Identifying the 5T mutation of the CFTR gene using buccal cells from human test subjects and allele specific PCR









LB 145 Cell and Molecular Biology

Tuesday and Thursday 10:20 AM

Olivia Franklin, Imani Harris and Skyler Kerr

11/21/2017

Abstract

Written by: B815

Revised By: B850

Finalized by: B870

Many mutations of the CFTR gene, including the 5T variant, account for around 2% of cystic fibrosis (CF) cases (Cashman et al, 1995), with the 5T mutation located on the poly-T tract in intron 8 causing the deletion of exon 9 (Weimin et al, 2005). A PCR experiment was used to amplify strands of DNA containing the 5T mutation, and the 9T wild type for diagnostic purposes. DNA from the buccal cells of three human test subjects was extracted and purified before being added to a PCR cocktail. DNA known to contain the 5T mutation was obtained from a research facility and two PCR trials were completed. Both sets of DNA were put into two cocktails, one with primers designed to anneal to DNA with the 5T mutation, and one with primers designed to anneal to the 9T variant. The cocktails were then analyzed using gel electrophoresis to determine the length of the amplified DNA segment. It is hypothesized that PCR and gel electrophoresis would be able to prove the presence of the 5T mutation in DNA received from researchers, or in DNA from test subjects without CF symptoms, using published and designed primers. The 5T allele could be present in test subjects due to the fact that one could have the 5T mutation on one allele, meaning that they are a carrier of CF. The 9T variant, may or may not bind to test subject’s DNA (they could also have the 12T variant), suggesting that they do not have the 5T mutation on both alleles, and that the designed primers successfully annealed to the target DNA. It was predicted that the DNA of human cheek cells could be identified as mutant or wild type by the presence or absence of bands, where 5T was 424 base pairs long, and 9T was 428 base pairs long. PCR could be used as a noninvasive method of identifying the variant that has caused a patient’s cystic fibrosis or symptoms. This would also allow for future parents to be educated about the possibility of passing mutant alleles to their children through genetic counseling.

Figure

Amplification of DNA extracted from buccal cells using 5T mutant published primers and Lambda Rz Gene. Amplification products consisted of 6µl Kb+ Ladder in well labeled L; 6 µl of extracted and purified DNA from human epithelia using Chelex-100 along with increasing increments of 0.5 µl of MgSO4 starting at 1.5 microliters of MgSO4 in wells 1, 2, and 3; Lambda Rz gene in well 4; and 1 µl of bromophenol blue dye in each labeled well. Results of this PCR and gel electrophoresis show a successful Kb+ ladder and successful amplification of Lambda Rz gene, with some smearing. Bromophenol dye ran to 300 base pairs as expected. Wells 1-3 shows unsuccessful amplification of human DNA suggesting that test subjects were not carriers of the 5T mutation, or a possible error in PCR

Discussion

Written by: B815

Revised by: B850

Finalized by: B870

Experiment Summary

Cystic fibrosis (CF) is an inherited and sometimes fatal disease caused by various genetic defects in the CFTR gene (Welsh and Smith, 1995). This disease has many phenotypes due to a combination of missense/nonsense mutations and other mutation variants on the same allele (Farjadian et al, 2017). Among these are the 5-thymidine (5T) and 9-thymidine (9T) variants, which occur on intron 8 and in front of exon 9 of the CFTR gene, where the length of the Poly-T tract ranges from 5 to 12 thymines (,Farjadian et al, 2017). The relatively lower amount of thymines in the 5T variant of the Poly-T tract is known to cause the deletion of exon 9, whereas the higher quantity of thymines in the 9T variant allows for “normal mRNA splicing”, where exon 9 is not deleted (Tsui and Dorfman, 2013). Transcripts missing exon 9 causes a chloride channel defect resulting in a CFTR protein with no chloride channel activity (Massie et al). This is why the 5T variant, accompanied by higher class mutations, is known to cause mild forms of cystic fibrosis. The 9T variant has no effect on the CFTR protein, which is why it is considered our wild-type for this PCR experiment.

PCR tests are used to detect infections and disease, (Yoshimasa Yamamoto, 2002) but the focus is to see if it can detect the 5T and 9T variants in particular. It is hypothesized that PCR and gel electrophoresis would be able to detect the presence of the 5T mutation in DNA received from researchers, or in DNA from test subjects without CF symptoms, using published and designed primers. The 5T allele could be present in test subjects due to the fact that one could have the 5T mutation on one allele, meaning that they are a carrier of CF. The 9T variant, may or may not bind to test subject’s DNA (they could also have the 12T variant), suggesting that they do not have the 5T mutation on both alleles, and that the designed primers successfully annealed to the target DNA. It was predicted that the DNA of human cheek cells could be identified as mutant or wild type by the presence or absence of bands, where 5T was 424 base pairs long, and 9T was 428 base pairs long. The 9T variant of the poly-T tract mutation could be identified through gel electrophoresis, as well as the 5T mutation, because the designed primers have been written to anneal to the poly-T tract on intron 8 where the mutations are present, and collected DNA samples could have the mutation or variant (Chillon et al, 1995). The conducted research suggests that the wild-type primers are more likely to anneal effectively to the DNA extracted from healthy human test subjects’ cheek cells than the 5T mutant primers because of 9T is considered healthy DNA. This can possibly result in bands showing at the 428 base pair position for 9T as opposed to bands showing up at the 424 base pair position for 5T on the agarose gel (“Genome Browser Gateway”, 2013.). A successful amplification of the mutant and wild-type DNA will show that PCR could be used as a noninvasive method of identifying the variant that has caused a patient’s cystic fibrosis or symptoms. Running these tests can identify the if someone is a carrier of a possibly fatal mutation, allowing for future parents to be educated about the possibility of passing mutant alleles to their children through genetic counseling.

Original Predictions

In amplifying DNA from patients with CF from intron 8 using our forward and reverse primers, it is hypothesized that PCR and gel electrophoresis would be able to prove the presence of the 5T mutation in DNA received from researchers, or in DNA from test subjects without CF symptoms, using published and designed primers. 5T will cause the deletion of exon 9 which houses over 80 amino acids. The only time this will happen however, is if the patient is also a carrier of another CF causing mutation such as the missense R117H mutation or the nonsense ΔF508Del. (Lap-Chee Tsui, 1992.) In addition to the 5T mutation, forward and reverse primers were designed for the 9T variant to compare band sizes. Successful annealing was hypothesized to result in bands showing up for one mutation at a time. A lack of bands was hypothesized to suggest that there were errors made in the annealing processes, or errors in the concentration of MgSO4 in each sample.

Results and Ultimate Findings

Optimal PCR conditions were determined by calculating the annealing temperature for the forward and reverse primers of 5T and 9T used in this experiment (Tamburino et al, 2008.) and by determining the optimal concentration of magnesium chloride (MgSO4) which acts as a cofactor for taq polymerase during the reaction processes (Matthew Williams, 2017). Optimal MgSO4 concentration also depends on the specific template, buffer, DNA, and dNTP’s (Matthew Williams, 2017). If the concentration is too low, no PCR product will be seen. If the concentration is too high, undesired PCR products may be seen (Matthew Williams, 2017). In each trial, the agarose gel showed bands only under the well containing the Lambda Rz gene, and the Kb+ ladder. Due to the fact that the cheek cells were harvested from healthy test subjects without the 5T mutation, the designed and published primers for the 5T mutation would not anneal to the sample DNA. These findings demonstrate that the mutation was not present in the assay, concluding that DNA was not be amplified, and that PCR was not successful.

Future Directions

The first assay used PCR to identify the Lambda Rz virus, planning for it to act as the positive control. Different concentrations of MgSO4, ranging from 0.5 to 2.5 µL were used for 5 different trials. 9 µL of DNA and 3 µL of bromophenol blue dye were pipetted into each well and gel electrophoresis was ran for about 15 minutes. No bands could be seen, but the ladder was visible implying that there could be error in the DNA. After troubleshooting it was found that too much DNA was pipetted into the wells. PCR was run under the same conditions, but this time only 6 µL of DNA, and 1 µL of bromophenol blue dye was pipetted into each well. After gel electrophoresis was ran, successful bands were seen in wells containing 1.5, 2.0, and 2.5 µL of MgSO4. Through further research, it was concluded that the Lambda Rz virus is at approximately 324 base pairs. Our second assay attempted to amplify segments of DNA from human test subjects. This was completed by using a specific Chelex-100 protocol, and the successful concentrations of MgSO4 were used for each trial. Gel electrophoresis was ran for approximately 15 minutes, and no bands were seen under UV light. After troubleshooting it was concluded that experimental measures were correct, and that it was the PCR that failed. Given another attempt at this assay, PCR temperatures would be altered to accommodate the specific DNA. Given a chance to re-design this experiment, the Lambda Rz would still be used as a positive control under the same conditions, and genomic purification methods would remain unchanged. A new protocol would have 9T primers obtained first, as a second positive control, and they would then be tested with genomic DNA. 9T would act as a second positive control, because it is likely that one of the three healthy test subjects could have the 9T variant, giving a much better chance of annealing, and leading to successful bands, than the 5T mutant-type that was attempted. The 5T primers would then be obtained to anneal with genomic DNA containing the 5T mutation, giving a very high percentage for positive bands, and resulting in completion and success of this designed experiment.