Showing posts with label PCR. Show all posts
Showing posts with label PCR. Show all posts

Tuesday 24 September 2024

New Video: What is the most significant change I've seen in the biosciences during my career?

In the video What is the most significant change I've seen in the biosciences during my career?, I look at what I consider to be some of the biggest changes I have seen.

This video is part of my "You Ask, I Answer" series.

Over my career in bioscience, I’ve witnessed some incredible changes that have significantly changed how we do biomedical sciences and how cells work.

Recently, I was asked what I considered the most significant change in the biosciences I had seen. At first, I thought of the rise of omics technologies like genomics and proteomics, but after I thought about it some more, a few key innovations and changes in our thinking truly stood out.

In this blog post, I’ll tell you what I consider to be the major changes I have seen.

The PCR Revolution

When I first encountered PCR in the late 1980s, it seemed almost like a magic trick. The idea that you could heat a solution containing enzymes to 90°C and still get a reaction was mind-blowing. As a biochemist, the method seemed to go against what I had been taught on my degree - enzymes start to denature and stop working once you get above 40 ºC. 

Now, PCR is everywhere in the biosciences. It's used for research, criminal forensics, and disease diagnosis, and the ability to amplify DNA has changed the way we do science.

PCR Videos:

Mass Spectrometry and Proteomics

Another major technological leap I saw was the rise of easy-to-use mass spectrometers. Early in my career, mass spectrometry was a complex and inaccessible tool. However, the introduction of user-friendly mass spectrometers transformed lab work. Suddenly, we could easily measure the monoisotopic mass of peptides and carry out peptide mass fingerprinting to identify proteins with ease. 

This shift was a game-changer for proteomics and molecular biology, enabling us to quickly identify proteins. The combination of advanced instruments and accessible databases allowed for faster, more detailed analyses that were once considered out of reach for most labs.

Bioinformatics

Bioinformatics has completely altered the way we approach science. Finding a scientific paper or sequence information in the past required extensive library work and manual searches. Now, with databases full of genetic sequences, protein structures, and published studies, the challenge has shifted from finding data to filtering and making sense of it. 

When I started, labs had no computers at all. Now, every researcher has a personal computer linked to a global network of scientific knowledge and a vast array of powerful bioinformatic tools. 

Adipocytes - the cells that changed

Beyond technological advances, our understanding of biology has also changed. 

When I first started studying bioscience, adipocytes (fat cells) were considered passive storage units for energy. Today, they are recognised as dynamic endocrine organs that secrete a range of adipokines, influencing metabolism and overall health.

As we age, we tend to gain more fat cells, effectively adding to the number of these endocrine cells in our bodies. This shift in understanding has implications for obesity research, diabetes treatment, and metabolic health,

Mitochondria - not just a powerhouse

Our understanding of the mitochondrion—the "powerhouse" of the cell—has also changed.

Mitochondria were initially viewed as static structures within cells; however, we now know that mitochondria are dynamic organelles. They fuse, move, and interact with other cellular components, forming complex networks that help regulate cellular energy and even apoptosis (programmed cell death).

Mitochondria Videos

The Golgi

Like the mitochondria, our view of how the Golgi works has also changed. Previously, the Golgi was viewed as a series of membranes that looked like a stack of dinner plates, with the proteins being processed moving through the stack. Now, the machinery processing the proteins comes to the proteins, a radical change in our thinking.

My top picks

So, which do I view as the top change? Well, you will have to watch the video to find out!

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Monday 19 August 2024

New Video Posted: PCR Primer Design: Tips for Accurate DNA Amplification

I have posted a video on PCR primer design - PCR Primer Design: Tips for Accurate DNA Amplification.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Polymerase Chain Reaction (PCR) is an important lab technique that allows scientists to amplify specific DNA sequences. However, the success of a PCR largely depends on correctly designing the PCR primers. 

Why Primer Design Matters

The importance of primer design in PCR cannot be overstated. If your primers are poorly designed, the DNA amplified during the reaction might not be the correct region of DNA, or you will get a low product yield. To avoid such issues, you must carefully consider three primary factors when designing primers: 

  1. Melting temperature (Tm)
  2. Disruptive secondary structures
  3. Specificity of the primers

1. Melting and Annealing Temperatures

Two crucial temperatures play a role in PCR primer design:

  1. the melting temperature (Tm)
  2. the annealing temperature
The Tm is the temperature at which 50% of the primer would dissociate from a double-stranded to a single-stranded form. The primers bind to the denatured DNA template during each PCR cycle at the annealing temperature. Typically, the ideal Tm is 55-65 °C, and the annealing temperature is set 3-5 °C below the Tm to ensure optimal primer binding.

In the lab, you would use a computer program to determine the Tm of a primer. The program usually uses the nearest neighbour method to calculate the Tm accurately. This method considers the specific sequence of bases and the concentrations of components in the reaction.

2. Disruptive secondary structures

It is essential to ensure your primers won’t form problematic secondary structures like hairpin loops or primer-dimer pairs. Hairpin loops occur when a primer folds back on itself, creating a loop that can be difficult to melt during the PCR process, leading to inefficient amplification. Primer-dimer pairs form when two primers bind to each other instead of the target DNA. This can result in an inefficient reaction and the production of the wrong DNA.

In the lab, you would use a computer program to test for hairpin loop formation and primer-dimer pairs.

3. Specificity of the primers

A standard PCR primer is usually 20 to 30 bases long and has an ideal Tm of around 55-65 °C. The Tm and the length of the primer help ensure specificity. 

Finally, it is advisable to perform a BLAST search with your primer sequences, which can help confirm that they do not have unintended matches with other sequences in the species. If the DNA species is not human, a BLAST search against a human database should be performed to check for the possibility of a result if the reaction is contaminated with human DNA.

Advanced Techniques: Overcoming Challenges in PCR

In some cases, even with well-designed primers, you might need help getting the correct DNA to amplify, especially when dealing with low amounts of template DNA or needing to add restriction sites to your product. To address this, you can use a nested primer approach. This involves using two sets of primers, one set falling inside the other, to improve specificity and yield through a two-round PCR process.

For adding restriction sites, primers are designed with the desired restriction site sequences at the 5’ end. However, this introduces a mismatch between the primer and the template DNA. To compensate, the initial PCR cycles are performed at a lower annealing temperature to help the primers bind more effectively. Once the restriction site is incorporated into the amplified DNA, the annealing temperature is raised to the standard level for subsequent cycles. A GC clamp at the 3’ end of the primer, where the last two bases are guanine (G) or cytosine (C), can also help improve binding due to the stronger hydrogen bonds these bases form.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Additional Reading

The video was produced with help from the following resources:

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Friday 3 May 2024

New video posted: Understanding Nucleic Acid Hybridisation: Methods & Applications Explained

In this video, I look at Nucleic Acid Hybridisation and how it is the underlying principle for several lab techniques, such as PCR (Polymerase Chain Reaction), dot blots, colony blot hybridisation, chromosome in situ hybridisation (FISH), microarrays, Southern and Northern blotting, and CRISPR/Cas9 gene editing.

If you would like to support my blogging efforts, then please feel free to buy me a coffee at https://www.buymeacoffee.com/drnickm

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.
 

Additional Reading

The video was produced with help from the following resources:

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Wednesday 1 May 2024

New video posted: DNA Cloning - how do we clone DNA in the lab?

In this video, I give a brief introduction to the subject of DNA cloning.

DNA cloning is an important lab skill that all life and biomedical science students should possess. In the video, I provide a summary of in vivo and in vitro cloning, along with the key steps, tools and methods you would use.

If you would like to support my blogging efforts, then please feel free to buy me a coffee at https://www.buymeacoffee.com/drnickm

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.
 

Additional Reading

The video was produced with help from the following resources:

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