Traditional Primer Method Confirms the Absence of the G542X Mutation in IB3 Cells Using PCR and Gel Electrophoresis

 

 

 

 

 

By:

Katie Courville, Aaron Robins, Justin Ruszkowski, and Tim VandenBerg

 

 

 

Abstract

The G542X mutation that causes cystic fibrosis is a single point mutation on the 542nd base pair on exon 11 where a guanine is switched to a thymine (Haenisch et al., 2009).  The presence or absence of the G542X mutation was detected in DNA extracted from IB3 cells by PCR using forward wild-type (WT) and mutant primers (MT) and a reverse WT primer followed by gel electrophoresis.  A gel band of 502 base pairs for PCR product containing either the traditional MT primer or both traditional WT primers was predicted since the 1 base pair mismatch was placed at the 3’ end of the forward primers and the DNA template was 502bp long (Dieffenbach, 1993).  A band at 502bp using gel electrophoresis of PCR products with WT primers confirmed the IB3 DNA to have an absence of the G542X mutation.  Concentrations of KCl as well as MgCl2 were varied within PCR cocktail and were predicted to yield superior intensity at 10mM KCl concentrations and between 1mM and 2mM MgCl2 concentrations (Gelfand 1989, Kidd et al., 1995).  High concentrations of KCl have been shown to introduce great steric hindrance affecting the necessary bonding of a sufficient concentration of MgCl2 to Taq polymerase as well as to dNTPs during PCR (Haenisch et al., 2009).  10mM KCl as well as 1-2mM MgCl2 produced the brightest bands via gel electrophoresis.  Understanding the raw chemical aspects of how different components of a PCR cocktail react, especially regarding specific concentrations of various ions, could significantly improve modern applications of PCR including genetic testing.       

 

Discussion

           

The G542X mutation results in a switch of the genetic code “G” to a “T” at nucleotide 1756, which changes the 542nd amino acid of the CFTR gene from a Glycene to a stop codon (Kristidis et al, 1992). To diagnose our IB3 human bronchial epithelial cells for the G542X mutation, we ran numerous polymerase chain reaction tests on the purified DNA from the cells. A PCR test is essential to perform because it simply synthesizes a specific portion of DNA (Henegariu et al, 1997).  By designing three different primers: forward primer WT, forward primer MT, and reverse primer WT our PCR product proved that the G542X mutation was not present in our given DNA. The forward primer WT was used when a band was shown, therefore confirming the absence of the mutation. The band received was around the 500 base pair line, demonstrated by the ladder, proving the forward wild-type primer annealed properly. The primers were designed to be 502 base pairs in length.

Within the lab, we were able to compare and contrast two different styles for designing primers. The ultimate goal when designing a primer is to optimize specificity and efficiency of amplification (Dieffenbach, 1993). One procedure is known as the traditional PRC primer design procedure; where there is a one base pair mismatch at the mutation point. The other procedure is known as the Yaku primer design method. This is where there is a three base pair mismatch at the 3’ end of the allele, starting two base pairs away from the mutation site. This specific allele amplification method is supposed to yield a more specific band due to the three base pair mismatching, which decreases dNTP binding efficiency to the DNA (Yaku et al, 2008). By using the traditional primer design protocol, a faint band was confirmed around the 500 base pair line on the first try. When repeated, a second band, fairly brighter than the first, was confirmed at the same length; thus proving that for identifying the absence or presence of the G542X mutation, using traditional primer design is superior to the Yaku method.

Using the amplified DNA target region for the PCR, different concentrations of MgCl2 was used to make PCR buffers in hopes of optimizing band fluorescence, due to early success with using standard PCR buffer. A chemistry approach was taken when working with these buffer concentrations. The MgCl2 buffers were made with higher and lower concentrations of MgCl2, by varying intervals. The MgCl2 concentrations were varied by a spectrum of 0-6 mM. When lowering the MgCl2 concentrations, predictions were made that there should be no band or a very faint band yielded. This is because when there are small amounts of Mg2+ there are not enough of this divalent cation to torque and hold the dNTP’s negative tail (Haenisch, 2009; Dr. Sweeder). The torque of the tail opens up the nucleophile at the alpha site, which allows for the substrate to attack backside (Dr. Sweeder). High amounts of MgCl2 will result in no band because the DNA will not break their bonds for Taq to polymerize (Haenisch, 2009).

When MgCl2 is increased or decreased, it effects the binding of the dNTPs to the phosphate backbone of DNA. The dNTP has a negatively charged tail, which attracts Mg2+, a divalent cation. The molecular size of Mg2+ also plays a significant role in the torque of the dNTP tail (Haenisch, 2009; Dr. Sweeder). Once Mg2+ has formed bonds with the dNTP, the negatively charged tail bends which opens up its Alpha site (Haenisch, 2009; Dr. Sweeder). When the dNTP’s bind to the DNA phosphate backbone, it is done so in SN2 reaction form (Dr. Sweeder). The dNTP acts as the nucleophile while DNA’s phosphate backbone is the substrate, attacking the dNTP. By increasing the MgCl2 to a certain extent has been shown to have positive effects (Henegariu et al, 1997).  However, when too much MgCl2 is added to a PCR cocktail, the dNTP’s may not bind to DNA because the Mg2+ does not allow for the DNA double strand to separate (Kidd et al, 1995). Excess MgCl2 makes DNA’s noncovalent hydrogen bonds stronger, therefore not separating during denaturation (Kidd et al, 1995).

While experimenting with MgCl2 concentrations, the results received contradicted one another. The two broad spectrum gels showed two different bands at different concentrations. We hypothesize that this is due to different people making the cocktails, therefore there is no control. Also, our DNA was diluted one-fourth. We started with 57-59 ng/mL, and was then diluted to about 15 ng/m. This would effect how Mg2+ reacts within the cocktail

The KCl concentrations were increased by 1mM, starting at 0mM and ending at 5mM. As for KCl, studies supports evidence to show that primers which yield shorter amplification products do better in higher salt concentrations (Henegariu et al, 1997). Since our product is fairly short (502 base pairs), we predicted that increasing the KCl concentration would decrease band efficiency, where as decreasing KCl concentration would increasing band efficiency. When tested, results proved to support our hypothesis. The low concentration of KCl yielded a band, where as high concentrations did not. We attribute this to the KCl slowing down the rate of Taq-polymerase during PCR. A KCl molecule has a large molecular size, which effects how Taq can move within the aqueous solution of the cocktail. For Taq to move swiftly through the solution and polymerize DNA, it must be able to move sufficiently. Adding higher amounts of KCl will inhibit the Taq to move through the solution, therefore slowing down the rate that Taq can polymerize. High amounts of KCl are shown to increase the inhibition of Taq because Taq slows down in high salt concentrations (Gelfand, 1989).

 Another buffer molecule was researched in order to better understand buffer chemistry. Triton X-100 is a non-ionic surfactant which acts like a detergent molecule by grouping around Taq-polymerase, in the PCR cocktail, so Taq does not precipitate during denaturation (Dr. Sweeder; Gushchin and Deryugin, 1978) . The triton x-100 is separated by the polarities of the molecule when grouping around Taq.

To further support our product and to show that our primers were successful on our first try, we ran a restriction enzyme digestion procedure. Our band was 502 base pairs, which was cut at 215 base pairs, giving bands at 215 and 287 base pair sites. This was found using the NEBcutter V2.0, compliments of New England Biolabs. When cut, two smeared, but distinct bands appeared around the expected lengths. Smearing is accounted for when not using a blunt cut, resulting in sticky ends (Roberts et al, 1991).

This is relevant to science because in science, accuracy and efficiency is everything. Being able to optimize band fluorescence in PCR, due to a buffer, can be the difference in receiving convincing results or starting all over. For future directions we could look more into the physics and calculating the torque of the dNTP when it bends. Also, we could research more chemistry related subjects such as bond strength when different anions and cations attract to the DNA phosphate backbone or a dNTP,.Or, look at energy levels when bonds form or break during different reactions. But our main focus would be to gather significant data with the MgCl2 concentration tests.

 

 

 

 

 

 

 

 

 

 

 

Figures

 

 

 

 

 

 

 

 

                                                     

 

 

 

Figure 1: Gel confirming 502 bp band using 100 bp ladder

 Lane 1 consisted of 5 mL of 100 bp ladder. 100 bp ladder was used because we wanted to get a better band length estimate and the 100 bp ladder did exactly this. Lane 2 consisted of 10 mL of PCR product and dye. 10 mL product (with WT primer) and 2 mL dye was mixed and 10 mL of this was taken out. Lane 3 consisted of 10 mL control (MT primer) with 10 mL product and 2 mL dye (10 mL was taken). Our expected band is 502 bp and this band is pretty close to that. This band is extremely bright which means that our primers are definitely correct.