Biobanks or biorepositories are collections of biological material, most often from clinical patients whose medical history is well documented, used for the mining of genomic targets for drug discovery. Samples stored in biobanks can range from frozen tissue, buccal cells, blood, plasma, formalin-fixed paraffin embedded (FFPE) tissues and cultured cells. Biobanks are an invaluable source of information which allows researchers to study and draw conclusions about the role(s) of genes in the presentation of diseases.
Biobanking is so important that several European countries have national biobanking programs. Norway, Sweden, Estonia, Canada and the UK are among the most active biobanks targeting large populations of people. Biological samples and complete health records are collected from the volunteers to aid in the study of diseases and monitoring phenotypic and genotypic characteristics. A pan European biobank initiative (www.biobanks.eu) with 16 member organizations is underway to enhance therapy and to prevent common diseases such as cancer, heart disease, infectious and metabolic diseases.
Other biobanking efforts include collections of cohorts living in different environmental areas in North Carolina to investigate the effects of the environment on genetic diversity. Howard University collected biosamples from African-Americans to investigate diseases such as asthma, diabetes, obesity, and hypertension which affect African-American populations in large numbers. Disease specific biobanks have also been formed to study such diseases as attention-deficit hyperactivity disorder (ADHD), autism, schizophrenia, breast cancer, heart disease and diabetes to name but a few.
According to the National Cancer Institute:
“Following the mapping of the Human Genome in 2000, biological research has moved into what is called the “genomic age.” This designation refers to the ability of scientists to study disease at the most basic “molecular” level, by identifying genes and their function, and understanding the role genetics plays in the origin and progression of disease. Other emerging fields of study include proteomics – the study of the full set of proteins encoded by the genome – and pharmacogenomics, which seeks to link the human genome to variation in patient response to pharmaceuticals.
In addition to molecular information, scientists are also analyzing a vast amount of clinical information from patient records and clinical trials. From this data, it is possible to identify patterns that provide a pathway to understanding disease sub-types, and potential strategies for diagnosing and treating disease in new and more effective ways.
Human biospecimens can provide a bridge between emerging molecular information and clinical information, by enabling researchers to study the molecular characteristics of actual human disease, and then correlating those patterns with what is known about the clinical progression of the disease. Specifically, human biospecimens can be used to:
- Identify and validate drug targets (such as specific genes or gene products)
- Identify disease mechanisms
- Develop screening tests for “biomarkers” associated with certain sub-types of a disease
- Group patients based on their genetic characteristics and likelihood of positive response, for testing of new drugs
- Group patients based on the “biomarkers” of their disease to determine which treatment is appropriate”
National Cancer Institute, Office of Biorepositories and Biospecimen Research website, Frequently Asked Questions, http://biospecimens.cancer.gov/resources/faqs.asp
To serve the requirements of biobanking and pharmacogenomic studies there is a need for a method and device for rapid DNA collection, shipping, storage and purification. FTA Elute matrix devices were designed with solving those problems in mind. FTA Elute is a chemically coated matrix which reversibly traps DNA from biological samples. The chemicals within the matrix act as a DNA preservative such that the samples are stable for room temperature for extended periods of time which simplifies sample storage and shipping. Potentially harmful viruses and pathogens are inactivated by the matrix coating so that the samples are now safe to handle and free from biohazardous material. Just a few drops of blood from a finger-prick (approximately 10 – 40 µl) are required to provide enough DNA for many genetic analyses. FTA Elute used in combination with Whole Genome Amplification (WGA) technologies can create virtually unlimited supplies of DNA for a wide number of tests. Valuable biological samples can be archived or banked at ambient temperature, replacing the expensive and space consuming freezer banks.
FTA Elute matrix devices are a simple and rapid method for purifying DNA and can easily be automated for high throughput genotyping laboratories, saving time and money. To purify DNA from FTA Elute, a small disk (3mm) is placed in a microcentrifuge tube or multiwell plate and rinsed with water (Figure 1). 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.
Real Time PCR
DNA eluted from the FTA Elute matrix device was subjected to quantitative PCR to demonstrate the quality of the purified DNA. Blood samples were collected from 10 separate individuals onto the FTA Elute matrix. Disks (3.0mm) were extracted according to the water extraction protocol in a final volume of 100µl. Approximately 2.5µl of the purified DNA was added to the real time PCR mixture and amplified using the Yo-Pro 1 DNA binding dye (Invitrogen). Figure 2 shows a very tight grouping of curves with an average Ct of 26.58 which equals 22.14 ng of DNA in the 100µl final volume. The yield of DNA from FTA Elute is sample dependent. For blood and buccal samples, the yields are 55-70% and 60-75%, respectively.
DNA Sequencing
DNA sequencing is the “gold standard” for detecting polymorphisms in gene sequences. DNA purified with FTA Elute was tested as a template for sequencing a 1.05 kb fragment from exon 1 of the 2B15 variant of the UDP-glucuronosyltransferase (UGT) gene. The G/T polymorphism at the D85Y locus was the target SNP. Figure 3 shows an example of three sequences from individuals showing polymorphisms in the sequence. This profile indicates that the DNA extracted with FTA Elute yields excellent sequencing data.
Multiplex Gene Deletion Assay
Many labs perform multiplex gene deletion assays using end-point PCR and agarose gel electrophoresis as a means of genotyping. Figure 4 shows a multiplex PCR performed with DNA purified with FTA Elute detecting a deletion in the UCT2B17 gene. The wild type and mutant genes are demonstrated by bands of 316 bp and 884 bp, respectively. As can be seen, the individual in lane 7 is homozygous for the mutant form of the gene. The ability to multiplex with multiple primers requires high quality DNA to prevent mis-priming and non-specific banding patterns. DNA purified by FTA Elute is robust and less than 1 ng is required to yield clean strong bands in the multiplex PCR.
FTA Elute is a unique and dynamic product positioned to serve the pharmacogenomics and bio-banking markets. FTA Elute is a simple, easy to use, one step method to collect and stabilize DNA for genotyping and SNP analysis. A quick hot water elution method yields high quality DNA for a variety of genotyping methods such as Amplification Refractory Mutation Systems (ARMS™) Scorpion™ and allele specific Oligonucleotide (ASO) genotyping. DNA from FTA Elute has also served as a template for whole genome amplification (WGA) thus supplying an unlimited amount of DNA for extended genetic studies. The archiving ability of the FTA Elute matrix is beneficial in “look back” studies and for archiving samples for future genetic analysis as test panels become available.