological, and Emotional Aspects from Galactosemia Video on YouTube.

http://www.youtube.com/watch?v=cnSRlbOYr8Y