dtc genetic testing
TRANSCRIPT
The Technical Process of Direct-‐to-‐Consumer Genetic Testing
An Informative Description for Students in the Life Sciences
Laura Bruce ENGL 202C 03/27/2014
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Audience and Scope
The purpose of this document is to provide undergraduate students in the life sciences with a contemporary, informative description of the technical process behind direct-‐to-‐consumer (DTC) genetic testing. An emphasis is placed on the progression of the laboratory and analytical steps. Since this document does not include extensive molecular-‐level descriptions, basic background knowledge of biochemical concepts and laboratory techniques in biology will be beneficial for full comprehension. Thus, while this document can be useful to anyone who has an interest in genomics, it will be especially suited to students studying the life sciences, who likely have relevant prior knowledge in cell biology, biochemistry, and genetics. Notably, the information in this document is increasingly valuable, as the growing popularity of DTC genetic testing is pressuring the healthcare system to move towards a more personalized system. It is essential that students pursuing careers in the life sciences are aware of developments in biotechnology, including DTC genetic testing.
Introduction
As more individuals become interested in what their genome may uncover, genetic testing, a medical screening process that identifies variations in an individual’s chromosomes, genes, or proteins, has become increasingly prevalent. To some extent, genetic testing has been routinely used in forensics, genealogy, fetal and newborn screening, prenatal planning, and disease diagnosis. Only recently, however, has genetic testing become readily available for individuals.
In 2003, the Human Genome Project was completed, mapping the entire human genome and shedding light on how our genes influence our physiology and reveal our ancestral roots. As a result, scientists are increasingly capable of understanding the relationships between genotype, the molecular coding of genes for a particular trait, and phenotype, the physical manifestation of that particular trait, which can also be impacted by environmental factors. One specific way of measuring genotype and genetic variation between individuals is by analyzing their single nucleotide polymorphisms (SNPs). SNPs are naturally occurring mutations (changes) in the four possible nucleotide building blocks of DNA (C: cytosine, G: guanine, A: adenine, T: thymine). As shown in Figure 1, in the highlighted region of DNA, three individuals have three different nucleotides, which represent SNPs. Typically, an individual’s genome will contain about 10 million SNPs, most of which have no effect. SNPs only affect phenotype if they occur within a gene or its regulatory region. Phenotypically, these effects might be linked to traits like eye color, inherited conditions, or tendencies in drug metabolism. SNPs can also be used to track ancestry.
(Source: The Broad Institute, http://www.broadinstitute.org)
Figure 1. SNPs
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Direct-‐to-‐consumer (DTC) genetic testing is a type of health and ancestry genetic testing that is sold directly to individuals, without input from healthcare providers or insurance companies. This service relies on a technical process that accesses and analyzes all of the interpretable SNPs for an individual. The main purpose of DTC genetic testing is empowerment, allowing customers to obtain wide-‐ranging personal genetic information from a single source. Individuals may choose DTC genetic testing for a variety of reasons, including:
• Identifying ancestry, paternity, or ethnicity • Seeking disease-‐related risk information to complement healthcare and lifestyle choices • Fulfilling personal curiosity
The DTC genetic testing process is quick, affordable, and more accessible than it would be to seek such data from healthcare providers or other sources. Companies that offer DTC genetic testing are privately held, and can provide individuals with health and ancestry information at costs ranging from $99-‐$2000, with online data provided within 6-‐8 weeks.
Well-‐known DTC genetic testing companies include: • 23andMe, Inc. • BritainsDNA • DNADTC • Full Genomes Corporation • GeneDx
A schematic of a typical DTC genetic testing service, as provided by 23andMe, is depicted in Figure 1. After ordering, customers are sent an at-‐home saliva kit, which they must register, spit into, and mail back to the company. Since saliva contains both white blood cells and skin cells from the inner cheek, this provides a means for the company to access the customer’s DNA. After receiving the saliva sample, the company can begin laboratory processing towards SNP analysis of the DNA, which is the focus of this document. The raw and interpretative results are reported for the customer via an informational website that can include discussion boards and a means of connecting to relatives identified via the testing.
Figure 2. 23andMe’s DTC Genetic Testing Service
(Source: 23andMe, https://www.23andme.com) *Note: CLIA refers to the FDA’s Clinical Laboratory Improvement Amendments
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The Technical Process
How does the DTC genetic testing company establish health and ancestry information from the customer’s saliva sample? This is achieved via an elaborate technical process that occurs at the DTC genetic testing company site. Laboratory techniques and computer analyses are employed. As depicted by a schematic in Figure 3, the technical process involves four main steps, some of which contain sub-‐steps. Generally, the process includes DNA extraction, preparation for hybridization via amplification and labeling, hybridization, and analysis via laser scanning and normalization.
1. DNA Extraction:
The first step in the genetic testing process is to extract DNA (deoxyribonucleic acid), the molecule that contains all of an individual’s genetic information. The white blood cells and skins cells found in the customer’s saliva sample are suitable for this extraction. In this technique, DNA is purified via chemical and physical means within various test tubes. This method is essential in order to isolate the DNA from proteins, lipids, and other molecules that are in saliva, so that it may be further analyzed.
Figure 3. Technical Process Behind DTC Genetic Testing
Saliva sample
1. DNA Extraction
2. Preparation for Hybridization
3. Hybridization
4. Analysis
Scanning
Labeling (coupling)
Amplification (PCR)
Normalization
(Source: Wikipedia, http://wikipedia.org/wiki/)
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As shown in Figure 4, DNA extraction itself involves various steps. Initially, the sample must be combined with cell lysis solution (brand name FABG) and protein degradation enzymes (brand name Proteinase K) to disrupt the cell membranes and break down the proteins that make up the cell, respectively. This results in a heterogeneous solution, which is then moved into a special binding test tube for centrifugation, the use of a high-‐power spinning machine to separate biological molecules. Once centrifuged, DNA remains in the upper aqueous layer, binding to an inner portion of the special test tube, while proteins remain in the lower phenol layer, and are discarded (see Figure 3 for additional imagery). Afterwards, the DNA is centrifuged repeatedly with wash buffer (two times) and elution buffer (one time), to remove any leftover reagents and salts from the resulting phenol layers. In the end, pure genomic DNA remains.
2. Preparation of the DNA for
Hybridization:
The next main step in the process of DTC genetic testing is to prepare the extracted DNA so that it is in an exploitable form for hybridization. This requires amplification of the DNA so that there is enough to be analyzed, followed by labeling of the amplified DNA so that it can be monitored during subsequent analysis.
i. Amplification (PCR): A biochemical process know as polymerase chain reaction (PCR) is employed in order to exponentially copy the customer’s DNA for analysis (Figure 5). The DNA is mixed with enzymes, primers, and other stabilization agents in a small test tube. The enzymes serve to bind to the DNA molecule, and the primers provide a place where biochemical amplification can begin.
Figure 5. PCR Amplification
Figure 4. DNA Extraction
(Source: Science Creativity Quarterly, http://www.scq.ubc.ca)
(Source: Favorgen Biotech, http://www.favorgen.com)
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PCR occurs within a thermal cycler machine, which can increase and decrease temperature according to a specific schedule. First, the sample is heated, allowing the DNA double-‐helix to denature into two separate strands. Then, the sample is cooled slowly, which allows the enzymes and primers to bind to each strand. Finally, the sample is heated (incubated) to an ideal temperature for the primers, which begin extending each single strand into new double strands. After each cycle, the number of copies (complete double-‐helices) of DNA is doubled. For DTC genetic testing purposes, the thermal cycler is set to complete around 50 cycles, resulting in 250 copies of the customer’s DNA.
ii. Labeling (Coupling): Although a sufficient amount of DNA is produced by the PCR process, this DNA is not useful unless it is labeled with fluorescent tags that can be monitored during later steps (hybridization and analysis). Labeling is quite simple, and requires fluorescent tags in solution to be added directly to the test tube containing the amplified DNA. Typically, cyanide dyes known as Cy3 and Cy5 are added, since they have green and red fluorescent properties, respectively. Each dye binds to a different location on the DNA molecule, based on covalent binding properties. Since there are two dyes, the process of adding these dyes is known as coupling.
3. Hybridization of the Prepared DNA to a Microarray:
After the customer’s DNA has been isolated, amplified, and labeled, the actual “testing” aspect of genetic testing can begin. In this step, the identifiable SNPs in the DNA will be recognized. The process, known as hybridization, refers to placing the prepared DNA on microarray chips, and then incubating to allow the tagged DNA molecules to bind (hybridize) to the chips. These hybridized chips can be analyzed according to the binding pattern that occurs. Specifically, DTC genetic testing companies use microarray bead chips that are customized for SNP genotyping, as depicted in Figure 6. Each chip is essentially a small glass slide that is covered with approximately 1 million tiny immobile beads, one for each SNP that will be tested. Each bead has an attached probe, a chain of small allele-‐specific oligonucleotides (15-‐20 nucleotide base pairs). These nucleotide probes are pieces of DNA that are complementary to the sites on human DNA that represent SNPs. All probes are fluorescently labeled in the same way as the prepared DNA. Typically, DTC genetic testing companies simply purchase these SNP microarray bead chips from biotechnology companies. The purchased bead chips are designed to include probes for SNPs with associations to health-‐related traits, or SNPs that are ancestral markers.
Figure 6. SNP Microarray Chips
(Source: Science Creative Quarterly, http://www.scq.ubc.ca)
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Figure 7 shows the how the sample DNA hybridizes to the fixed probes on the bead chip. First, the customer’s DNA is washed over the chips and the chips are incubated, allowing for hydrogen-‐bonding to occur between the sample and the probes. The sample DNA will bind strongly to the nucleotides on the probe if the probe is fully complementary to it. Any partially complementary strands are weakly bonded and are easily washed off with buffers, leaving only perfectly complementary pairs attached. 4. Analysis of the Hybridized
Microarray:
Once the SNP microarray bead chips are subject to hybridization and washing, the chips are analyzed in order to determine which SNPs are present. This is accomplished via laser scanning and subsequent normalization by a computer program.
i. Laser Scanning: If the customer’s DNA is perfectly complementary to an SNP probe, the Cy5 and Cy3 fluorescent tags on the sample DNA and the probe will fluoresce in an equivalent way, indicating that the customer’s genome includes that specific SNP (Figure 7). The bead chips are scanned by a laser system, which measures the fluorescence as a light signal. Data appears as a cluster of green and red spots (Figure 8).
ii. Normalization: The fluorescent hybridization signals as provided by the laser scanner must be normalized, or processed as an overall image, to quantify which SNPs are in the customer’s genome. A high-‐power computer program normalizes the fluorescence data via a statistical system. Background noise is removed, and the system accounts for the location of the signal on the chip while noting the intensity of the signal. This normalized data is linked to SNPs and their associated traits or markers, which completes the technical process of DTC genetic testing. Finally, the data is interpreted into a form that has health and ancestral significance. These interpretations are displayed for the customer via the company’s online website.
Figure 7. DNA Hybridization
Figure 8. Laser Scanning Data
(Source: Wikipedia, http://wikipedia.org/wiki/)
(Source: Nature, http://www.nature.com)
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Conclusion
The goal of DTC genetic testing is to empower individuals to obtain reliable genetic information from a single source. After the customer’s saliva sample is received, technical processing employs laboratory and computer techniques to obtain, quantify, and analyze data before it is interpreted as health and ancestry information for the customer. In summary, there are four main steps to the technical processing, some of which contain sub-‐steps. DNA is extracted from the saliva sample, amplified via PCR, and labeled with fluorescent tags. The prepared DNA is subject to hybridization on an SNP microarray bead chip, which is quantified via laser scanning and normalized by a computer. The ensuing interpretation of the data with relation to ancestry and health risk is the most controversial aspect of the DTC genetic testing service, but interpretation accuracy will likely improve as the relationships between SNPs and phenotype are further deciphered. Overall, DTC genetic testing provides a glimpse at what biotechnology in genetics can accomplish, and is of high relevance to students studying the life sciences.
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Works Cited
“How it works.” 23andMe. 23andMe, Inc, n.d. Web. 24 March 2014. <https://www.23andme.com/howitworks/>. Jasmine, Farzana, et al. "Whole-‐genome amplification enables accurate genotyping for microarray-‐based high-‐density single nucleotide polymorphism array." Cancer Epidemiology Biomarkers & Prevention 17.12 (2008): 3499-‐3508. Su, Pascal. "Direct-‐to-‐Consumer Genetic Testing: A Comprehensive View. "The Yale journal of biology and medicine 86.3 (2013): 359. “What is direct-‐to-‐consumer genetic testing?” Genetics Home Reference. U.S. National Library of Medicine, 24 March 2014. Web. 24 March 2014. <http://ghr.nlm.nih.gov/ handbook/testing/directtoconsumer>. Image on title page. DNA double-‐helix. Digital Image. Creatia. n.p., n.d. Web. 24 March 2014. <http://creatia2013.files.wordpress.com/2013/03/dna.gif>. Figure 1. SNPs. Digital Image. Glossary. Broad Institute, 2014. Web. 24 March 2014. <http://www.broadinstitute.org/education/glossary/snp>. Figure 2. (cropped) 23andMe’s DTC Genetic Testing Service. Digital Image. How it Works. 23andMe, LLC, 2007-‐2014. Web. 24 March 2014. <https://www.23andme.com/howitworks/>. Figure 3. (cropped, labels edited) Technical Process Behind DTC Genetic Testing. Digital Image. DNA Microarray Experiment. Wikipedia, 5 March 2013. Web. 24 March 2014. <http://en.wikipedia.org/wiki/DNA_microarray_experiment>. Figure 4. DNA Extraction. Digital Image. Genomic DNA Extraction Blood DNA Mini / Maxi Kit. Favorgen Biotech Corporation, 2009. Web. 22 March 2014. <http://www.favorgen.com/products/nucleic_genomic_dna_extraction/blood_dna.html>. Figure 5. PCR Amplification. Digital Image. DNA Fingerprinting in the Standardization of Herbs and Nutraceuticals. Science Creativity Quarterly, 2014. Web. 22 March 2014. <http://www.scq.ubc.ca/dna-‐fingerprinting-‐in-‐the-‐standardization-‐of-‐herbs-‐and-‐ nutraceuticals/>. Figure 6. SNP Microarray Chips. Digital Image. Test Your DNA for Diseases — No Doctor Required. Time Magazine, 23 October 2012. Web. 25 March 2014. <http://healthland.time.com/2012/10/23/drugstore-‐genomes-‐ whos-‐pushing-‐the-‐sequencing-‐industry/>.
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Figure 7. (cropped, circles added) DNA Hybridization. Digital Image. DNA Hybridization. Wikipedia, 5 March 2013. Web. 24 March 2014. <http://upload.wikimedia.org/wikipedia/en/a/a8/NA_hybrid.svg>. Figure 8. (cropped) Laser Scanning Data. Digital Image. Cystic fibrosis carrier screening: Validation of a novel method using BeadChip technology. Nature, 13 May 2004. Web. 26 March 2014. <http://www.nature.com/gim/journal/v6/n5/fig_tab/gim200457f1.html>.