 By Kayle Noble, Nate Zeitler, Kayla Mosey, and Alli Pearson
Abstract 5382insc is one of the most common of a class of mutations located on the BRCA1 gene known to increase risk of breast cancer. Enhancing the test for this gene could improve public health. PCR and gel electrophoresis can be used to obtain visual proof of the presence or absence of 5382insC based on four specific oligonucleotide primers designed in sets of two to detect mutant and wild type DNA. Mutant primers produced a band of 500 bp in length if the mutant DNA was present, while the wild set produced a band of 1000 bp if the wild type was present. A survey was conducted to test the hypothesis that science literacy would affect opinions on genetic testing. T-tests showed .05 level significance for some questions, but the overall opinion of genetic testing was not significantly affected by science literacy. Discussion BRCA1 is a
tumor suppressor gene that codes for a protein which aids in the
repair of damaged DNA. When a mutation causes this gene to
malfunction, damaged DNA more easily replicates, leading to an
increased susceptibility to cancer (Starita, 2003). The BRCA1
mutation of interest, 5382insC, is the insertion of a cytosine base
at the 5382nd nucleotide. An accurate PCR test for this mutation can
be achieved through a four oligonucleotide primer PCR design. These
primers were designed with unique nucleotide sequences causing them
to be complementary only to mutant or wild type DNA. The specific
primers designed give a band of 1000 base pairs for the wild type and
500 base pairs for the mutant type. Since DNA that is homozygous
for the mutation is thought to be embryonically lethal, any DNA with
this mutation would be heterozygous. Because of this, the mutant DNA
is composed of both DNA with the mutation and wild type DNA therefore
it would also produce a band 1000 base pairs in length.
Multiple distinct bands appeared in the PCR tests conducted. A band appearing at 500 base pairs suggests but does not guarantee that the DNA sample is at least heterozygous for the 5382insC mutation because the primers used to target the mutant DNA sequence were 500 base pairs apart. Similarly, a band of 1000 base pairs indicates the wild type is present because the wild primers are 1000 base pairs apart. The presence of a 500 and 1000 base pair band would confirm the sample as heterozygote. Since the mutation is embryonically fatal in its homozygous state this is the predicted positive result. In this experiment known wild type DNA along with the wild type primer pair, and known mutant DNA along with the mutant primer pair were tested in a single gel. The presence of a 1000 base pair band in the wild type and the presence of both a 1000 and a 500 base pair band in the mutant type area strongly support the hypothesis that the primers designed are a viable test for the 5382insC mutation. Previously published research has had success in detecting the 5382insC mutation with PCR and gel electrophoresis. PCR and gel electrophoresis successfully found 38 mutations in the BRCA1 gene of 160 patients with a family history of breast cancer (Stoppa-Lyonnet et al., 1997). Also, a great deal of reviewed literature has used further advanced techniques. A high through-put automated PCR allelic discrimination assay has been used along with two different fluorescent dyes to distinguish wild type BRCA1 genes from mutant BRCA1 genes (Abbaszadegan et al., 1997). Gel electrophoresis is not required when using this technique. Another technique, HRM analysis, or high resolution melting analysis, is referred to as the best method for detecting mutations in DNA (Takano et al., 2008). HRM analysis compares the melting time of mutant DNA to wild type DNA in order to distinguish between the two types of DNA (Takano et al., 2008). PCR and gel electrophoresis were chosen to be used in this experiment because of their relative simplicity and low cost. The improvement of genetic testing methods has many implications on society. Currently, screening for breast cancer is expensive and only performed on individuals with a high risk based on family history. Increasing the efficiency and efficacy as well as decreasing the cost of genetic testing would be beneficial. This could increase the number of individuals who choose to receive genetic testing early in life and ultimately augment those who choose to make preventative lifestyle choices. Furthermore, testing a larger population provides data that can be used to project carrier frequencies in a population. Population carrier frequencies estimate how prevalent a specific mutation is in a population. This allows the population to have an understanding of their risk for breast cancer and which specific mutation they may be predisposed to. A significant weakness of this test is that it only identifies the presence of one mutation out of over a thousand seen on BRCA1(Easton et al., 2007) and therefore would be of limited clinical use. Most of these mutations, however, do not have any effect and 5382incC is one of the three most frequent BRCA1 mutations (Shattuck-Eidens et al., 1995). It would be a moot point to attempt to develop a wide scale genetic test for 5382insC if few people would agree to take it. This raises the question of what affects a person’s opinions of genetic testing. One promising candidate for a factor is science literacy. Scientific literacy is a measure of a person’s knowledge of scientific terms in order to understand the fundamental nature of contending arguments on a given controversy in science (Miller, 1998). It was predicted people who are at a higher level of scientific literacy may be more knowledgeable of genetic testing and value its importance. Therefore, it was presumed that they would rank benefits of genetic testing very high and its consequences very low and ultimately favor genetic testing. Conversely, it was predicted that the less scientifically literate will rank consequences genetic testing higher and benefits lower. T-tests for difference of means obtained from our survey comparing opinions of people with 5 different levels of scientific literacy suggest that there is a significant difference in ranking for the questions dealing with the consequences due to insurance (p-value .0177) and stress (p-value .000123), and the benefits of taking preventative measures (p-value .0266) and making family decisions (p-value .000041). No statistical significance was found in the question dealing with the risk of family discord (p-value .2635) and, most importantly, no significance was found in the rankings of feelings toward genetic testing in general (p-value .0715) (table 1). We thus reject the hypothesis that science literacy has an effect on a person’s overall view of genetic testing. The t-tests did show that there was some correlation between scientific literacy and some of the specific benefits and consequences of genetic testing. Further testing could be completed which would examine a larger sample size. A larger sample size would strengthen the survey because it would represent the opinions of more people therefore decreasing variability. Genetic
testing has a great clinical significance. Its reliability and
efficiency is of major consequence to patients who depend on the
results to make life decisions. If this experiment were continued, it
would be useful to design efficient PCR tests for the two other most
common BRCA1 mutations: 185 del AG and 4184 del 4 (Shattuck-Eidens et
al., 1995). It would also be important to investigate more advanced
methods of testing for mutations, in order to find the most
dependable and efficient testing method. Fortunately, science is
always progressing and testing methods for genetic mutations are
continually improving. FIGURE 1. The
above figure is a gel comparing different concentrations of magnesium
in the PCR cocktail used in testing for wild-type DNA. Lane 1
contains a 1 kb ladder. Lanes 2, 3, and 4 contain wild-type DNA and
the pair of wild-type primers. Lanes 5, 6, and 7 contain wild-type
DNA and the pair of mutant primers. Lanes 2 and 5 both contain 2
mgμL
of MgCl2,
lanes 3 and 6 contain 4 mgμL
of MgCl2,
and lanes 4 and 7 contain 6 mgμL
of MgCl2
in the buffer solution of the PCR cocktail. Bands are present in
lanes 2 and 3 at approximately 1000 base pairs. The presence of a
smear in lanes 2, 3, 4, 5, 6, and 7 show nonspecific binding. Less
nonspecific binding occurred in lanes 2 and 5, where the PCR cocktail
contained a lower concentration of magnesium. |
