Replication Basics

To gain a solid understanding of Molecular Diagnostics, one has to grasp the fundamentals of DNA Replication. The double helix nature of DNA requires numerous moving parts working together to produce two identical strands of DNA from one original DNA molecule.

Image courtesy of
Image courtesy of

The Origin of Replication

The semi-conservative process of DNA replication occurs in a 5’ to 3’ anti-parallel direction. The replication process is described as semi-conservative because the sequence of nucleotides is maintained through new generations of replication. An extremely important enzyme involved in the beginning stages of DNA replication, is called Topoisomerase. It is responsible for regulating the over-winding and under-winding of DNA just ahead of the replication fork. Topoisomerase binds to the DNA then “cuts” the phosphate backbone so that the DNA can be unwound then resealed at the end of replication. Also, before replication can begin, an enzyme called helicase must first unwind and untangle the double-stranded DNA. Single stranded binding proteins (ssbp) prevent premature binding as well as protect the single stranded DNA from being digested by nucleases.

Leading Strand vs. Lagging Strand

During replication, two separate strands of DNA are formed in different ways. The lagging strand exhibits discontinuous 3’ to 5’ growth away from the replication fork and requires primase to “prime” the synthesis of the lagging strand. An RNA primer is added to the lagging strand of the DNA by RNA polymerase. This RNA primer begins synthesis of the lagging strand. The separate fragments of the lagging strand are termed Okazaki fragments. It’s important to note that due to the discontinuous formation of the lagging strand, each Okazaki fragment requires its own, separate, RNA primer. Finally, DNA ligase forms phophodiester bonds between the existing DNA strands to join the Okazaki fragments together. Alternatively, the leading strand during replication grows towards the replication fork in a 5’ to 3’ direction. The leading strand only needs one single RNA primer to immediately begin replication and therefore does not require DNA ligase.

Toward Replication Fork Single RNA Primer
5’→ 3’
Continuous Growth
Away from Replication Fork Primase
3’→ 5’ Multiple RNA Primers
Discontinuous Growth DNA Ligase
Creation of Okazaki Fragments

DNA Polymerase III and its Role in Replication

While you should become familiar with the extensive list of DNA Polymerases (shown below), the core polymerase involved in DNA replication is DNA Polymerase III. It functions as a catalyst in the formation of the phosphodiester bonds between an incoming deoxyribose nucleotide triphosphate (dNTP) determined by hydrogen bonding to the template at the 3’ end of the primer.

DNA Polymerase I Recombination, Repair, Replication
DNA Polymerase II Repair
DNA Polymerase III Core Polymerase


DNA Polymerase IV and V Bypass DNA Damage (Y-Family DNA Polymerases)
Alpha (α) RNA Primase

Lagging Strand

Replication (Initiation, Okazaki Fragment Priming)

Beta (β) DNA Repair
Delta (δ) Leading Strand


Epsilon (ε) Sensor of DNA replication that coordinates transcription cycle


Gamma (γ) Mitochondrial Replication
RNA Polymerase I rRNA (ribosomal RNA)
RNA Polymerase II mRNA (messenger RNA)
RNA Polymerase III tRNA (transfer RNA)

sbRNA (small nuclear RNA)

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

Do Children of Teenage Fathers Have an Elevated Rate of DNA Mutations?

If you recall one of my previous posts about DNA mutations, I discussed briefly the difference between somatic and germ-line mutations as well as the various types of mutations and resulting consequences.

Recently, I came across a research article that suggests an elevated germ-line mutation rate in teenage fathers, thus leading to an unexpectedly high level of DNA mutations in the children born to teenage fathers.

Many studies have been conducted on the theory that male germ cells go through a higher number of cell divisions when compared to that of female germ cells, and that the higher number of paternal cell divisions leads to an increased DNA mutation rate. The paper suggests that the increased presence of DNA mutations in sperm cells of teenage boys could explain why their offspring might be at higher risk for a spectrum of disorders when compared to parents in their twenties.

It’s an interesting read! More information as well as the full article can be found at


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

A “Primer” on DNA and the Consequences of Mutation

We can’t talk about Molecular Diagnostics without possibly talking about DNA, and with DNA, comes mutation. Much of the work I do revolves around searching for specific variants in a patient’s DNA sequence. We all have mutations in our DNA, but does that mean we all are affected negatively? Absolutely not. Spontaneous mutations occur during normal process such as DNA replication and repair within our cells. They also can arise from exposure to ionizing radiation, UV exposure, and chemical agents. Some mutations are passed down through reproductive cells. Mutations can be categorized as harmless or sometimes hurtful giving rise to gene defects, copy number variants, metabolic deficiencies, and cancer, while others result in positive effects.

Where do mutations occur?

Somatic mutations occur in cells that aren’t reproductive in nature. These mutations for the most part do not have a blatant effect on the organism because our normal body cells are able to counteract the mutated cells. However, sometimes mutations in somatic cells can affect division of cells, which has the potential to result in forms of cancer. On the contrary, germ-line mutations occur in reproductive cells and have the possibility of being passed down through generations, resulting in the presence of the mutation in all of the organism’s cells.

Understanding DNA

A T G C bases, along with a sugar, and a phosphate group combine to form a polymer of nucleotides. This polymer backbone of alternating sugar and phosphates is what we call DNA (Deoxyribonucleic Acid). DNA is transcribed to RNA (Ribonucleic Acid) which instead of Thymine (T) contains a Uracil (U). Finally, RNA is translated to protein.

Pyrimidines       Purines
Single-ringed organic bases Double-ringed organic bases
Thymine (T)

Cytosine (C)

Uracil (U)

Adenine (A)

Guanine (G)

Hydrogen Bonds Between Complimentary Strands of DNA
Adenine bonds Thymine   A = T(U)

Guanine bonds Cytosine   G ≡ C

Two Hydrogen Bonds

Three Hydrogen Bonds

Nucleic acid→ triplet codon (amino acids)→ polypeptides→ proteins (approx 50 amino acids in length)

Amino acids are the triplet codons that make up proteins. It is extremely important to become familiar with the codon table. You don’t need to memorize it, however you should at a minimum know what triplets code for START and STOP codons. You will quickly see that there are multiple codes for a single amino acid. This becomes important when considering the effects of mutation:


Like I mentioned earlier, many of the tests I perform involve screening for variants in DNA sequence. I accomplish this through different methods, which I will touch on in future blog posts. We could easily discuss mutations for hours and hours, but for now, it’s probably most important to gain a quick (and simple) understanding of the types of mutations and what the resulting effects can be. For the sake of demonstration, let us consider the following normal (wild type) sequence pattern:

NOTE: Uracil replaces Thymine because of transcription from DNA to single stranded messenger RNA (mRNA)
NOTE: Uracil replaces Thymine because of transcription from DNA to single stranded messenger RNA (mRNA)

Transition Mutations occur when mispairing results in a purine being replaced with a different purine, or a pyrimidine replaced with a different pyrimidine:



Transversion Mutations occur when a purine is replaced with a pyrimidine or vice versa:



Missense Mutations have the ability to result in a change in the amino acid. This happens when the codon triplet is altered, and the amino acid sequence of the encoded protein is changed:


Sometimes, Missense Mutations are silent:

Notice how the structure of the gene product is unchanged. This happens because all amino acids can be encoded by more than one triplet codon.
Notice how the structure of the gene product is unchanged. This happens because all amino acids can be encoded by more than one triplet codon.

Frame shift mutations can result from either a single base insertion or a single base deletion.


Nonsense mutations are exactly what they are called… Nonsense! Here, a base substitution changes an amino acid into a stop codon. Nonsense mutations result in abnormal termination of translation and the protein product is shortened:


Chromosomal Abnormalities

Sometimes changes in the actual chromosomal structure or number take place in the cells. These changes can result in deletions, duplications, inversions, and translocations of chromosomes. Chromosomal deletions and insertions are simply the loss or gain of chromosomal material. Inversions result from the removal, flipping, and then reconnection of the chromosomal material within the same chromosome.

Translocations are more complicated and involve switching of genetic material between two chromosomes. A reciprocal translocation occurs when parts from two different chromosomes exchange. We call a reciprocal translocation “balanced” when there isn’t a gain or loss of genetic material between the chromosomes (both chromosomes remain fully functional). An “unbalanced” reciprocal translocation affects an offspring’s phenotype due to extra or missing genes.

This information I have touched on is very general, however, it doesn’t mean it is not of importance. These basic concepts are the fundamentals needed to begin understanding mutation as they will provide us with the framework for recognizing the needs and importance of molecular diagnostic testing!

Test your Molecular Knowledge

  1. What amino acids do the following codons code for:
    1. GAU
    2. AUG
    3. UAG
    4. CAG
    5. UGA
  2. Classify the following mutations:
    1. T→A
    2. T→C
    3. AGA→UGA
    4. AGA→AAA
    5. AGA→CGA


1 a) Aspartic Acid b) Methionine (start) c) Stop codon d) Glutamine e) Stop codon. 2 a)Transversion b) Transition c) Nonsense d) Missense e) Silent

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

The Path to Molecular Diagnostics – MB(ASCP) Certification

While I have been working in the field of all things “Molecular” for close to twelve years, it wasn’t until early this year that I decided to actually sit for the ASCP certification exam in Molecular Biology. With over five years working in research and currently going on seven years in a clinical lab, I was feeling pretty confident about my knowledge and background, but then panic set in. Would I be able to pass a test? I haven’t taken an exam since my final years of college! I have two young children who are extremely active in extracurricular activities, a husband who also works full time. Needless to say, any extra time I had for myself would be spent studying. It didn’t take long to find out that while there was a very general outline of topics covered and an extensive list of textbooks to serve as a starting point, there were not specific details on where to focus my efforts. There was no single study guide, no tangible tools I could utilize to make exam preparation fit into my busy life any easier.

Fast forward to the present, where all of the panic was for not. In February I did pass (with flying colors) and happily sport MB (ASCP)CM after my name. When I was recently approached about writing on a regular basis for Lablogatory, I knew it would be an amazing opportunity to educate others on the exciting field of Molecular Diagnostics as well as combat the fear that comes with taking the Molecular Biology exam. I plan to focus on more specific areas in the field of Molecular Diagnostics that I came across in my studies, which will help others in their preparation for the MB(ASCP) exam. I will cover theory, applications, techniques, and practices. Also, keep an eye out for case study questions that I will provide to stretch your thinking through interpretation of molecular results, at the same time, keeping you up to date on hot topics in the field.

With all that said, the first most important suggestion I have is: spend time preparing to study. While it might seem silly to some, having a plan BEFORE you jump into study mode will actually set you up for success. All too often, just reading everything you can on every single topic will result in information overload and ultimately burn you out. You will quickly find yourself jumping all over the place frantically trying to memorize every detail you come across.

First, browse the content outline and choose some texts to review. Notice how I say, “Choose some?” Don’t feel like you have to read all of them. I went online and reviewed the texts, I asked colleagues for recommendations, then decided to purchase two books. You might find that you need more or less, just don’t go overboard.


Next organize a binder. I am extremely visual and hands on so I study best with things in front of me that aren’t electronic based. I divided my binder based on the content outline. You will come across papers, technical notes, and procedures that you will want to keep as study tools. Having a binder for all of these notes from multiple sources will keep you on track. It will also serve as a great reference guide for you as you move through your career.


Now is where you can get super retro. You may have noticed in my photos that I made flash cards! It is a little archaic and time consuming, but I knew I was going to be busy with my daughter’s traveling basketball team; therefore, I needed something small I could take with me to study while sitting on the bleachers. I am aware of software programs that allow you to make electronic based flash cards, so if that works better for you feel free to try that route.

My take home message is, obtain and prepare whatever tools you need to get yourself ready to study. Organization is key and setting aside time each day, even if only an hour to focus on one application or theory will definitely be to your benefit. Reach out to colleagues and gather as much information as you can. Once all of your tools are in place, it’s time to get to work! For anyone interested in obtaining their MB(ASCP) certification, I urge you to check out the content outline and list of texts on the ASCP Board of Certification site.

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

Enterovirus D68

Over the past few weeks, hospitals around the country have seen a sharp uptick in cases of respiratory distress in children. The majority of patients test positive for Enterovirus D68, and most seem to have a history of asthma.

Only select laboratories test for this strain of enterovirus. If a suspected case comes to your facility, contact the CDC or your local health department for information about specimen collection and shipping.



Viral DNA Testing, Cervical Cancer, and Cytology

An FDA panel recently recommended use of Roche’s Cobas HPV DNA test as the initial screening tool for cervical cancer instead of the ubiquitous Pap smear. The panel found that “A negative HPV result predicted a lower 3-year risk of ≥CIN3 than did a negative cytology result, suggesting that using HPV as the primary test is superior to cytology for cervical cancer screening.The low 3-year CIR for a negative HPV result also confirmed the safety of a 3-year interval for HPV primary screening and officer clinicians more confiedence in a negative HPV result than a negative cytology result.”

While this isn’t a final FDA guideline, it’s conceivable that clinicians could alter their practice based on these findings. Of course, this will affect the cytology and molecular diagnostics departments, as well. What do you think? Is HPV viral testing the way to go? Or should we stick with the test that’s been around for decades?

Are Pathologists and Primary Care Physicians Ready for the Genomic Era and Personalized Medicine?

I was reading about the FDA’s recent crackdown on 23andme to stop marketing their saliva based whole genome testing and interpretation service. Rather than resist, 23andme decided to comply and is currently in “talks” with the FDA so that they can complete the process for FDA validation and again begin to market their kits and testing. For now, they can continue to provide their genealogy testing and whole genome sequencing without interpretations.

Currently, in some academic research centers, whole genome or exome sequencing via next generation sequencing (NGS) methods is utilized on a limited basis by researchers and clinicians to identify pathogenic mutations. NGS and bioinformatic analysis methods continue to steadily improve and costs have been decreasing. However, there are limitations and barriers to widespread use at this point. These include but are not limited to: 1) widely used databases such as the Human Gene Mutation Database (HGMD) and the Online Mendelian Inheritance in Man (OMIM) still only contain information that only covers a fraction of the human genome, 2) more research is still needed to identify more variants mutation-disease associations, and 3) most mutations identified fall under the category of “unknown clinical significance”.

Tools such as NGS, despite its improvement over previous technology, still cannot identify large deletions or copy number variations (CNV) and is a technology not accessible, cost-wise and support-wise, to most health care institutions. Despite all of this, primary care physicians, even now, still may be confronted with patients who bring them their genomic screening results, whether obtained from commercial services provided by companies like 23andme or through molecular testing through a health care institution. But today’s physicians, including primary care physicians and pathologists, were not trained in medical school to understand how this testing is performed or the significance of these results. But the time is coming, and maybe sooner than we realize, when we will have to deal with such testing on a daily basis.

So, it is imperative that we train our doctors and doctors-in-training now to be ready for when that time comes. But, my question this week is “How should we go about it?” Additionally, who should compose the health care team to provide guidance and counseling to patients once results are available? And who should regulate how testing should be done and what information should be included in results reporting? Leave me a comment if you have an opinion or any ideas.


Betty Chung, DO, MPH, MA is a second year resident physician at the University of Illinois Hospital and Health Sciences System in Chicago, IL.