Potent Technology Developed in the Form of FTA - Essay Example

Running Head: FTA Cards as DNA Collection and Storage Mechanism in Molecular Analysis with a special focus on GWAS Analysis The FTA Panacea FTA Cards as DNA Collection and Storage Mechanism in Molecular Analysis with a special focus on GWAS Analysis Name University Abstract This essay explores a potent technology developed in the form of FTA; studying its principle mechanism of functioning and the various applications it has been used for so far. Illustrated with the help of many examples, this essay has attempted to touch many of the crucial research fields where FTA Card technology has been invaluable in analysis and studies impacting Human life greatly. FTA Cards as DNA Collection and Storage Mechanism in Molecular Analysis Flinders Technology Associates (FTA) technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. Fujita (2006). It comprises of a cellulose-based matrix containing chemicals for cell lysis and nucleic acid preservation.  The chemicals are activated when a biological fluid contacts the surface.  An additional feature of this chemical treatment is bacterial and viral inactivation.  Not only are the biosamples protected from microbial growth contamination, but the user is also protected from any potential biohazards present in the biosample.  These features make FTA paper an ideal medium for transporting bioamples at room temperature without the requirement of a biohazard shipping label. FTA paper is a commonly used substrate for DNA storage in a number of industries such as pharmaceutics, law enforcement, agriculture and governmental regulatory agencies.  This medium has been available commercially for a number of years by Whatman Inc., who have demonstrated that DNA stored on FTA paper has a long, useful lifetime.  In fact, suitability for use of DNA recovered from up to seventeen year-old biosamples in human identification assays has been demonstrated, inarguably. According to experience in medical epidemiology, biobanks in which human bodily substances are collected and linked to information on donors’ states of health or lifestyle, as well as on their working and environmental conditions, may be expected to contribute to identifying the causes and mechanisms of diseases. By investigating the frequency and distribution of diseases among the population, epidemiologists have in many cases established correlations between environmental factors and the incidence of disease. For example, epidemiological methods have established links between a number of cancers and chemical substances to which sufferers were exposed at work (e.g. pulmonary tumors and uranium; bladder tumors and aniline dyes; or liver tumours and vinyl chloride). The high prevalence of limb malformations in newborns in the late 1950s was ultimately found to be due to the mothers’ ingestion of thalidomide during pregnancy. German National Ethics Council (2004). However genetic epidemiology studies not individuals but population groups. Biobanks serve as large molecular repositories where a large amount of data in the form of DNA from diverse sources can be compared. For example, the United Kingdom BioBank intends to archive the genetic material of 5,00,000 individuals as mentioned in UK Biobank literature. Large series of samples from donors (several hundred to several thousand) with a given multifactorial hereditary condition – such as hypertension, cancer, diabetes, asthma or epilepsy – are compared with corresponding series from healthy donors. The distributions of a large number of genetic markers (genotypes) in chromosomal regions where prior studies suggest the presence of genes relevant to the disease in question are compared in samples from patients and from healthy subjects. By means of a specifically directed but highly complex strategy, comparison of markers permits an ever closer, step-by-step approach to a disease-relevant gene whereby it can ultimately be identified. The investigation of large groups is necessary because genetic factors involved in the causation of multifactorial diseases can only ever supply partial explanations. There is only a certain probability that genetic factors will result in a given multifactorial disease, so that statistical conclusions are all that is possible. Now, as the sample space increases the probability becomes more accurate. Current systems are small and are capable of storing nearly 40,000 samples in about one tenth of the space required by a −80 °C (−112 °F) freezer. FTA Elute matrix devices were designed to serve these large scale storage, shipment and preservation requirements of Biobanks and Pharmacogenomics studies. FTA Elute is a chemically coated matrix which reversibly traps DNA from biological samples. To purify DNA from FTA Elute, a small disk (3mm) is placed in a microcentrifuge tube or multiwell plate and rinsed with water. The wash is removed then fresh elution water is added and the disk heated for 30 minutes at 95°C. DNA is released from the matrix while proteins, impurities and inhibitors remain bound to the matrix. The DNA is now ready for use in a number of genotyping methodologies. FTA Elute used in combination with Whole Genome Amplification (WGA) technologies can create virtually unlimited supplies of DNA for a wide number of tests as described officially in the Whatman literature. Valuable biological samples can be archived or banked at ambient temperature, replacing the expensive and space consuming freezer banks. Eventually, the identification of genetic factors in pathogenesis may prove significant not only for prevention but also for therapy of a disease. This information may also provide a basis for the identification of molecular drug targets. To quote a few examples of such work being carried out for disease identification include cytodiagnosis where FTA Paper has been found to be useful in extended period storage of tumor cells for PCR based DNA analysis as investigated by Dobbs, Madigan et al (2002) or as an alternative to liquid based detection of Human papilloma Virus (HPV) as studied by Lindell et al (2009) Single Nucleotide Polymorphisms (SNP’s) are small variations that can occur in a person’s genetic code. On average, SNPs occur in the human population more than 1 percent of the time. Because only about 3 to 5 percent of a persons DNA sequence codes for the production of proteins, most SNPs are found outside of "coding sequences". SNPs found within a coding sequence are of particular interest to researchers because they are more likely to alter the biological function of a protein. Although many SNPs do not produce physical changes in people, scientists believe that other SNPs may predispose people to disease and even influence their response to drug regimens. The advent of high-throughput SNP genotyping has revolutionized our ability to obtain high density genotypes, however, a key issue remains; the need to access, store, and extract DNA from each individual. While DNA collected for SNP analysis needs to be of sufficient quality to ensure high genotype call rates, the method of collection used in the field needs to be straightforward. Previous research has shown that multiple genomic sources, including lymphocytes, buccal cells, whole genome amplified samples, and fingernails can be used to generate high-density SNP data provided the DNA sample is of adequate quality and quantity. While venous blood is often considered an optimal source for DNA, the invasiveness and cost of obtaining venous blood samples can be prohibitive, especially for large-scale studies or those that deal with livestock and wild animals. Additionally, fresh samples collected in the field may experience degradation before they can be processed. The ease of collection, transportation, storage, and protection from degradation of samples stored on FTA cards alleviates many of these issues as described by McClure, McKay, Schnabel, Taylor. (2009) The number of punches that could be obtained from a single specimen were usually a limitation; insufficient for testing the large number of loci required to identify genetic factors that control human susceptibility or resistance to multifactorial diseases. However a recently carried out process through an improved technique allowed to perform up to 15,000 genotypes from one buccal cell sample as studied by Hongbin (2006). The availability of PCR-based methods in plant molecular research has increased dramatically. Applications of these methods alone, or in combination with other tools, range from marker-assisted selection and molecular mapping, to variety identification and phylogeny research, genomics and chip technology. PCR is also being used routinely in the detection of genetically modified (GM) organisms. Screening can be done at various stages starting with callus cells, then small seedlings, and finally the propagated plants. When screening many samples, a fast and easy DNA preparation method could be beneficial. Whatman FTA Card technology provides a simple, matrix-based DNA purification procedure. A good example of application of FTA Card in Plant biotechnology is described by Yadav et al (2005) Plant viral diseases present major constraints to crop production. Effective sampling of the viruses infecting plants is required to facilitate their molecular study and is essential for the development of crop protection and improvement programs. Retaining integrity of viral pathogens within sampled plant tissues is often a limiting factor in this process, most especially when sample sizes are large and when operating in developing counties and regions remote from laboratory facilities. : DNA and RNA viruses were successfully recovered from leaf tissues of maize, cassava, tomato and tobacco pressed into FTA Classic Cards. Viral nucleic acids eluted from FTA cards were found to be suitable for diagnostic molecular analysis by PCR-based techniques and restriction analysis, and for cloning and nucleotide sequencing in a manner equivalent to that offered by traditional isolation methods. On similar lines Successful application of FTA Classic Card technology was carried out and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes by Owor et al (2007). FTA Technology has also been used in a number of Animal Tissue culture applications. For example, Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. When FTA cards were evaluated for the use of isolation and storage of viral RNA, they were found satisfactory by Muthukrishnan et al (2008) Similarly another example where the issues of tropical temperatures and lack of a cold chain were overcome by FTA Paper technology include the rapid sample collection and molecular detection and genotyping of two animal viruses; African swine fever , a large double stranded DNA and another Peste des Petits Ruminants, a negative single-stranded RNA virus as researched by Michaud et al (2007). Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus. The combination of Flinders Technology Associates filter papers (FTA cards) and real-time PCR was examined to establish a simple and rapid technique for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) from whole pig blood. A modified live PRRS vaccine was diluted with either sterilised saline or pig whole blood, and the suspensions were applied onto the FTA cards. The expected PCR product was successfully amplified from either saline diluted or pig whole blood diluted vaccine. The same PCR ampliocon was detected from all blood samples assayed in this study. This study suggested that the combination of an FTA card and real-time PCR is a rapid and easy technique for the detection of PRRSV. This technique can remarkably shorten the time required for PRRSV detection from whole blood and makes the procedure much easier as discovered by Inoue (2006) May it be Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on large California dairies by Berghaus et al (2006) or Using FTA cards to store avian blood samples for genetic studies and their application in sex determination by Arruga (2002), the numerous manifestations of FTA technology in various important fields of research are too long to list. Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. The utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores was explored. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples as concluded by Lampell et al (2004) FTA Paper has is gaining a crucial position in Evidence collection and preservation in Forensic medicine also. So much so that its importance was discussed at the “DNA Forensics” Conference held by the National Forensic Technology Center in 2002. It was observed that the utilization of FTA technology for the archiving and analysis of nucleic acid within the forensic setting where "out of laboratory" sample acquisition and the need for efficient DNA databasing was prevalent. In addition, emphasis was laid on automation of the processes involving FTA technology. Just to cite an example to understand the application of FTA in forensics, recently extraction of sperm DNA from mixed body fluids containing semen was carried out. This proved to be a rapid and simple method of extracting DNA from sperm when body fluids mixed with semen were collected using FTA cards. After proteinase K digestion of the sperm and body fluid mixture, the washed pellet suspension as the sperm fraction and the concentrated supernatant as the epithelial cell fraction were respectively applied to FTA cards containing DTT. The FTA cards were dried, then directly added to a polymerase chain reaction (PCR) mix and processed by PCR. The time required from separation of the mixed fluid into sperm and epithelial origin DNA extractions was only about 2.5-3h. Furthermore, the procedure was extremely simple. Genome Wide Association Studies (GWAS) Genome-Wide Association Study (GWAS) is an approach that involves rapidly scanning markers across the complete sets of DNA, or genomes, of many people to find genetic variations associated with a particular disease. These genetic variations could be Insertions, deletions or Single Nucleotide Polymorphisms. SNP’s are the most vastly annotated of these variations post the Human Genome Project and HapMap Project in 2005 After genotyping, the genotype relative risk is estimated. The definition of genotype relative risk (GRR) is depends on the disease model. If f0, f1, f2 are the probabilities of being affected for individuals with 0, 1, or 2 copies of the risk allele, then GRR is defined as follows then, Multiplicative GRR= f1/f0= f2/f1 The sample population is divided into two groups viz Cases and Controls for a defined set of alleles. The probabilities are then laid in a 2*2 or a 3*2 contingency table and a variety of statistical tools like the chi square test of independence, Logistic regression, Binomial proportions or the robust trend test can be used ot calculate significant associations as described by Skol (2007) Once new genetic associations are identified, researchers can use the information to develop better strategies to detect, treat and prevent the disease. Such studies are particularly useful in finding genetic variations that contribute to common, complex diseases, such as asthma, cancer, diabetes, heart disease and mental illnesses. Current genome-wide association studies assay a very dense set of markers (>100,000) across the genome in individuals affected and unaffected by a disease using one of the commercially available genotyping chips. The simplest analysis strategy involves carrying out a test of association at each assayed SNP stated by Marchini (2007). 11 million SNP’s are currently archived in dbSNP and 4 million SNP’s genotyped in HapMap There is the presence of 1 SNP every 750 base pairs. However it is a herculean task to genotype all of these on corresponding markers. High throughput and massively multiplexed genotyping technology now enable GWAS studies due to chip based genotyping. Researchers already have reported considerable success using this new strategy. For example, in 2005, three independent studies found that a common form of blindness is associated with variation in the gene for complement factor H, which produces a protein involved in regulating inflammation. Few previously thought that inflammation might contribute so significantly to this type of blindness, which is called age-related macular degeneration which was put forth by Klein (2005). This was followed by a genome wide association study to study the association between the IL23R gene and Crohns disease by Richard (2006) which concluded that IL-23 should be prioritized as the signaling pathway as a therapeutic target in inflammatory bowel disease. To accomplish these goals of Comprehensive genome scans involving many thousands of SNP assays will however require significant amounts of genomic DNA from each sample. Moreover the DNA samples are required in PCR amplification compatible form. FTA provides the classic solution to both these classic needs. As claimed by the manufacturer, “the samples can be stored for years at room temperature before analysis (14 years to date for blood on FTA).” There is considerable interest in noninvasive and cost effective methods for obtaining DNA in large-scale studies. FTA cards show compatibility with virtually all cell types. In the study performed by Taylor (2009) While previous studies have shown that DNA harvested from FTA cards is suitable for genotyping 1,516 SNP on the Illumina GoldenGate platform and 10,000 SNP on the Affymetrix 10 K GeneChip, it has now be proven that FTA Cards are suitable for high-throughput genotyping on the Illumina iSelect platform, which currently assays up to 200,000 SNP. It was concluded that FTA cards provide an excellent medium for harvesting DNA from multiple tissue types, and that when assayed using the Illumina iSelect technology, yield high genotype call rates and reproducibility, particularly when the DNA is extracted using the GenSolve kit. It was thus demonstrated that high quality and repeatable genotypes can be obtained from DNA stored on FTA cards by McClure. These FTA Cards could be extended to be used in Illumina Golden Gate Bead Array Assay which has been employed in ovarian cancer studies to assess its performance in Multiple Displacement Association WGA study performed by Cunningham (2008). Thus the observation that Association studies designed to identify the genetic determinants underlying complex disease increasingly require sustainable high-quality DNA resources for largescale single-nucleotide polymorphism (SNP) genotyping by Paynter (2006) is satisfied by FTA cards in the form of variable sample and genotyping platform compatibility. The point would be further validated by incorporating the example of Hunt BioSciences. Hunt BioSciences comprised of a population based epidemiological health study in Norway initiated as early as 1984. They target several issues like diabetes, breast cancer in Norwegian cohorts. At that time there were 75000 participants with a participation rate of 88% hardly matched by some studies. In their third ongoing survey HUNT3 where 10000 samples of DNA were collected and preserved on FTA paper as given in their BioBank publication (2008). It was also demonstrated that blood spots on FTA cards are a more efficient source of DNA for Improved Primer Extension Polymorphism (I-PEP) as compared to MDA, especially for STR analysis by Sun in 2005. A large yield of DNA for Whole Genome microarray analysis from neonatal blood cards where 10 year old DNA samples stored on Guthrie cards were successfully extracted using modified FTA technology known as GenSolve in comparison to the traditional procedures of strong alkali or heat treatment which compromised on the physical and chemical integrity by Hardin in 2009. In conclusion, FTA Cards capture nucleic acid in one easy step. Captured nucleic acid is ready for downstream applications in less than 30 minutes. Nucleic acids collected on FTA Cards are stable for years at room temperature. FTA Cards are stored at room temperature before and after sample application, reducing the need for laboratory freezers. They are Suitable for virtually any cell type and any genotyping platform. Indicating FTA Cards change color upon sample application to facilitate handling of colorless samples. They are available in a variety of configurations to meet application requirements. It has found extremely wide applications in Forensics, Transgenics, Transfusion Medicine, Plasmid Screening, Food & Agriculture Testing, Drug Discovery, Genomics, STR Analysis, Animal Identification, Diagnostics, Pharmacogenomics and Molecular Biology. Thus FTA Cards have in the true sense of the term proved to be a panacea for the high density DNA extraction and preservation needs posed by the advent of the Genome Wide Association Studies. References Fujita Y, Kubo S (2006) Application of FTA® technology to extraction of sperm DNA from mixed body fluids containing semen Legal Medicine, Volume 8, Issue 1, Pages 43-47 German National Ethics Council (2004) Biobanks for Research Opinion, National Ethikrat Pages 26-27 Whatman Ltd (2007-2009) < http://www.whatman.com/CatBioBankingDNARepositories.aspx> Dobbs, L. J.; Madigan, M. N.; Carter, A. B.; Earls, L. (2002) Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing Arch Pathol Lab Med, Vol (126), Issue (1), Pages (56-63) Lindell et al (2009). Use of FTA card for dry collection, transportation and storage of cervical cell specimen to detect high-risk HPV. Journal of Clinical Virology, doi:10.1016/j.jcv.2009.06.021 National Center for Biotechnology Information (2007) < http://www.ncbi.nlm.nih.gov/About/primer/snps.html> Hongbin He (2006). Improved technique that allows the performance of large-scale SNP genotyping on DNA immobilized by FTA® technology. Infection, Genetics and Evolution. Vol (7) Issue (1) Pages 128-132 Yadav et al (2005) Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues. Virology Journal Vol (2) Page:45 Owor (2007) Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes. Journal of Virological Methods. 140(1-2):100-5 Muthukrishnan et al (2008) Evaluation of FTA® cards as a laboratory and field sampling device for the detection of foot-and-mouth disease virus and serotyping by RT-PCR and real-time RT-PCR Journal of Virological Methods Vol (151) Issue (2) Pages 311-316 Michaud, V. et al.(2007) Long-term storage at tropical temperature of dried-blood filter papers for detection and genotyping of RNA and DNA viruses by direct PCR, J. Virol. Methods, doi:10.1016/j.jviromet.2007.07.006 Inoue et al (2006) Simple and rapid detection of the porcine reproductive and respiratory syndrome virus from pig whole blood using filter paper Journal of Virological Methods. Vol(141) Issue (1) Pages 102-106 Berghaus RD, Farver TB, Anderson RJ, Jaravata CC and Gardner IA (2006) Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on large California dairies J Dairy Sci 89:963-970. Gutierrez-Corchero F, Arruga MV, Sanz L, Garcia C, Hernandez MA and Campos F (2002) Using FTA cards to store avian blood samples for genetic studies. Their application in sex determination Molecular Ecology Notes 2: 75-77 Lampel et al (2004) Detection of Bacillus Spores Using PCR and FTA Filters Journal of Food Protection Vol (67) Issue (5) Pages 1036-1038 Smith Martin, Whatman (2002) Inc “ Utility of FTA Technology for Archiving and Analysis of DNA” EVIDENCE COLLECTION AND PRESERVATION DNA Forensics < http://www.healthtech.com/2002/fdx/index.htm> Fujita Y, Kubo S (2006) Application of FTA® technology to extraction of sperm DNA from mixed body fluids containing semen Legal Medicine, Volume 8, Issue 1, Pages 43-47 Whatman (2003) Application Note Skol, A. (2007), "Two-stage genome-wide association designs", in Weale, M. (ed.), Genetic Epidemiology II: Latest Developments, The Biomedical & Life Sciences Collection, Henry Stewart Talks Ltd, London (online at http://www.hstalks.com/bio) Klein, R. J., Zeiss, C., Chew, E. Y., Tsai, J. Y., Sackler, R. S., Haynes, C., Henning, A. K., Sangiovanni, J. P., Mane, S. M., Mayne, S. T., Bracken, M. B., Ferris, F. L., Ott, J., Barnstable, C., and Hoh, J. (2005). Complement factor h polymorphism in age-related macular degeneration. Science, 308(5720):385-389. McClure Mathew et al (2009) Assessment of DNA extracted from FTA® cards for use on the Illumina iSelect BeadChip BMC Res Notes. Volume 2, Page: 107 Cunningham, J. M., Sellers, T. A., Schildkraut, J. M., Fredericksen, Z. S., Vierkant, R. A., Kelemen, L. E., Gadre, M., Phelan, C. M., Huang, Y., Meyer, J. G., Pankratz, V. S., and Goode, E. L. (2008). Performance of amplified dna in an illumina goldengate beadarray assay. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 17(7):1781-1789. Paynter et al (2006) Accuracy of Multiplexed Illumina Platform-Based Single- Nucleotide Polymorphism Genotyping Compared between Genomic and Whole Genome Amplified DNA Collected from Multiple Sources Cancer Epidemiol Biomarkers;15(12):2533–6) Hunt Biosciences (2008) Non Confidential Information Hunt Biobank < www.ntnu.no/eksternweb/.../HUNT_Biosciences_Inf_43487a.pdf> Sun. G et al (2005) Whole-genome amplification: relative efficiencies of the current methods Legal Medicine, Volume 7, Issue 5, Pages 279-286 Hardin, J., Finnell, R., Wong, D., Hogan, M., Horovitz, J., Shu, J., and Shaw, G. (2009). Whole genome microarray analysis, from neonatal blood cards. BMC Genetics, 10(1):38+. Read More