Viral Genome Detection Using Polymerase Chain Reaction - Lab Report Example

Viral genome detection using polymerase chain reaction (PCR) Laihla Middle Initial. Sur Polymerase chain reaction (PCR), a molecular technique that amplifies a specific gene fragment offers a more rapid, sensitive and specific method of detecting and identifying pathogens. The purpose of this experiment is to detect the presence of viral genome in the genomic DNA of HeLa and MRC5 cell lines. Human papilloma virus (HPV) 18-specific primers were used to amplify viral DNA. After 30 PCR cycles, the PCR products were subjected to agarose gel electrophoresis (AGE). Results showed that the genomic DNA of HeLa cells is contaminated with HPV-18 DNA as indicated by the presence of a band in the agarose gel. However, no viral DNA was detected in MRC5 cell line. In conclusion, PCR is an effective tool for the detection of viral genome in infected host cells. Introduction Conventional laboratory methods of identifying disease-causing pathogens often involve morphological characterization and antigen detection (Lee et al., 2009; Nitzan et al., 2009; Cicek et al., 2007). However, the traditional morphological examination which relies heavily on culture techniques, presents a serious laggard, on top of contamination problems (Candrian 1995). The emergence of molecular diagnostic tools have circumvented and altered the limitations brought about by the conventional diagnostic techniques (Lion et al., 2006). Specifically, genome-based techniques are increasingly becoming popular due to their high specificity and sensitivity. One tool that has revolutionized the field of clinical diagnostics is polymerase chain reaction (PCR), an enzymatic procedure which amplifies a specific fragment of DNA or RNA (Lion et al., 2006). Since PCR makes use of nucleic acids to detect the presence of organisms, it is extremely useful in the identification of notoriously difficult-to-detect pathogens like viruses. It has been established that human papilloma virus (HPV) infection is a necessary precursor for the onset of cervical cancer (Stanley 2010). In fact, approximately 90% of cervical cancer cases can be accounted for by HPV types 16 and 18. Since HPV infection is very common with a lifetime risk of infection of 50-80%, its accurate diagnosis is very crucial (Stanley 2010). . The purpose of this experiment is to detect the presence of viral genome using polymerase chain reaction. Specifically, the experiment seeks to compare the genomic DNA extracted from HeLa cells, a cervical cancer cell line and MRC5 cells derived from a 14-week old fetal lung tissue in order to confirm that HeLa cells contain human papilloma virus (HPV) 18 DNA. The HeLa cell line, which was derived from the cancer patient Henrietta Lacks, was among the first cell lines to be derived from humans. Materials and Methods Kindly refer to the module booklet. Results In this experiment, HPV 18 specific primers were used to amplify gene fragments containing HPV 18 DNA sequences. After 30 rounds of PCR cycle, the amplicons were then subjected to agarose gel electrophoresis (AGE) to confirm the presence of amplified DNA. Results of the gel electrophoresis revealed a single band in the HeLa sample (lane 1), indicating that a DNA fragment has been amplified (Figure 1). Since the primers used were specific for HPV18 DNA, it can be said that the observed band is a viral DNA. Based on the molecular marker, the molecular weight of the amplified DNA is approximately 766 bp. Meanwhile, no band was observed for the MRC5, the normal lung cell line sample similar to the negative control. This indicates that MRC5 genomic DNA is not contaminated with HPV 18 genome. Discussion Polymerase chain reaction is an in vitro, enzymatic procedure that makes use of a primer, polymerase enzyme, and dNTPs to produce 108 or even more copies of a specific portion of DNA or RNA without having to use cells (Campbell and Reece, 2004). According to Campbell and Reece (2004), because of the specificity of PCR, only minute amounts of DNA need to be present in the starting material. Moreover, impure DNA can also be used as starting material. Since PCR exponentially increases a particular DNA portion, tasks involving DNA as the sample for analysis such as DNA sequencing, gene expression studies, DNA fingerprinting, taxonomic classification, mutagenesis experiments, identification of microorganisms and pathogens are facilitated. In this laboratory exercise, viral DNA transcipt was detected in HeLa cells, a cervical cancer cell line while none was detected in the MRC5 cell line. This result is consistent with the study conducted by Lesnikova et al. (2010). In their study entitled “Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma”, the authors were able to detect the presence of HPV18 gene in HeLa cells and in cervical cancer tissue blocks using PCR. On the other hand, the absence of bands in the MRC5 sample is to be expected since this cell line is derived from a normal lung tissue not infected with HPV18. According to Stanley (2010), human papillomaviruses are a large family of small double-stranded DNA viruses that infect squamous epithelia or cells with the potential for squamous maturation. Currently, there are about 130 HPV types but types 16 and 18 are the most common, high-risk HPVs which cause cervical cancer in women (Stanley 2010). Statistically, HPV 16 DNA sequences are found in 50-70% of cervical cancer while HPV18 accounts for 7-20% of cases. Furthermore, the HPV genome can be divided into 3 domains: a noncoding upstream regulatory region; an early region with 6 genes namely E1, E2, E4 E5, E6, and E7; and a late region encoding for genes L1 and L2 (Stanley 2010). HPV infection is established first, through microabrasion of skin, specifically the genital epithelium. Then, HPV binds to the basement membrane by virtue of a L1 receptor before entering the keratinocytes. Once inside the cell, the virus can now replicate its DNA and may then be incorporated into the host genome (Stanley 2010). HeLa cells are among the HPV-positive cell lines which was derived from Henrietta Lacks in 1951, a patient with cervical tumor (Racaniello 2009). This cell line is specifically useful in the field of virology. Studies have shown that HPV DNA are integrated into the chromosomal genome of HeLa cells. Specifically, HPV18 DNA is found to have 5 incorporation sites in HeLa chromosomes, making it possible to detect HPV genome in HeLa chromosome. References Campbell, N., and Reece, J. 2004. Biology. 6th ed. Benjamin Cummings, San Francisco, California. pp. 382-383 Candrian, U. 1995. Polymerase chain reaction in food microbiology. Journal of Microbiological Methods 23: 89-103 Cicek et al. 2007. Comparison of the “ProDect BCS RV CHIP” assay with the combination of shell vial cell culture and immunofluorescence antibody test for the detection of respiratory viruses. Journal of Virological Methods. 143: 161–168 Lee et al. 2009. Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay. Journal of Virological Methods 156: 111–116 Lesnikova et al. 2010. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma Infectious Agents and Cancer. 5:2 Lion et al. 2006. Detection and monitoring of virus infections by real-time PCR. Molecular Aspects of Medicine 27 \: 254–298 Nitzan et al. 2009. Identification of pathogenic bacteria in blood cultures: comparison between conventional and PCR methods. Journal of Microbiological Methods. 78 (3): 325-330 Racaniello, V. 2009. The amazing HeLa cells of Henrietta Lacks. Accessed at: http://www.virology.ws/2009/02/09/the-amazing-hela-cells-of-henrietta-lacks/ Date accessed: Oct. 31, 2010 Stanley, M. 2010. Pathology and epidemiology of HPV infection in females. Gynecologic Oncology 117: S5–S10 Read More