molecular diagnostics – Page 5

The Exciting World of Molecular Diagnostics

Hello everyone! I am Sharleen Rapp and I’m a Molecular Diagnostics Coordinator at Nebraska Medicine. I feel lucky to be able to discuss all about the exciting world of Molecular Diagnostics. For my first post, I’d like to give you a little background about myself and why I feel I am lucky to be in the career that I’m in.

Ever since I was little, science has intrigued me. Perhaps it was the experiments my Dad performed in our kitchen as practice for his labs for his high school chemistry classes (who doesn’t enjoy watching salt crystals “grow” on string in peanut butter jars?) or watching my brother set up his fruit fly experiment for his high school science class, but I’ve always enjoyed learning about how things work.

I went to a small parochial school in the middle of Nebraska, and unfortunately we didn’t have the funds for elaborate science class labs. Interestingly enough, the event that clinched science for me was a project that I did for my government class. We were responsible for writing, essentially, a textbook, complete with chapters, endnotes, quizzes and tests, on a topic of our choosing. I chose to write about the Human Genome Project. I wrote this in the year 2000, when the Project was in full swing. I had read about it in the previous years, and I was completely amazed by what it accomplished. In the middle of the school year, in fact, Time magazine came out with an issue titled “The Future of Medicine – How genetic engineering will change us in the next century.” It contained nineteen different articles, all focused on how the information from the Human Genome Project would impact the future – one of which discussed the way pharmaceutical companies were designing drugs to combat the mutations in different types of cancer. I knew then I would be a part of that future; I just didn’t know how. At this time, I had no idea how I could go about working in this field. I had never heard of the discipline “Molecular Diagnostics” or medical technology.

I went off to college and got a degree in Biological Sciences with the intent to go to graduate school and study in Genetics, but I still had no real idea about how to get into the field of study of DNA. Through some interesting twists and turns, including working in a fruit fly lab in college and an amazing internship at Washington University under Elaine Mardis, I ended up at a small private company where my job was to sequence mitochondrial DNA and mitochondrial-related genes, and in doing this, I knew I had found my career. I am a self-proclaimed science nerd and I love sequencing, the whole process from wet bench to analysis, more than anything that I have ever done. When I moved over to Nebraska Medicine and began working in the Molecular Diagnostics lab, I was amazed at the work that was being done there. I’ve had some amazing opportunities to work with all different types of sequencing – dideoxy sequencing, pyrosequencing, and now, massively parallel (aka, next generation) sequencing. I am so excited to be sharing some of my experiences and case studies from the work that we do in our lab in future posts.

Thanks for reading!!

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-Sharleen Rapp, BS, MB (ASCP)^CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.

Blotting and Probing Techniques

“Blotting,” in relation to molecular diagnostics, is a term that refers to the process of detecting the presence and quantity of DNA, RNA, or protein in cells. There are three main types of blotting procedures that those in the field should be familiar with: Southern, Northern, and Western. Three additional blotting procedures are termed Southwestern, Eastern, and Far-Eastern. These are also summarized in the table below.

Southern Blot Steps

DNA is isolated and cut with restriction enzymes.
The DNA fragments are then analyzed by gel electrophoresis and separated by size (see previous blog post on Separation and Detection).
Depurination – Gel is soaked in hydrogen chloride (HCl) to remove the purine bases from the sugar-phosphate backbone. This loosens up larger fragments before denaturation.
Denaturation – The DNA is denatured by exposing the gel to sodium hydroxide (NaOH). Denaturation breaks the hydrogen bonds that hold the DNA strands together.
Blotting – The denatured DNA is transferred to a solid substrate (nitrocellulose) that helps to facilitate probe binding and signal detection.
Pre-hybridization – Prevents non-specific binding of the probe to other sites on the membrane surface.
The membrane is exposed to the hybridization probe, usually a single DNA fragment with a specific sequence to the target DNA. The probe DNA is labelled either with radioactivity or fluorescent dyes.

Importance of the Membrane

Nitrocellulose and nylon membranes are best for smaller sized single stranded DNA fragments. It is compatible with many types of buffers and transfer systems. These membranes work well with protein and nucleic acids.

METHODS OF TRANSFER
Capillary Transfer	Utilizes capillary movement of the buffer from a soaked paper to the dry paper Denatured DNA moves from the gel to the membrane
Electrophoretic Transfer	Electric current moves the DNA from the gel to the membrane
Vacuum Transfer	The force from suction moves the DNA from the gel to the membrane

Northern Blots

Northern blots are used in the laboratory to look at RNA structure and quantity. It’s a powerful method that can measure levels of gene expression, as well as structural abnormalities in RNA.

Needs to take place in an RNase-free environment.
The samples are applied directly to an agarose gel.
The sample is cut out from the gel, soaked in ammonium acetate to remove the denaturant (denaturant is inhibitory to the binding of RNA to nitrocellulose membranes), and stained with acridine orange or ethidium bromide.

Western Blots

Western blots detect proteins and separates them according to their molecular weight or charge

Run using a polyacrylamide gel with molecular weight standards / markers.
Utilizes capillary or electrophoretic transfer methods.
Membrane must be blocked with a solution to prevent binding of the primary antibody probe to the membrane.

TYPES OF PROBES
DNA Probes	Southern Blots	Complementary to the target gene
RNA Probes	Northern Blots	Complementary to the target sequence
Protein Probes	Western Blots	Antibodies bind to the target protein

SUMMARY OF HYBRIDIZATION TECHNIQUES
Method	Target	Probe	Purpose
Southern Blot	DNA	Nucleic Acid	· Gene structure
Northern Blot	RNA	Nucleic Acid	· RNA transcript structure, processing, and gene expression
Western Blot	Protein	Protein	· Protein processing and gene expression
Southwestern Blot	Protein	DNA	· DNA binding proteins and gene regulation
Eastern Blot	Protein	Protein	· Modification to western blot using enzymatic detection · Detection of specific agriculturally important proteins
Far-Eastern Blot	Lipids	None	· Transfer of HPLC-separated lipids to PVDF membranes for analysis by mass spectrometry

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-LeAnne Noll, BS, MB(ASCP)^CM is a molecular technologist in Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.

New Zika Test on the Horizon?

According to a recent press release, Rheonix is pursuing a rapid Zika Virus diagnostic test. Lablogatory recently discussed this test with the senior vice president for scientific and clinical affairs at Rheonix.

Lablogatory: I understand this test is a so-called “self-confirming” assay; it corroborates serological results with a molecular confirmation. Can you tell readers a bit about the methodology behind this?

Richard Montagna, PhD, FACB: It works much like the “dual assay” for HIV that we recently developed. Using microfluidics, the sample will be split between two sections of the same cartridge. One section will test for antibodies in a methodology similar to ELIZA. The other section will use LAMP technology to lyse, extract, purify, and amplify Zika-specific RNA sequences.

Lab: Sounds efficient! How long do you anticipate the assay will take to run?

RM: We expect it would take less than an hour to perform. Using our existing equipment base, we anticipate the capacity to perform 24 tests in an hour.

Lab: Given the timeline of the impending outbreak, will you seek Emergency Use Authorization (EUA) from the FDA?

RM: Once development is complete, we’ll discuss EUA with the FDA to determine if that approach is feasible.

Molecular Diagnostics Survey

If you’re involved with molecular diagnostics, then L. J. Lee’s team at the Ohio State University would like your input. They’ve developed a new technology using molecular beacons and would love for lab directors, managers, and bench technologists to answer this short survey.