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. 

Probe Structure for the Molecular Laboratory Professional

The purpose of real-time PCR is to perform efficient amplification of a target sequence and quantify the PCR products in “real time” by employing the use of a fluorescent reporter.  Fluorescent reporters can found in the form of DNA-binding dyes or fluorescently labeled primers or probes.  It is extremely important to understand the difference between DNA-binding dyes, and the various fluorescent primer and probe based chemistries.  The best way to grasp these theories is often to have a visual illustration of each of the different chemistries.

DNA-binding Dyes

  1. SYBR Green Dye – SYBR Green I is a fluorescent DNA binding dye that is commonly used as it binds to all double-stranded DNA.
    • SYBR Green is detected by quantifying the increase in fluorescence during PCR.
    • Advantages to using SYBR Green are that it is inexpensive, easy to use, and easily incorporated into the PCR reaction.
    • Disadvantages of using SYBR Green are that there is usually an increase in background and non-specific binding that can lead to detection of false positive results.
probe1
Image courtesy of: http://www.sigmaaldrich.com/technical-documents/protocols/biology/sybr-green-qpcr.html

 Fluorescent PCR Primer and Probe Based Chemistries

  1. Taqman Chemistry – Utilizes 5’ – 3’ exonuclease activity of Taq Polymerase (enzyme that copies DNA and necessary for PCR) to generate a signal.
    • The probe is composed of a single stranded DNA oligonucleotide which is complementary to the specific target sequence of the PCR template.
    • The probe has a modification to the 3’ end so that the polymerase cannot extend the sequence.
    • The 5’ end has the fluorescent dye and the 3’ end contains the quencher
    • During DNA synthesis, the exonuclease activity of the Taq Polymerase will degrade the probe, thus resulting in release of the reporter from the quencher.
probe2
Image courtesy of: https://es.wikipedia.org/wiki/TaqMan#/media/File:TaqMan_GX_cartoon.jpg
  1. Fluorescent Resonance Energy Transfer (FRET) – Energy is transferred between two light sensitive molecules.
    • Increase in target à More probes bind à Increase in fluorescence
    • The 5’ end is the donor (catalyst) and the 3’ end is the acceptor (fluorophore)
    • The energy is detected in the form of heat or fluorescence emission.
    • If probes bind, energy is transferred from donor to acceptor and generates the signal.
probe3
Image courtesy of: http://www.cdc.gov/meningitis/lab-manual/images/chapt10-figure01.gif

 

  1. Molecular Beacon – This type of chemistry measures the accumulation of product during the annealing phase of PCR.
    • Signal is detected only when probes are bound to the template before displacement by the polymerase.
    • A chemical modification prevents degradation during the extension step of PCR.
    • The 5’ end contains the reporter fluorophore and the 3’ end contains the quencher.
    • The amount of fluorescence is directly related to the amount of initial template available for binding and inversely proportional to the cycle threshold (CT) value.
    • During extension, the probe is displaced by Taq Polymerase and the hair-pin (non-fluorescent) structure is restored.
    • Unbound molecular beacon probe à reporter is too close to quencher à no signal is generated.
    • Beacon probe binds to target à reporter is separated à signal is generated.
probe4
Image courtesy of: http://www.bio-rad.com/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/real-time-pcr-detection-standard-pcr-primer-and-molecular-beacon.gif
  1. Scorpion – Scorpion probes use two PCR primers, where one serves as a probe and once contains a stem-loop structure.
    • The stem-loop structure contains a 5’ fluorescent reporter and a 3’ quencher.
    • The loop of the scorpion probe contains complementary sequence to the internal portion of the target sequence.
    • If the primer binds and extends, the reporter is separated from the quencher and a signal is given off.
probe5
Image courtesy of: http://www.bio-rad.com/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/real-time-pcr-detection-scorpions-pcr-primer-probe.gif

Understanding the various primer-probe chemistries including the interactions between the reporters and quenchers will provide some basic groundwork for those interested in pursuing a career in molecular biology.

 

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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.

International Lung Cancer Experts Seek Public Comments on Revised Molecular Testing Guideline

From the press release:

BETHESDA, MD. June 28, 2016 — The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) announced today the open comment period for the revised evidence-based guideline, “Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors.”

The open comment period begins today and will close on August 2, 2016. The online format provides an opportunity for public review of new draft recommendations for several key topics, as well as recommendation statements that have been reaffirmed since the initial guideline was jointly published online in April 2013 by Archives of Pathology & Laboratory Medicine, The Journal of Thoracic Oncology, and The Journal of Molecular Diagnostics.

The guideline revisions are designed to provide state-of-the-art molecular testing of lung cancer recommendations for pathologists, oncologists, and other cancer and molecular diagnostic laboratory professionals. The revisions are all based on evidence from an unbiased review of published experimental literature since 2013 and include the recommendations from an expert panel of renowned worldwide leaders in the field. The final recommendations will be approved and jointly published after consideration of the public comments, further panel discussion, and a complete evidence analysis. For more information and to provide comments, visit www.amp.org/LBGOCP.

Hybridization Conditions and Melting Temperature

Stringency is a term that many molecular technologists are all very familiar with. It is a term that describes the combination of conditions under which a target is exposed to the probe. Typically, conditions that exhibit high stringency are more demanding of probe to target complementarity and length. Low stringency conditions are much more forgiving.

  • If conditions of stringency are too HIGH → Probe doesn’t bind to the target
  • If conditions of stringency are too LOW → Probe binds to unrelated targets

 

Important Factors That Affect Stringency and Hybridization

  • Temperature of hybridization and salt concentration
    • Increasing the hybridization temperature or decreasing the amount of salt in the buffer increases probe specificity and decreases hybridization of the probe to sequences that are not 100% the same.
  • Concentration of the denaturant in the buffer
    • For example: Deionized Formamide and SDS can be used to reduce non-specific binding of the probe
  • Length and nature of the probe sequence
STRINGENCY AND BINDING
– Long Probe

 

– Probe has increased number of G and C bases

 

Binding occurs under more stringent conditions
– Short Probe

 

– Probe has increased number of A and T bases

 

Binding occurs under less stringent conditions

Melting Temperature (Tm) Long Probes

  • The ideal hybridization conditions are estimated from the calculation of the Tm.
  • The Tm of the probe sequence is a way to express the amount of energy required to separate the hybridized strands of a given sequence.
  • At the Tm: Half of the sequence is double stranded and half of the sequence is single stranded.
  • Tm = 81.5°C + 16.6logM + 0.41(%G+C) – 0.61(%formamide) – (600/n)

Where M = Sodium concentration in mol/L

n = number of base pairs in smallest duplex

  • If we keep in mind that RNA is single stranded (ss) and DNA is double stranded (ds), then the following must be true:

 

RNA : DNA Hybrids   More stable

DNA : RNA Hybrids        ↓

DNA : DNA Hybrids    Less stable

 

  • Tm of RNA probes is higher, therefore RNA : DNA hybrids increase the Tm by 20 – 25°C

 

Calculating the Tm for Short Probes (14 – 20 base pairs)

  • Tm = 4°C x number of G/C pairs + 2°C x number of A/T pairs
  • The hybridization temperature (annealing temp) of oligonucleotide probes is approximately 5°C below the melting temperature.

melt-temp

Sequence Complexity (Cot)

  • Sequence complexity refers to the length of unique, non-repetitive nucleotide sequences.
  • Cot = Initial DNA Concentration (Co) x time required to reanneal it (t)
  • Cot1/2 = Time required for half of the double-stranded sequence to anneal under a given set of conditions.
  • Short probes can hybridize in 1 – 2 hours, where long probes require more time.

 

Test Your Knowledge

  1. Calculate the melting temperature of the DNA sequence below:

ATCTGCGAAATCAGTCCCGG
TAGACGCTTTAGTCAGGGCC

 

Answer
If the number of G/C pairs = 11, and the number of A/T pairs = 9. The calculation is as follows:
4(11) + 2(9) = X
X = 62°C

L Noll Image_small

-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.

 

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

  1. DNA is isolated and cut with restriction enzymes.
  2. The DNA fragments are then analyzed by gel electrophoresis and separated by size (see previous blog post on Separation and Detection).
  3. 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.
  4. Denaturation – The DNA is denatured by exposing the gel to sodium hydroxide (NaOH). Denaturation breaks the hydrogen bonds that hold the DNA strands together.
  5. Blotting – The denatured DNA is transferred to a solid substrate (nitrocellulose) that helps to facilitate probe binding and signal detection.
  6. Pre-hybridization – Prevents non-specific binding of the probe to other sites on the membrane surface.
  7. 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 Ÿ Utilizes capillary movement of the buffer from a soaked paper to the dry paper

Ÿ Denatured DNA moves from the gel to the membrane

Electrophoretic Transfer Ÿ Electric current moves the DNA from the gel to the membrane
Vacuum Transfer Ÿ 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.

Separation and Detection of Nucleic Acids via Electrophoresis Methods

Analysis of a DNA sequence can be accomplished via a method called electrophoresis. Electrophoresis is a term that basically describes the movement of molecules by way of an electric current and separation of those molecules based on size. This process occurs through agarose or polyacrylamide gels, which serve as a way to limit migration of molecules as they move from the negative anode to positive anode. Small molecules move through the gel matrix faster than larger molecules.

How does it work?

Each phosphate group from a DNA molecule is ionized

DNA becomes negatively charged

DNA migrates towards the positive pole (anode)

 

Factors Affecting Electrophoretic Separation

  • Ÿ Strength of the electric current (voltage)
  • Ÿ Concentration and type of the buffer
  • Ÿ Gel density
  • Ÿ Size of the DNA
AGAROSE CONCENTRATION AND SEPARATION RANGES
Agarose Concentration (%) Separation Range (base pair size)
0.3 5,000 – 60,000
0.6 1,000 – 20,000
0.8 800 – 10,000
1.0 400 – 8,000
1.2 300 – 7,000
1.5 200 – 4,000
2.0 100 – 3,000

As agarose concentration increases, the separation range decreases

 

TYPES OF ELECTROPHORESIS SYSTEMS
Pulsed Field Electrophoresis
  • Ÿ Best for very large DNA molecules
  • Ÿ Current is applied in alternating directions
Field Inversion Gel Electrophoresis

(FIGE)

  • Ÿ Alternates the + and – electrodes
  • Ÿ Requires temperature controls and a switching mechanism
Polyacrylamide Gel Electrophoresis

(PAGE)

  • Ÿ Best for very small DNA fragments
  • Ÿ Initially used for protein separation, but can also be used for high resolution of nucleic acids
Capillary Electrophoresis
  • Ÿ Molecules are separated by size and charge
  • Ÿ Small Molecules = Fast
  • Large Molecules = Slow
  • Negatively Charged = Fast
  • Positively Charged = Slow
  • Ÿ Utilizes a polymer inside of a capillary instead of a gel
  • Ÿ Increased sensitivity

Understanding Buffer Systems

In order to change the pH of a buffered solution by one point, either the acidic or basic form of the buffer must be brought to a concentration 1/10th that of the other form.

Test Your Knowledge

  1. Based on the diagram, determine the sizes (to the best approximation) of the DNA fragments for each of the samples:

 

 

electro1

Answer:

  • Sample A: 200bp, 700bp
  • Sample B: 90bp, 600bp, 875bp
  • Sample C: 400bp

 

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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.