DNA Sequencing and Healthcare

DNA sequencing is a powerful tool with a wide range of applications in healthcare. With Sequencing we can determine the exact sequence of nucleotide bases in a DNA segment. Determining the exact sequence of DNA bases adenine (A), cytosine (C), guanine (G), and thymine (T), is key to understanding the genetic basis for many diseases.

DNA sequencing methods first emerged in the 1970s, with what is now called first-generation technology. More efficient and cost effective techniques to sequence DNA, known as next-generation sequencing (NGS), have been developed since then. In addition, it is now possible for computers to decode the whole genome using DNA sequencing. In addition to being efficient, these next-generation sequencing are comprehensive and easy to understand.

Next Generation sequencing

The first step to DNA sequencing is fragmentation, splitting the DNA into fragments called reads. Fragmentation enables the sequencing of the entire DNA sequence using techniques like sonication, nebulization, or various enzymes.

1. Sonication is using sound energy to break apart the DNA strands.
2. Nebulization is the procedure of forcing a DNA strand through a small hole in a nebulizer to create a fine mist, which is then collected. The size of the fragment is determined by speed, the pressure of the gas going through the nebulizer, temperature, viscosity , or friction between liquids in the solution. Nebulization is easier and quicker and only requires small amounts of DNA.
3. Enzymes , on the other hand, molecularly fragment DNA into many pieces.

One of the most comprehensive forms of next-generation DNA sequencing is sequencing by synthesis. This technique was developed by the biotechnology company Illumina.

The technology for sequencing by synthesis involves having one adapter placed on one end of a DNA fragment, and another at the other. Then a flow cell surface, a glass slide with lanes, is used. In a flow cell surface, the inside is lined with oligos, or short single strands of DNA/RNA. To use the cell, DNA fragments are added as a template to be sequenced. Half of the oligos are complementary to one of the adapters that are added onto the DNA fragments, and the other half are complementary to the other adapter. Once the fragment gets added into the cell, it attaches to one of the oligos due to its complementary adapters. Then, a polymerase creates a complement of the attached fragment cell and the original fragment gets washed away, i.e new complement is left behind. The fragments are then cloned, for which the complement must bend over and attach its adapter to the other type of complementary oligo. Then the polymerase again creates the complement for the bent strand, forming a "bridge" that is double-stranded. The bridge is then split into two single-stranded copies of the molecule attached to the flow cell. This duplicates them many times. After this process is over, the reverse strands are taken away so only the forward strands remain.

After the flow cell is prepared with many cloned DNA strands, the actual sequencing begins. First, fluorescently tagged nucleotides are added in, and based on the sequence of the fragment, only one complementary type of nucleotide can attach to each amino acid. After all the nucleotides have fluorescent nucleotides paired with them, a light is shined on the strand and a fluorescent signal is released, revealing the sequence of the DNA. Sequencing technology is adopted for research and development in several fields.

Healthcare

One of the most common uses of DNA sequencing is in oncology , for treating malignant tumors. Using the genome sequence of a healthy human from the Human Genome Project as a baseline for comparison, scientists can use NGS to analyze a specific patient's genome and identify their particular DNA mutations. Doctors can then use this information to design a personalized treatment plan for the patient. This application of DNA sequencing works because the specific type of DNA mutations in a patient's DNA may play a role in how they respond to a particular drug. Because of this, two patients with the same type of cancer may need completely different treatment pathways based on which DNA errors caused cancer (Barua et. al). This information can completely change how cancer is treated, and so the knowledge of the gene sequence can potentially save someone who previously could not be treated.

Another valuable application of DNA sequencing in the healthcare field is the usage of whole genome sequencing, or WGS, to analyze underlying genetic causes of diseases that are otherwise very challenging to diagnose and have no known risk factors. An example of this would be using WGS of patients to search for variations that might be causing the disease. If used properly, this information could be used to help diagnose and treat the disease. However, the biggest drawback of this application of DNA sequencing is that any variants found using WGS may not have a well-established clinical significance, which could lead to unnecessary anxiety and expense (Phillips et. al).

Forensics

DNA sequencing has revolutionized people's lives in the past decade. Forensic science has also benefited from this technology. Previously, investigations were based on witnesses, footprints, and other inconclusive pieces of evidence to prove if a suspect was guilty or innocent. DNA sequencing technology has enabled law enforcement to use science to find the perpetrator of a crime. Through DNA sequencing, DNA found at the crime scene is compared with the DNA of the suspects. A matching DNA confirms the suspect is guilty of the crime beyond doubt. These tests have a ninety-nine percent accuracy because people do not share the same DNA short tandem repeats (STRs).

There are about two hundred laboratories that send DNA from crime scenes and former criminals to a database known as the Combined DNA Index System ( CODIS ). Despite the difficulties in keeping up with the sequencing requests, the information in the database is important because criminals have a higher chance of committing crimes again. The database has approximately 16 million convicted offenders and arrestees and data from 750,000 crime scenes ( Arnaud ).

With the growing number of requests to sequence DNA from crime scenes, laboratories need to find more efficient ways of sequencing to keep up with the demand. The Minnesota Department of Public Safety first uses Phenol-Chloroform or "Maxwell 16" to separate the DNA from other materials. Then, minute quantities of DNA are faithfully amplified via Polymerase Chain Reaction (PCR). Next, Capillary Electrophoresis (CE) is carried out to separate the DNA molecules based on size. People often use the process of Short Tandem Repeat analysis because of their "technical robustness and high variation among individuals"; furthermore, by using " different fluorochromes ," the profile of the person is created by showing their genotypes (qtd. in Jordan and Mills). Challenging forensic samples, such as very degraded ones, can benefit from kinship analyses and family reconstructions for missing persons and unidentified human remains. These also are useful for providing investigative leads in some cases without a suspect and no genetic profile match in CODIS (Bruce and Daal).

The use of DNA sequencing in forensics will continue to grow as new discoveries of DNA are made and technology advances. In addition, as the technology continues to grow, the DNA sequencing systems will be faster and more accessible. For instance, 23andMe is available for comparing the DNA of the user with the DNA of other people to find a person's genetic ancestry.

The Future of Sequencing

DNA sequencing has already had a profound impact on the healthcare field, and can be expected to continue to do so. While sequencing may be slightly inaccurate and expensive today, in the future, the technology will likely be improved and can result in cheaper, faster, and more accurate sequencing. Armed with game-changing information from this sequencing, in the future, scientists can potentially treat previously untreatable diseases and prevent others entirely.

Article By:

• Annika Thakur (Oak Park High)

• Arya Sule (Dougherty Valley High)

• Jonathan Brose (Bellarmine College Preparatory)

  (External students, post on the request of Mentors)

References​

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