New Video Posted: DNA Sequencing: Sanger Method and Beyond Explained

In the video DNA Sequencing: Sanger Method and Beyond Explained, I explain the Sanger method and look at some of the new approaches and methods that can speed up the DNA sequencing process.

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

DNA sequencing allows scientists to decode the genetic information that dictates everything from the colour of our eyes to how our cells function. One of the most widely used laboratory methods for DNA sequencing is the Sanger method, also known as the chain-terminating dideoxynucleotide method.

The Basics of Sanger Sequencing

The Sanger sequencing process begins like a PCR (Polymerase Chain Reaction), with a template DNA that you want to sequence, a single primer (and not two primers as in PCR), DNA polymerase, and nucleotides (dNTPs). However, the method also includes special nucleotides called dideoxynucleotides (ddNTPs), which play a key role in the sequencing process.

In the sequencing method, the reaction goes through 25-30 cycles of denaturing, annealing and extension, just as you would in PCR. The DNA polymerase builds new DNA strands during the reaction and adds nucleotides to the growing chain. The twist comes with the ddNTPs; when one of these is incorporated, the chain is terminated, and no further nucleotides can be added. Each ddNTP is tagged with a different fluorescent marker corresponding to one of the four DNA bases (A, T, C, or G). This allows the sequence to be read by analysing the fluorescent tags after the reaction.

Once the reaction is completed, the next step is separation. The DNA fragments are separated using gel electrophoresis. As the fragments move through the gel, a laser scans the gel for the fluorescent tags, and the sequence of the DNA is determined by the fluorescence colour at each position.

While the Sanger method has been incredibly valuable, it has limitations. It can only read between 500 to 1000 bases per reaction, making it labour-intensive and unsuitable for large-scale projects.

Moving Beyond Sanger: Advanced Sequencing Methods

As the need for faster and more efficient sequencing has grown, new methods have been developed. One such method is shotgun sequencing, where the genome is broken into random segments. Each segment is sequenced individually using the Sanger method, and then the fragments are pieced together like a puzzle to reconstruct the full genome.

Another significant advancement was Next-Generation Sequencing (NGS), also known as massively parallel sequencing. NGS technologies have revolutionised genomics by allowing the simultaneous sequencing of many DNA molecules. This high-throughput approach can detect the sequence using either fluorescent nucleotides or pH changes.

Finally, we have Third-Generation Sequencing methods, which include techniques like nanopore sequencing. In nanopore sequencing, DNA is passed through a nanopore, and the sequence can be determined by detecting changes in electrical current.

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

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)
•  📗 - Catchup Chemistry - (affiliate link)

New Video Posted: DNA Libraries: Genomic vs. cDNA - Key Differences and Applications

 I have posted a video on the key differences between genomic DNA (gDNA) and complementary DNA (cDNA) libraries - DNA Libraries: Genomic vs. cDNA - Key Differences and Applications.

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

DNA libraries are essential tools for researchers to study and manipulate genetic material. If you're working in a lab, you'll likely encounter two main types of DNA libraries: genomic DNA (gDNA) libraries and complementary DNA (cDNA) libraries. Though both serve important roles in research, they are fundamentally different in their composition, creation, and applications. 

What Are DNA Libraries?

A DNA library is a collection of DNA sequences cloned into vectors, small pieces of DNA that can carry foreign DNA into a host cell, such as bacteria. These libraries allow scientists to store, access, and manipulate specific DNA sequences for various research purposes.

Genomic DNA (gDNA) Libraries

A genomic DNA library is created from the complete genomic DNA of an organism. This means it contains all the genetic material necessary to build that organism. In eukaryotes, this includes both the coding regions (exons) and the non-coding regions (introns) of genes. 

How is a gDNA Library Made?

1. DNA Extraction: Genomic DNA is extracted from cells.
2. DNA Fragmentation: The extracted DNA is cut into smaller fragments using restriction enzymes.
3. Cloning: These fragments are then cloned into plasmids, which are circular DNA molecules.
4. Transformation: The plasmids are inserted into bacteria, which replicate the DNA fragments, creating a library.

When to Use a gDNA Library?

gDNA libraries are ideal when studying the full structure of genes, including regulatory elements, or when exploring gene functions across the genome.

However, there are some limitations. Because a gDNA library contains both introns and exons, it can complicate the task of isolating and studying the sequences that actually code for proteins. Additionally, genes in a gDNA library may be fragmented across multiple clones, making it challenging to reconstruct the complete gene sequence through sequence alignment.

Complementary DNA (cDNA) Libraries

cDNA libraries are derived from messenger RNA (mRNA) in a cell. This means that a cDNA library only contains the sequences actively expressed as proteins when the library is made. Therefore, the content of a cDNA library depends on the type of cell, the time of day, and the cell's conditions.

How is a cDNA Library Made?

1. mRNA Isolation: Cells are lysed, and the mRNA is purified from the lysate using affinity chromatography, which often involves oligo-dT beads that bind to the poly-A tails of mRNA molecules.
2. Reverse Transcription: Using reverse transcriptase, the mRNA is used as a template to synthesise complementary DNA (cDNA). This gives a mRNA/DNA molecule.
3. RNA Removal: The original mRNA is removed from the mRNA/DNA molecule using an enzyme (RNase H). The now single-stranded cDNA is converted into a double-stranded DNA molecule using DNA polymerase.
4. Cloning: The cDNA is cloned into plasmids and inserted into bacteria.

Isolating mRNA can be tricky because it is easily degraded by enzymes released during cell lysis or by RNases in the environment. Moreover, because cDNA libraries only reflect the genes being expressed at a specific time, they may not provide a complete picture of an organism's genome.

When to Use a cDNA Library?

cDNA libraries are particularly useful for studying gene expression, identifying specific mRNA sequences, and producing recombinant proteins.

Choosing the Right Library

It is essential to understand the distinctions between gDNA and cDNA libraries. A gDNA library is the right choice if the goal is to study the entire genome or understand gene regulation. However, a cDNA library must be used if you're interested in the proteins a cell produces or need to work with specific mRNA sequences.

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:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

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:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: Bacterial Transformation: Natural vs. Artificial Methods Explained

In this video - Bacterial Transformation: Natural vs. Artificial Methods Explained - I look at how bacteria can take up DNA from the environment and how we can get bacteria to take up DNA in the lab.

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

Bacterial transformation is where bacteria take up external DNA, which can lead to genetic changes within the cell. This process can occur naturally or be induced artificially in a laboratory setting. 

Natural Transformation

In natural transformation, bacteria acquire DNA from their surroundings, typically from nearby bacteria that have lysed (broken apart). The free-floating DNA can then be integrated into the bacterial chromosome or replace an existing gene, leading to potential new traits or functions. This ability to naturally take up DNA is a trait of "competent" cells, which possess specific genes that encode the machinery necessary for DNA uptake.

Artificial Transformation 

In a lab, we can induce transformation using artificial methods. Unlike natural transformation, this process requires deliberate manipulation of the bacteria to make them more likely to accept new DNA. First, the bacteria must be made competent so they can take up the DNA. Techniques such as heat shock, electroporation, or polycations are then used to encourage bacteria to take up the DNA. Each method works differently but ultimately serves the same purpose: introducing new genetic material into bacterial cells, enabling researchers to study gene function, produce recombinant proteins, or create genetically modified organisms.

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:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: How Bacteriophages Transfer DNA Between Bacteria

In this video - Understanding Transduction: How Bacteriophages Transfer DNA Between Bacteria - I look at the process of DNA exchange, transdcution, between bacteria by bacteriophages.

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

Transduction is driven by bacteriophages, which are viruses that specifically target and infect bacterial cells. In the video, I explain the mechanics of transduction and look at how bacteriophages can inadvertently transfer DNA from one bacterium to another.

Bacteriophages operate through two distinct life cycles: the lytic and lysogenic cycles. During the lytic cycle, the bacteriophage attaches to a bacterial cell, injects its DNA, and takes over the bacterium to produce new viruses. This ultimately leads to the bursting (lysis) of the bacterial cell, releasing the newly formed phages to continue the infection cycle. In the lysogenic cycle, the bacteriophage DNA is integrated into the bacterial chromosome, where it can lie dormant until conditions favour a return to the lytic cycle.

Some bacteriophages, such as the P22 phage, can mistakenly package bacterial DNA instead of their own during the assembly of new viruses. When these phages go on to infect other bacteria, they transfer the captured bacterial DNA, effectively driving genetic exchange between bacteria. This mechanism contributes to bacterial evolution in the wild and serves as a valuable tool in the lab for gene mapping and studying bacterial genetics.

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:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: Gene Mapping in Bacteria Using Conjugation and Interrupted Mating

Gene mapping in bacteria is a process that has helped scientists understand the order and location of genes on a bacterial chromosome. 

In this post, I will look at a method of gene mapping using bacterial conjugation, specifically focusing on a technique known as interrupted mating. Although this technique is historical and has been largely replaced by genome sequencing, it remains an important method that sheds light on genetic exchange in bacteria.

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

 

The Role of Hfr Cells in Gene Mapping

The process of using conjugation to map genes in bacteria was developed using a specific type of bacteria discovered in the 1950s, known as Hfr (High Frequency of Recombination) cells. These cells are particularly efficient at transferring genes during conjugation, a process where DNA is exchanged between two bacterial cells.

In Hfr cells, the F (fertility) factor, which is normally an independent plasmid, gets incorporated into the bacterial chromosome. This integration means that during conjugation, the F factor and parts of the bacterial chromosome can be transferred to a recipient cell.

Two outcomes can occur during this process:

1. F' Factors: If the F factor is excised from the chromosome, it may carry with it some bacterial genes, forming what is known as F' factors.
2. Chromosome Transfer: Alternatively, the entire bacterial chromosome, including the F factor, can be transferred to the recipient cell.

The Concept of Interrupted Mating

Interrupted mating is a technique that was used for gene mapping. To understand how it works, let’s consider an example involving two bacterial strains:

• Donor Bacteria (Hfr positive): This strain has the genes to produce amino acids leucine and threonine but is sensitive to the antibiotic ampicillin.
• Recipient Bacteria (Hfr negative): This strain lacks the genes for leucine and threonine production but is resistant to ampicillin.

In this experiment, the donor and recipient bacteria are mixed and allowed to conjugate. The goal is to map the location of the genes that produce leucine and threonine on the donor’s chromosome. Here’s how the process unfolds:

1. Conjugation Begins: The bacteria are mixed, and DNA transfer begins from the Hfr donor to the recipient.
2. Interrupted Mating: At specific time intervals, the mating process is interrupted, effectively stopping the transfer of DNA.
3. Plating on Selective Media: After interrupting the mating, the bacteria are plated on media that either lacks leucine or threonine and contains ampicillin. The donor bacteria, which are sensitive to ampicillin, will die. The recipient bacteria will only grow if they have received and integrated the necessary genes from the donor to produce the missing amino acids.
4. Mapping Gene Order: By examining which plates show bacterial growth at different time points, scientists can determine the order in which the genes were transferred. For instance, if bacteria start growing on the leucine-lacking plate before the threonine-lacking plate, it indicates that the leucine gene is closer to the origin of transfer than the threonine gene.

Applications and Limitations of the Technique

This method of mapping was useful because it allows scientists to estimate the relative positions of genes on a bacterial chromosome. For example, using this technique, researchers were able to map the E. coli K12 genetic map into a timeline of 100 minutes, representing the time required for the full chromosome to transfer at 37 °C.

However, the technique has limitations:

• It is mostly applicable to E. coli and closely related bacteria.
• Mapping the entire chromosome is rare and works best for genes that are close to each other.
• The F plasmids involved are large, of low copy number, and may not be ideal for genetic manipulation.

Despite these limitations, the interrupted mating technique provides valuable insights into genetic exchange mechanisms in bacteria. While modern approaches like genome sequencing have largely replaced it, understanding this historical method helps us appreciate the exchange of DNA between bacteria.

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:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: Genetic Exchange in Bacteria: Conjugation, Transduction, and Transformation

In this video, I look at the three methods bacteria use to share genetic information: Conjugation, Transduction, and Transformation.

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

Bacteria typically increase their numbers through a process akin to cloning; that is, they make exact copies of themselves. While this method is efficient, it presents a significant evolutionary limitation: genetic diversity can only arise through random mutations. In the absence of a mechanism for exchanging genes, bacteria would be stuck in a genetic standstill, unable to benefit from the rapid spread of advantageous traits. Bacteria solve this problem by horizontal gene transfer (HGT), which is also called lateral gene transfer (LGT).

To help the spread of advantageous genes in a bacteria population, the bacteria use three methods to exchange DNA:

1. Conjugation: This process involves direct physical contact between two bacteria through a structure called a sex pilus. A donor bacterium with an F plasmid (F+) forms a bridge to a recipient bacterium (F-), transferring a copy of its DNA. This method not only promotes genetic diversity but also allows for the rapid spread of advantageous traits such as antibiotic resistance.
2. Transduction: In this method, bacteriophages (viruses that infect bacteria) play a crucial role. When a virus infects a bacterium, it sometimes incorporates fragments of the host's DNA into its own genetic material. As the virus infects new bacterial cells, it transfers these DNA fragments, facilitating genetic exchange.
3. Transformation: This process occurs when bacteria take up free-floating DNA from their environment, often released by dead bacterial cells. The acquired DNA is then incorporated into the recipient's genome, providing new genetic traits that can be beneficial for survival and adaptation.

In the video, I examine the three methods.

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:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)