PCR test built to amplify exon 20 of CFTR gene in purified cheek cells with an Rz gene control test

By: Alexis Anderson, Nicole Curtis, and Sage Wood

LB 145 Cell and Molecular Biology

Tuesday and Thursday 3:00-4:50 PM

Marla Nazee and Hayden Stoub

11/21/2017

(Title page written by: B915, revised by: B780 and B940)

Abstract

Written By: B780

Revised by: B940

Finalized by: B915

The purpose of our experiment was to design a diagnostic assay using PCR to detect the W1282X mutation of the CFTR gene that causes cystic fibrosis in the Ashkenazi Jewish population. It was hypothesized that creating one base pair mismatch on a primer to a mutant DNA sequence and, therefore, two base pair mismatches for a wild-type DNA sequence (or vice versa) creates discrimination against the differing DNA sequences when running a polymerase chain reaction assay, resulting in amplification of only the target DNA. This will occur, because one base pair mismatch can still continue the extension process, while two base pair mismatches results in too many mistakes for full extension, based on previous research by Yaku (Yaku et al, 2008) (Figure 2). For our experiment, we first amplified the Rz gene of bacteriophage lambda, using the 1Rz1F and 1R primers in a PCR cocktail. We then purified human DNA using Chelex-100 Resin beads and DNA from our saliva. Next, we used the extracted DNA and primers from previously published research to amplify the DNA segment containing the W1282X mutation, located on exon 20 of the CFTR gene. The resulting base pair length for the Lambda Rz gene PCR reaction was 351 base pairs. The primers were designed to amplify a region of 394 base pairs long. A total of 0.0008034 mg of DNA was collected and the average concentration was 0.16125mg/mL. The length of our published primers will be 470 base pairs, because the forward primer will start annealing at nucleotide base pair 45961 and the reverse primer will start annealing at nucleotide base pair 46533 (Sanger et al, 1982). Researchers in the future may be able to continue on from our research using our proposed primer concentrations, annealing temperatures and designed primers to detect the W1282X mutation in cystic fibrosis patients. Our research could build upon the limited existing research on this mutation to give a more efficient method for polymerase chain reaction.

Discussion

Written By: B780

Revised by: B940 and Finalized by: B915

Experiment Summary

Cystic fibrosis is a disease caused by a homozygous autosomal recessive gene mutation on the seventh chromosome of the human genome (Zhao et al, 2017). The Ashkenazi Jewish population suffers from a unique mutation of the disease, W1282X, which makes up 4% of cystic fibrosis patients worldwide (Kerem et al, 1990). No CFTR protein is expressed in the cell membrane because a stop codon causes a poorly functioning truncated protein to be made (Haggie et al, 2016). This disease causes a buildup of thick mucus producing symptoms including difficulty breathing, salty sweat, disrupted liver function, and increased risk for infection, making day-to-day life extremely difficult (Welsh and Smith, 1995).

The purpose of this experiment was to develop a diagnostic assay to amplify the region of the CFTR gene where the W1282X mutation is located. Combining recommendations of annealing temperatures, primer concentrations and primer design from three sources of previous research will produce a reliable PCR diagnostic assay to identify the genotype of those with the W1282X cystic fibrosis mutation (Kerem et al, 1990) (Yaku et al, 2008) (Shoshani et al, 1992).

Original Predictions

The first step in this experiment was testing the controls. For the positive control, the primer pair of 1Rz1F and 1Rz1R targeted a portion Rz gene that is associated with the lambda virus. We predicted the length of the target region would be 394 base pairs, because the forward primer would start annealing at nucleotide position 45961 and the reverse primer starts annealing at nucleotide position 46355 (Sanger et al, 1982). For the next step in the experiment, we predicted that the product of purified DNA samples will range from 2.37 μg to 29.07 μg, because that was the average range of product when tested using Chelex 100 resin and approximately 2 ml of saliva (Rogers et al, 2007). The next step to this experiment was testing the published primers Kerem et al. used to amplify exon 20 of the CFTR gene, which includes the W1282X mutation. We predicted the length of this product will be 470 base pairs long, because the forward primer will start annealing at nucleotide position 163905 on the CFTR gene and the reverse primer will start annealing at nucleotide position 164375 on the CFTR gene (Zielenski et al, 1991).

Using the Yaku-Bonczyk method, the designed primers for this experiment were designed to amplify the target region of the DNA, either mutant or wild-type, through PCR. We predicted the length of the PCR products, either of the W1282X mutation or the wild-type, will be 1,025 base pairs long, because the forward primers of both the mutant primer (primer 1) and the wild-type primer (primer 2) will start annealing at nucleotide position 164175 on the CFTR gene and the reverse primer, which is the same for both mutant and wild-type DNA, will start annealing at nucleotide position 165200 on the CFTR gene (Zielenski et at,1991).

When designing these primers, only specific primers will anneal to specific DNA. We predicted forward primer 1 will only anneal to the target DNA that contains the W1282X mutation, because the primer only includes one intentional mismatch near the 3’ end, but the one mismatch is not enough to prevent extension during PCR (Figure 2) (Yaku et al, 2008). The same explanation will apply to the wild-type DNA and forward primer 2. One of the main characteristics of the Yaku-Bonczyk model is the mismatches that either allow the primer to anneal to the target DNA, or prevent the primer from annealing to non-target DNA. This will be observed when testing forward primer 2 with DNA that is wild-type, which does not have the W1282X mutation. We predicted that primer 1 will not anneal to the wild-type DNA and no band will appear in the gel, because the primer and the DNA will have two mismatches on the 3’ end, which, as previously studied, with multiple mismatches towards the 3’ end of a primer the primer will not be able to anneal and extend the target DNA (Figure 2) (Ghedira et al, 2009).

Ultimate Findings and Implications

For our positive control using the lambda Rz gene, we obtained a band length of 351 base pairs. This experimental value is smaller than the actual band length should have been, which was 394 base pairs. The amount of DNA that was purified was 0.0008034 mg, which is in a 206 µL solution of cheek cells and chelex resin. The purpose of the chelex was to bind and remove ions from the DNA, as well as protect the DNA during the incubation at 100℃ (Polski et al, 1998). The purity of our sample was 1.438, which is below the desired purity of 1.7-2.

The final stage of this experiment was testing the published primers with the extracted DNA that was purified. After running PCR with the published primers and DNA, no bands were obtained. The first stage in troubleshooting was to see if the purification of the DNA was done incorrectly. During the first trial of purification, the cheek cells and chelex solution was initially incubated for 10 minutes with temperature ranging from 70-90℃ and then incubated for another 10 minutes at the desired 100℃. The initial incubation was problematic, because it was not at the right temperature and was done in a limited amount of time. The second incubation was done at the full time to ensure the DNA was released from the cells. Since bands were not obtained using the first trial of DNA, DNA was purified again, following the protocol exactly, and then amplified through PCR. This troubleshooting method did not succeed, as there were no bands at the desired length, but primer dimers appeared when running the PCR through gel electrophoresis. Next, the published primers and the primer dimers were troubleshooted by adjusting the annealing temperature to 45℃, rather than the 55℃ the published paper used. This adjustment was made, because both the forward and reverse primer’s annealing temperature was calculated, using the Thermo Fisher ™ Calculator and NEB Tm calculator, to be 45℃. When running this trial through PCR, no bands appeared. For trial 3 of troubleshooting, previous research has reported that high specificity amplification results at temperatures above the calculated annealing temperature, but below the lowest melting temperature of the primers (Hecker and Roux, 1996). This was tested by creating a temperature gradient that ranged from 55-60℃. When this method was ran through gel electrophoresis, no bands appeared. The final troubleshooting method that was tested was increasing the amount of DNA used. Since the purity of the DNA was not as high as it should be, it was hypothesized that the amount of DNA used in the PCR reaction should increase. When increasing the amount of DNA in the PCR and using the same protocol the published paper used, no bands appeared.

Figure 5: Amplification of the CFTR gene using PCR, with analysis through gel electrophoresis. The target DNA sequence of 470 base pairs of the CFTR gene was amplified using PCR and 1P1F forward primer and 1P1R reverse primer. The PCR cocktails contained nuclease free water, 10X PCR buffer, 10 mM dNTPs, 1P1F forward primer, 1P1R reverse primer, Human DNA, Taq polymerase, and MgSO₄. The PCR cocktails was ran in a thermocycler for 30 cycles. The PCR products were analyzed using a 1.5% agarose gel, which included Invitrogen agarose powder, 20X LB (Lithium Borate) buffer, DI water, and SYBR-Safe red dye. This was ran at 180 V for 12 minutes. Well 1 contained 1 Kb+ DNA Ladder and 6X blue loading dye. Well 2 contained PCR product using an increased amount of DNA (10uL) and recommended annealing time (30 seconds), and the loading dye. Well 3 contained PCR product using the increased amount of DNA and increased annealing time (45 secs), and loading dye. Well 4 contained PCR product using smaller amount of DNA (7uL) and increased annealing time (45 secs), and loading dye. The gel was then observed under ultraviolet light to see if any bands appeared, and no PCR products were observed.