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Deoxyribonucleic acid (DNA) sequencing, the process of determining the nucleotide order of a given DNA fragment, is extremely beneficial in molecular diagnostics. Sequencing can be used to identify and diagnose genetic and contagious disease. Research with sequencing can even lead to the development of new medicines and treatments for disease. With the many possibilities of product and service applications, sequencing is a technique you may want to bring into your molecular lab.
“Sequencing is considered the gold standard for molecular diagnostics,” said Dr. Kejian Zhang, Director, Molecular Genetics Laboratory, Division of Human Genetics at Cincinnati Children’s Hospital Medical Center. “For a lab to grow or survive, doing targeted genotypes alone is not enough.”
A New Direction
By performing sequencing in house, you can offer a more comprehensive diagnostic service to your medical community. Before taking this step, Dr. Zhang recommends looking at the goals and positions of your lab. What do you want to achieve? Where do you want to be in the molecular diagnosis field? Sequencing can give new purpose to your lab, such as:
• Specializing in the diagnosis of certain contagious diseases, such as HIV or Hepatitis C
• Better managing public health cases, like the H1N1 flu outbreak
• Specializing in inherited diseases, genetic defects or variations such as Cystic Fibrosis or
Severe Combined Immunodeficiency Syndrome (SCID)
• Aiding in the personalized treatment of oncology patients, i.e. BRCA1 and BRCA2
• Or combining multiple areas for a diversity of testing.
By determining your goals, looking at your patient population, and understanding what your test volume may be, you can plan for your lab’s sequencing needs.
Looking for a Change
DNA is the fundamental building block of information for genetic material. The genetic code is a blueprint for every living organism. At the nucleotide level you can see deletions, insertions, and base substitutions defects. Sequencing helps identify that change or defect at the nucleotide level. Because it is so sensitive, you can even examine how mutations affect only a single base. According to Dr. Zhang, 90-plus percent of all human inherited disorders are caused by just a tiny change in a chromosome. “Sequencing can identify the issue tied to the specific change that caused the defect or variation. You can’t have higher resolution than sequencing to detect a mutation,” she said.
Many diagnostic labs start with a handful of mutations to detect. Targeting specific mutations, or genotyping, is used to determine specific abnormalities. Some changes only exist in a few families, but in majority of the cases, or a particular family may carry a private mutation that is typically uncommon.
“Common mutation strategies won’t be sensitive enough to detect all possible mutations. This is why many labs are moving toward a sequencing-based assay for the best screening approach for the entire protein coding region. The test is very sensitive, and detects the majority of any type of mutations,” said Dr. Zhang.
Discovering Sanger’s Method
In 1977, Frederick Sanger and his colleagues developed the dideoxy DNA sequencing procedure. This is the most common sequencing method used today, especially for very long strands of DNA. Sanger and his team were awarded the Nobel Prize for chemistry in 1980, “for their contributions concerning the determination of base sequences in nucleic acids.”1 With full automation, the method can produce more than 1,000 bases of DNA sequence per second. Scientists can now sequence even large genomes within years, rather than decades.
Before Sanger’s method, scientists used protein sequencing. This slow and expensive process could take a year or more to sequence a protein of 500 amino acids. With current methods, the protein sequencing from DNA analyses can be completed in a few days.
You only need a small amount of DNA, such as a drop of blood, to use as a template and to amplify the region you are interested in. “The human genome is made up of about 3 billion base pairs,” said Dr. Zhang. “It’s not possible, and not necessary to examine them all. If you know your patient has cystic fibrosis, you know that a certain chromosome in a certain region contains the specific coding sequence. You can use PCR to make millions of copies of the target.”
Plan for Success
Sequencing could be the pathway to unprecedented advances in diagnostics and medicine. When you bring sequencing in-house, success depends on profit margin, what the market can pay, your cost structure, lab support, personnel, marketing support and more. “Small labs can be profitable,” said Dr. Zhang. “If you can build your reputation around your services, people will recognize you and keep coming back.”