Proposal Summary
We propose to develop a lab-on-a-chip device for the detection of DNA double strand breaks in situ. Current technology requires laborious manipulation of the cell sample by fixation and staining with antibody and an optical-based detection. All in all, the process may take up to several days before results are retrieved. The development of an on-site, immediate monitoring system will greatly benefit our understanding of DNA damage causes and prevention as well assessment of radiation risk by tracking such events instantaneously. Our proposed device is based on individually addressable carbon-nanotube (CNT)- array capable of multiplexing for detection of multiple analytes and targets from a biological sample. The impact of this device in our information-based world is manifold. Not only will the device make it accessible for the user to track, assess and follow trends of their own DNA damage levels but also empowers the user in making more informed lifestyle choices. Introduction
With rising urbanization, the DNA in our cells is increasingly being exposed to DNA damaging agents such as UV light, mutagenic chemicals, reactive oxygen species generated by Ionizing Radiation (IR) or redox cycling by heavy metal ions and radio-mimetic drugs etc. DNA Double-strand breaks (DSBs) occur when reactive oxygen species react with DNA bases causing lesions in the chromosomes and the formation of foci known as Radiation-induced foci (RFI). RFI are marked by the
DNA Profiling exists in blood, bone, hair follicles, saliva, semen, skin and sweat. They are the same in every cell and retain their distinctiveness throughout an individual’s lifetime.
As our cells divide and our DNA replicates, our telomeres provide a start and an end point for the enzyme DNA polymerase to attach to our chromosomes and replicate our DNA. By using a repeated, non-coding section of DNA as the starting point for this replication, the telomeres allow our cells to replicate without damaging important genes at the attachment point of the replicating enzyme. As this occurs though, our telomere begin to lose base pairs
In this lab, we used two different strains of Saccharomyces cerevisiae (yeast) cells. One of them was, Saccharomyces cerevisiae, UV-Sensitive Strain, G948-1C/U, alpha, rad1 rad18 phr1 ura3 mutant in excision repair (Haploid), while the other was a wild type strain of this yeast. We chose to do this, because it is important to compare UV sensitive yeast cells, which have a mutation that yields them unable to repair themselves, to a wild type that is able to repair itself after UV radiation exposure. According to Conconi, “monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Δ) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after
The main problem with chromosome instability produced by these breaks is the susceptibility to translocations and thus oncogene activation.
By helping to repair DNA, the BRCA2 protein plays a critical role in maintaining the stability of a cell 's genetic information. The cancer risk caused by BRCA2 mutations is inherited in a dominant fashion, even though usually only one mutated allele is directly inherited. This is because people with the mutation are likely to acquire a second mutation, leading to the dominant expression of the cancer. A mutated BRCA gene can be inherited from either parent. Because they are inherited from the parents, they are classified as hereditary or germline mutations. Because humans have a diploid genome, each cell has two copies of the gene (one from each biological parent). Typically only one copy contains a disabling, inherited mutation, so the affected person is heterozygous for the mutation. If the functional copy is harmed, however, then the cell is forced to use alternate DNA repair mechanisms, which are more error-prone. The loss of the functional copy is called loss of heterozygosity (LOH). Any resulting errors in DNA repair may result in cell death or a cancerous transformation of the cell.
On September 12, 1016, Belmont University graciously allowed Dr. Katherine Friedman from Vanderbilt University to come and talk to a crowd of students about the tendencies of how deoxyribose double stranded breaks can during cell replication and the elements required to hopefully repair this ordeal. She began the session by discussing what chromosomes are composed of and how they are produced, accompanied by visual and statistical representations. Moving on, she touched on how double strand breaks are a huge threat to a cell's, an organisms, stability. Correspondingly, she described what can cause these breaks; chemical factors, as well as inner cell disruptions during replication that are sometimes hard to remedy. However, she also stated that this breaks can occur on purpose, mostly in the immune system in efforts to make antibodies.
A mutation is any type of alteration or change in DNA. There are many types of mutations that can occur. Depurination and deamination are common mutations that happen spontaneously. Depurination is a hydrolysis reaction that leads to the loss of purines in DNA. Deamination is also a hydrolysis reaction, and the cause of this reaction is an amino group gets detached. These types of mutations cause an alteration in the base sequence of amino acids and also effect the way a gene reads a protein. Another cause of DNA mutations may be environmental elements such as: chemicals or radiation. (pg. 567) A common chemical that is a mutagen is cigarettes and a example of
After the spike in DNA discoveries and confirmations that could be compared to the 1849 California gold rush, scientists began to try to find other uses for DNA. Since then, DNA has been used for many things such as finding criminals and confirming paternity/maternity. Also DNA has been used to track diseases and problems that start at the molecular level. Three of the newer advances in DNA technology are DNA Fingerprinting, Recombinant DNA (rDNA) and Paternity/Maternity Tests.
Although DNA remains mostly stable, it is constantly subject to chemical modification from a variety of different sources. Numerous forms of DNA damage have been identified resulting from different factors such as alkylating agents, hydrolytic deamination, free radicals, and mismatching base pairs. Alkylating agents cause mutations of legitimate bases, transforming them in to either a non-coding or lethal lesion in the DNA. Hydrolytic deamination changes on base on the DNA double helix into another. (Sinha & Häder, 2002) Oxygen radicals are generated during cellular production of O2 gas is known to either attack the bases of DNA (A, T C, or G) or the Deoxyribose to damage the DNA bases or break strands. (Marnett, 2000) DNA remains under threat by other cellular factors, but it does have certain mechanisms in place to repair certain
Micronucleus test is considered as one of the main cytogenetic parameters, which record partial or whole chromosome breakage during mitosis [17]. Therefore, the efficacy of 5-ALA/PDT to induce DNA damage, and release chromatin during mitosis, was detected by MN test. When MCF-7 and HepG2 cell lines were treated with 5-ALA/PDT at 633nm, the ratio of micronuclei was increased significantly in comparison to non-treated groups. Some research studies demonstrated that not all sensitizers have the ability to induce DNA damage or even cause cytotoxicity when applied with laser irradiation, such studies include the observations of Zenzen and Zankl [23] who demonstrated that h-ALA/PDT did not induce significant chromosome aberrations or micronuclei neither in tumor cell line RPMI 2650 nor fibroblast cell line WS1, however, it caused cytotoxicity to RPMI 2650 tumor cell line. Also, the results of Horinouchi and Arimoto-Kobayashi [27] found that 5-ALA/PDT did not induce photogenotoxicity in keratinocyte NCTC2544 cells as evaluated by MN test, but PpIX disodium salt-induced genotoxicity after exposing the cells to 5-ALA/PDT with UV irradiation of 5J/cm2
Skin tumor in human body proceeds through three major steeps, initiation, promotion, and progression. When UV exposed DNA are not repaired, photoproducts will form through the gene giving raise to the mutation in coding region of tumor suppressor gene, p53. This mutation is thus considered as the initiation step of multistep carcinogenesis. Following initiation, if mutated genes get over exposed with UV light, promotion of benign tumor will take place. Furthermore, if those cells are continuously in contact with UV irradiation, inhibition of apoptosis will eventually lead to tumor progression. Those mutated genes will increase in copy number causing additional mutation and gene rearrangement that result in genetically unstable malignant skin cancer (Daya-Grosjean and Sarasin, 2005).
In addition, it is also shown that γ-irradiation causes DNA lesions that are not well repaired in HGPS cells4. As the study shows, molecular details about how repair proteins and checkpoints are responsible for DNA repair mechanism are still unknown4. To unravel the complications in deficiency of DNA repair mechanisms and their relationship with H3K9me3, immunofluorescence staining and confocal microscopy were carried out and it was found that γ-H2AX/53BP1 foci were associated with regions enriched with H3K9me34. Moreover, to analyze the hypothesis that H3K9me3 poses a barrier for defective DNA repair, SUV39 was knocked down by siRNA and it was revealed that DNA damage foci decreased significantly which proved the hypothesis tested4. In addition to this hypothesis, it should be also proven if SUV39H1 levels directly contribute to deficiency of DNA repair and checkpoint response proteins in progeria. For this purpose, levels of SUV39H1mRNA in cells with ectopic progerin were analyzed and it was concluded that SUV39H1 levels were significantly increased in mouse embryonic fibroblasts (MEFs) in comparison to the wild type control4. Based on previous hypotheses, it can be implied that since H3K9me3 is mainly catalyzed by SUV39H1 methyltransferase, levels of H3K9me3 must be higher in Zmpste24 - / -
First, 4:4 can occur when recombination doesn’t happen. Secondly, 2:2:2:2 can happen when all of the pairs of chromosomes are ordered in line and then recombine. Lastly, 2:4:2 recombination occurs due to independent assortment. So both 2:2:2:2 type and 2:4:2 types occur through recombination. The effects of radiation on DNA are cell killing and mutagenesis. Continued exposure to radiation can cause bad effects on living matter. “Radiation either ionizes or excites atoms or molecules in living cells, leading to the dissociation of molecules within an organism. The most destructive effect radiation has on living matter is ionizing radiation on DNA. Damage to DNA can cause cellular death, mutagenesis (the process by which genetic information is modified by radiation or chemicals), and genetic transformation. “ (Joseph Chao, Linda Su(date not found).The Effects of Radiation on Matter. Retrieved
Therefore in measuring the effects of microwave radiation on DNA one is measuring the effects of a portion of solar radiation on DNA. With this stated the hypothesis was correct due to longer periods under microwave radiation weakening the DNA more than shorter periods. This means that the longer period of time spent under solar radiation, the more damage inflicted upon one’s cells and thus DNA and the higher one’s risk of developing cancer.
Modification of damaged DNA seems to be an understudied subject, there is much to understand on the restoration of DNA damage, repair and DNA methylation. Genomic DNA can be modified by methylation but much of it is affected on a gene when silenced. When epigenetic modification has been implicated with cancer and aging it causes DNA methylation to also have an impact on the double strand of DNA analysis. Modification as such provoke deteriorating changes like aging found in multicellular organisms and DNA damage may magnify biochemical pathways that regulate a cells growth or control DNA replication with DNA repair. In the article “DNA Damage, Homology-Directed Repair, and DNA Methylation” written by Concetta Cuozzo, Antonio Porcellini, Tiziana Angrisano, et al. they hypothesize how DNA damage and gene silencing may induce a DNA double-strand break within a genome as well as when DNA methylation is induced by homologous recombination that it may somewhat mark its reparation through a DNA segment and protect its cells against any unregulated gene expression that may be followed by DNA damage. The experiments used to demonstration how gene conversion can modify methylation pattern of repaired DNA and when that occurs methylation is able to silence the recombined gene. When exploring the molecular mechanisms that link DNA damage and the silencing gene then there is an induced double strand break that can be found at a specific location or DNA sequence in where the