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MOLECULAR GENETIC

Def: Molecular genetics is a discipline of biology that is interested in genes from the molecular point of view, that is, the structure and organization of DNA, the expression of genes for protein synthesis, the way in which genetic information is transmitted from the parental generation to the descendant generation, the mechanisms of mutation, DNA repair during replication and the regulation of gene expression in organisms.

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ENZIMES 

Helicase: Ur-coils the doble heliz structure. Separates the two stands 

SSBP: Stablish the DNA molecule 

Topoisomerasa: Un-coils the doble helix 

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The nucleobases

Adenine,

Thymine,

Guanine,

Cytosine

(In DNA and uracil in RNA) constitute the “building blocks” of genetic information. They are paired in a specific way (adenine and thymine (or uracil in RNA) and guanine and cytosine). These pairs form the “rungs” of the DNA double helix and provide guarantees that the genetic information is accurate.

In replication, the process consists of unwinding the DNA and having the two strands serve as templates. DNA polymerases are the enzymes responsible for adding complementary nucleotides according to the pairing rules (adenine, thymine, guanine and cytosine). In this manner, two identical pieces of DNA are made, ensuring that each daughter cell contains the same genetic structure.

Central dogma of molecular biology:

DNA -> Trascription -> RNA -> Translocation-> Proteine

(Replication)

Explanation:

Central Dogma of Molecular Biology is like a blueprint of how genetic information flows in cells. It is the explanation of how DNA is decoded to make proteins, the workers of our bodies. This occurs in two distinct phases. The following steps occur:

The first step involves transcription: the process by which DNA is converted into RNA-a messenger carrying the necessary instructions.

The second step is translation: this is combining the amino acids together by reading this RNA using ribosomes to build proteins with those instructions derived from the DNA.

Hence, genetic info is "written" into RNA, and that "manuscript" is "translated" into proteins. An important stage is known to occur; it's like transforming the recipe into a dish-the very reason we live.

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REPLICATION

Glossary:

Primase: Enzyme that place a primer

Primer: Short sequences of nucleotide places by a primer

DNA plimerase III: Syntlantizes the new stans can only 5'-> 3'

Polymerase: enzyme that synthesizes or duplicates nucleic acids, either DNA or RNA, the introduction of new strands of nucleic acids, one nucleotide at a time, from nucleotides on the basis of the existing strand serving as a template.

Leading stand: 3' move in the same direction

Lagging stand: It's move on a different direction.

Short explanation in 4 steps:

1- Separation of the DNA strands: The DNA double helix opens like a zipper, separating the two strands.

2-Strand preparation: Proteins are incorporated to keep the strands open so that they do not close.

3- DNA copying: New strands are created by adding pieces of DNA, which are attached to the original strands like pieces of a puzzle. One is copied quickly and the other in short fragments.

4- Revision and completion: Gluing the fragments of the slower strand, checking that everything is correct and finishing the process with an exact copy of the DNA, so that we finally have two equal copies of DNA.

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Part 3 of replication

Lagging strand: Move in a different direction, that could be a problem because; as soon as they are separating the lagging strand collapses.

Primers are RNA: It is more volatile than DNA and disintegrates more easily over time.

Okazaki: This discovery was honored to the Japanese molecular scientist couple Okazaki (1962 or 1963).

Def: Okazaki fragments are small DNA sequences that form on the discontinuous strand during replication, since DNA polymerase can only synthesize in one direction. These fragments are then joined together to complete the new strand.

DNA polymerase I: Changes RNA primers for DNA nucleotides.

It is then folded to make a double molecule.

Ligase: Enzyme that closes the gaps in the laggin stand

DNA polymerase II: Proofrealing, mostly in lagging stand.

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TRANSCRIPTION

DNA transcription is the process by which an enzyme called RNA polymerase copies a DNA sequence into a messenger RNA (mRNA) molecule. This mRNA carries the genetic information from the DNA from the nucleus to the ribosomes, where it will be translated into a protein. The process has three stages: initiation, where RNA polymerase binds to DNA; elongation, where the mRNA strand is synthesized; and termination, when transcription is complete and the mRNA is released..

DIFFERENCES

DNA RNA

Can't leave the nucle - Can

Deoxytibose - Ribose

2 strands (double helix) - 1 strand

Thymine - Uracil

Stable - Reactive

STEPS FOR TRANSCRIPTION

1. Initiation

RNA polymerase binds to the promoter region and unwinds DNA, creating a transcription bubble.

The coding strand remains free while the template strand works to build the mRNA.

2. Elongation

RNA polymerase moves along the transcription area, adding RNA nucleotides to form, mRNA being complementary through the template strand.

3. Termination

RNA polymerase reaches a termination sequence, releases mRNA, and detaches.

DNA rewinds, and mRNA is released from the RNA polymerase to move to a ribosome for translation.

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Imagine that in our cells there is a process called transcription, that is like writing a draft of a recipe on a piece of paper (the pre-mRNA). But before you can use that recipe to cook (create proteins), you have to correct it and prepare it well.

1. 5' cap → A special structure is added to the beginning of the mRNA to protect it and help it to be read correctly.

2. Splicing → Parts that are not helpful (introns) are removed and useful parts (exons) are joined together, making sure that the information carries meaning. Sometimes, exons can be combined in different ways in alternative splicing, allowing different versions of the protein to be generated from the same gene.

3. Poly-A → Tail many adenines are added to the end of the mRNA to make it more stable and last longer inside the cell.

These changes ensure that the message is clear and functional so that the cell can make proteins without errors.

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Termination is the last step in the transcription process, where RNA polymerase ceases RNA synthesis and dissociates from DNA. This process varies between prokaryotic and eukaryotic cells.

Termination ensures that transcription stops at the appropriate time and location, allowing the resulting RNA to be used for protein synthesis or fulfill other roles in the cell.

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This drawing is a clear representation of the transcription process, showing its three key parts: initiation (promoter), elongation (when the RNA is copied), and termination (when transcription ends).

Explanation of the drawing:

The DNA double helix is represented by black lines and base pairs (colored bars inside the DNA).

The yellow area (promoter) indicates where transcription begins.

RNA polymerase (not shown directly, but can be inferred from the arrow and the process at the transcription site) runs along the strand and copies the information into RNA.

Finally, transcription stops at the blue part (termination signal).

Elements of the picture:

Promoter:

This is the DNA sequence to which RNA polymerase binds to start transcription.

It marks the start of transcription.

Transcription site:

This is the specific point where RNA synthesis begins.

The DNA opens at this site to allow the information to be copied.

Transcript termination signal:

The DNA sequence that indicates the end of transcription. When RNA polymerase arrives here, it stops and releases the newly synthe RNA.

_________________________________________Short notes_________

"DNA polymerase detects the termination signal and cleaves."

"A.S. > A piece of RNA can encode information depending on how it is processed."

"You can have instructions on how to do something, and it can change everything."

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(AUG) -> Metionine: 1º aminoacide of a protein-> Starding codon-> Sequence of 3 nucleotides

20 aminoacids in humans-> 9 ensential (ones that you have to eat them)

TRNA: Transfere RNA-> Transference -> anti codon

Aminiacid (aa) (looks like a triangle)

___________________________________________How it's work_______

SUMARY

Think of the factory where a boss works, the ribosome, who has a secret recipe, the messenger RNA, to make a special dish: a protein. But the boss doesn't cook alone: he needs the delivery men, the transfer RNAs, who bring him the ingredients, the amino acids. Each delivery man has a card with a special three-letter code that matches the one on the recipe. When the boss reads a section of the recipe, a codon, he calls the corresponding delivery person, receives the ingredient, and adds it to the satisfaction dish.

So, bit by bit, he assembles the protein, as if it were a pizza with many toppings. When it reaches the end of the recipe (STOP codon), the pizza is ready and served, and your body makes everything it needs.

_______________________With details____________________________

Protein synthesis is a fundamental process in cellular biology, and recent research in renowned laboratories has brought significant advances in its understanding and application.

Advances in Protein Structure Design and Prediction

In 2024, the Nobel Prize in Chemistry was awarded to David Baker, Demis Hassabis and John Jumper for revolutionizing the study of proteins using computational biology techniques. Their developments in artificial intelligence have made it possible to predict the three-dimensional structure of proteins with unprecedented accuracy, accelerating research in biotechnology and medicine.

Mayo Clinic Research in Immunotherapy and Colorectal Cancer

Mayo Clinic researchers have published key findings on cellular proteins that may determine the efficacy of immunotherapy against colon cancer. Their study highlights the use of spatial analysis of specific proteins as a prediction tool to select suitable candidates for PD-1 blockade immunotherapy in colorectal cancer, thereby improving treatment outcomes and reducing unnecessary treatments.

New Applications in Clinical Proteomics

Proteomics has opened new perspectives in clinical research, especially in the search for biomarkers for diagnosis, prognosis and response to pharmacological treatments. These applications are fundamental for cardiovascular, neurological, oncological and metabolic diseases, among others.

Innovations in the Production of Recombinant Proteins

Companies such as Cocoon Bioscience are innovating in the production of recombinant proteins on an industrial scale using moth chrysalises. This method significantly reduces costs compared to traditional methods and has applications in the production of vaccines and cultured meat.

These advances reflect the dynamism and importance of research in protein synthesis, with direct implications in biotechnology, medicine and other scientific areas.

Bibliografy:

Corbella, J. (2024, 9 octubre). La biología computacional que ha revolucionado el estudio de las proteínas gana el premio Nobel de Química. La Vanguardia. https://www.lavanguardia.com/ciencia/20241009/10007534/biologia-computacional-revolucionado-estudio-proteinas-gana-premio-nobel-quimica.html?utm_source

Ocronos Revista Médica y de Enfermería. (2024). El factor RHO y la terminación de la síntesis del RNA. Ocronos - Editorial Científico-Técnica. https://revistamedica.com/factor-rho-terminacion-sintesis-rna/

Pepinosa, J. (2024, 9 octubre). Nobel de Química 2024: cómo la inteligencia artificial ha revolucionado el estudio de las proteínas. Xataka Colombia. https://www.xataka.com.co/investigacion/nobel-quimica-2024-como-inteligencia-artificial-ha-revolucionado-estudio-proteinas

Theimer, S. (2023, 7 septiembre). Investigadores de Mayo Clinic publican hallazgos clave sobre proteínas celulares para determinar la eficacia de la inmunoterapia contra el cáncer de colon - Red de noticias de Mayo Clinic. Red de Noticias de Mayo Clinic. https://newsnetwork.mayoclinic.org/es/2023/09/07/investigadores-de-mayo-clinic-publican-hallazgos-clave-sobre-proteinas-celulares-para-determinar-la-eficacia-de-la-inmunoterapia-contra-el-cancer-de-colon/?utm_source=chatgpt.com

________________________________________________Ribosome___

E: Exit

P: Peptide bond is formed with the aa in site A

A: New TRNA with complimentary anticodon & aa arrives.

When UAA/UAG/UGA (stop codon) arrives, no new tRNA is bound. The A site becomes empty, the ribosomal complex dissociates, and the polypeptide chain is released, thus forming a functional protein.

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Mutations: The Basis of Genetic Variation

Mutations are changes that occur in the genetic material of an organism. They arise due to mistakes during DNA replication or cell division, leading to a new sequence of DNA. These changes can have a range of effects, from being completely harmless to causing significant alterations in an organism’s characteristics. Understanding mutations is crucial, as they play a fundamental role in evolution, genetic diversity, and even the development of certain diseases.

Causes of Mutations

Mutations can be caused by a variety of factors. One of the primary causes is errors during DNA replication. During cell division, the DNA must be copied so that each new cell receives an identical set of genetic instructions. Occasionally, the molecular machinery responsible for copying DNA makes mistakes, resulting in the insertion, deletion, or substitution of nucleotide bases. While some of these errors are corrected by repair enzymes, others persist, leading to permanent mutations.

External environmental factors can also induce mutations. Exposure to ultraviolet (UV) radiation from the sun, for instance, can cause changes in the DNA sequence of skin cells, sometimes leading to conditions such as skin cancer. Similarly, harmful chemicals, including those found in tobacco smoke or pollutants, can damage DNA and lead to mutations. Additionally, certain viruses integrate their genetic material into the host’s genome, potentially disrupting normal cellular functions.

Types of Mutations

Mutations can be classified into different types based on how they affect the genetic sequence. Point mutations involve a single nucleotide change, which may be a substitution, deletion, or insertion of a base pair. A substitution mutation occurs when one nucleotide is replaced by another, sometimes resulting in a different amino acid being incorporated into a protein. If the change does not alter the function of the protein, it is considered a silent mutation. However, if it leads to a malfunctioning protein, it can have severe consequences.

Frameshift mutations occur when nucleotides are inserted or deleted from the DNA sequence. Since the genetic code is read in sets of three bases (codons), adding or removing nucleotides shifts the reading frame, potentially leading to a completely different and nonfunctional protein. These mutations can be particularly harmful as they disrupt the entire sequence of amino acids in a protein.

Effects of Mutations

The effects of mutations vary widely. Some mutations are beneficial, providing an advantage that helps an organism survive and reproduce. These beneficial mutations drive evolution, as seen in the development of antibiotic resistance in bacteria. When a mutation gives a bacterial cell resistance to an antibiotic, it can survive treatment and multiply, leading to the spread of resistant strains.

Other mutations can be neutral, having no noticeable effect on the organism. These mutations persist in the genetic code without altering protein function or contributing to any significant changes in the organism’s traits.

However, some mutations can be harmful, leading to genetic disorders or diseases. For example, mutations in the BRCA1 and BRCA2 genes increase the risk of breast and ovarian cancer. Similarly, sickle cell anemia is caused by a mutation in the hemoglobin gene, resulting in misshapen red blood cells that can cause serious health problems.

Conclusion

Mutations are a natural and essential part of genetic variation. While they can sometimes lead to diseases, they also drive evolution and allow organisms to adapt to changing environments. Understanding the causes, types, and effects of mutations provides valuable insights into genetics, medicine, and biology as a whole. With continued research, scientists aim to find ways to repair harmful mutations and harness beneficial ones to improve human health and biodiversity.

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DEF:Mutagens are agents that cause mutations in the DNA of living organisms. These agents can be physical, chemical, or biological factors that alter the genetic material, leading to changes in the structure or sequence of DNA. Mutagens increase the frequency of mutations beyond the natural rate of occurrence.

Mutagens can be classified into three main types based on their source and mechanism of action:

1. Physical Mutagens

These mutagens cause physical damage to DNA, leading to mutations. Examples include:

Ultraviolet (UV) Radiation: Found in sunlight, it causes thymine dimers, where two thymine bases bond together, disrupting normal DNA replication. This can lead to skin cancer.

Ionizing Radiation: Includes X-rays, gamma rays, and radioactive particles. These high-energy waves break DNA strands, leading to chromosomal damage and mutations.

2. Chemical Mutagens

These substances interact with DNA, altering its structure or function. Some major types include:

Base Analogues: Chemicals that resemble normal DNA bases but pair incorrectly, causing mutations (e.g., 5-bromouracil).

Alkylating Agents: Add alkyl groups to DNA bases, changing their structure and leading to incorrect base pairing (e.g., mustard gas, ethyl methanesulfonate).

Deaminating Agents: Remove amino groups from DNA bases, altering their pairing behavior (e.g., nitrous acid).

Intercalating Agents: Insert themselves between base pairs in DNA, causing frameshift mutations (e.g., acridine dyes, ethidium bromide).

Oxidizing Agents: Cause oxidative damage to DNA, leading to mutations (e.g., hydrogen peroxide, free radicals).

3. Biological Mutagens

These are living organisms or viruses that introduce mutations into DNA. Examples include:

Viruses: Some viruses integrate their genetic material into the host DNA, disrupting normal gene function (e.g., Human Papillomavirus (HPV) can lead to cancer).

Bacteria: Some bacteria release toxins that damage DNA or interfere with its repair mechanisms (e.g., Helicobacter pylori, which is linked to stomach cancer).

Transposons (Jumping Genes): Certain DNA sequences can move within the genome, disrupting genes and causing mutations.

Conclusion

Each type of mutagen affects DNA differently, leading to various consequences such as cancer, genetic disorders, or evolutionary changes. Understanding these types helps in developing protective measures against harmful exposures and advancing medical research.

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1. Base substitution

Description Type of point mutation where another replaces one nucleotide in the DNA sequence. This can result in different types of mutations depending on how it affects the amino acid sequence of the resulting protein.

Missense acid mutation

Description Changes one amino acid in the protein

Possible Effect : May be mild or severe (e.g., sickle cell anemia)

Nonesense mutation

Description Converts an amino acid codon into a stop codon.

Possible Effect : Leads to a truncated, usually nonfunctional protein

Silent mutation amino acids

Description: Changes a nucleotide but does not alter the amino acid

Possible Effect : There is no effect on the protein

2. Insertion mutation

Description occurs when one or more extra nucleotides are added to a DNA sequence.

3. Deletion mutation

Description:Nucleotides are removed from the DNA sequence

4. Frameshift mutations

Description :Shifts the reading frame of codons

Possible Effect : Produces completely different, often nonfunctional proteins

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Genetic engineering is a field of biology that involves the direct manipulation of an organism's DNA to modify its characteristics. Instead of waiting for natural processes to evolve, scientists can change specific genes to achieve faster or more accurate results. This technology is used in many areas, such as medicine, agriculture and research.

For example, in medicine, genetic engineering can be used to create treatments for diseases that were previously incurable. Genes can be modified to correct genetic defects or to produce drugs more efficiently. In agriculture, crops are modified to be more resistant to pests or extreme weather conditions, which helps improve food production.

However, there are also concerns about the risks and ethical problems of genetic engineering. Some people wonder if it's okay to alter the genes of living beings, and if we could, for example, change the genes of humans, where would that go? It is an issue that generates debate about what limits should be set.

In short, genetic engineering is a powerful tool with great potential, but it also raises important questions about how and when to use it.

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Polymerase Chain Reaction (PCR) is a fundamental technique in molecular biology that allows millions of copies of a specific DNA sequence to be amplified in a few hours. This tool is essential in various applications, such as medical diagnoses, forensic investigations and genetic studies.

Principle of operation of the PCR:

PCR is based on three main stages:

1. Denaturation: The DNA sample is heated to approximately 96 °C to separate the two chains of the double helix, obtaining simple DNA strands.

2. Alignment (Annealing): The mixture is cooled to a temperature of about 50–65 °C to allow the primers (short fragments of DNA) to join the flanking regions of the target DNA.

3. Elongation: The temperature increases to approximately 75–80 °C, where the DNA polymerase synthesizes new complementary chains from the firsts, doubling the amount of DNA.

These cycles are repeated multiple times, exponentially doubling the amount of target DNA in each cycle.

Essential components of PCR:

• DNA template: The sample that contains the DNA that you want to amplify.

• Primers: Short DNA sequences that delimit the region to be amplified.

• DNA polymerase: An enzyme that synthesizes new DNA strands. Taq polymerase is commonly used due to its heat resistance.

• dNTPs: Nucleotides (adenine, thymine, cytosine and guanine) that serve as building blocks for the new DNA chains.

• Buffers and divalent cations: They provide the optimal environment for DNA polymerase activity.

Applications of PCR:

PCR has revolutionized molecular biology and has multiple applications, including:

• Detection of genetic diseases: Identification of mutations or alterations in specific genes.

• Diagnosis of infections: Detection of pathogens such as bacteria and viruses in clinical samples.

• Forensic identification: Analysis of biological samples at crime scenes to identify individuals.

• Biodiversity studies: Detection and classification of species from environmental samples.

Considerations and precautions:

Although PCR is a powerful technique, it is susceptible to contamination that can lead to false positive results. It is crucial to maintain strict working conditions, use adequate controls and employ decontamination techniques, such as the use of bleach (bleach) to clean surfaces and equipment.

References in APA format:

Thermo Fisher Scientific. (s.f.). PCR Basics. Thermo Fisher Scientific. Recuperado el 3 de marzo de 2025, de https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-basics.html

Bitesize Bio. (2021, 12 de octubre). PCR: The right way to decontaminate and eliminate false positives. Bitesize Bio. Recuperado el 3 de marzo de 2025, de https://bitesizebio.com/3473/pcr-the-right-way-to-decontaminate-and-eliminate-false-positives/?utm_source=chatgpt.com

Mullis, K., & Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 155, 335–350. https://doi.org/10.1016/0076-6879(87)55023-6

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Electrophoresis: the key to sequencing

Electrophoresis is a fundamental laboratory technique in molecular biology that allows separating fragments of DNA, RNA or proteins according to their size and electrical charge. This technique is widely used in DNA sequencing to analyze and order the fragments of genetic sequences, thus facilitating the identification of specific sequences within the genome.

Basic principle of electrophoresis:

Electrophoresis is based on the use of an electric field to move the charged molecules (such as DNA) through a gel, typically agarose or polyacrylamide. Because DNA molecules have a negative charge (due to their phosphate groups), when applying an electric field, the molecules move towards the positive electrode.

The gel acts as a network of pores that slows the passage of larger molecules, while smaller molecules can move more quickly. This results in the separation of DNA fragments according to their size, which is crucial for DNA sequencing.

Application in DNA sequencing:

Electrophoresis is a key tool in the DNA sequencing process, especially in Sanger sequencing techniques. In this technique, DNA is amplified by PCR and then fragmented with enzymes. These DNA fragments are then marked with fluorescence or radioactive isotopes and passed through an electrophoresis gel. At the end of the process, the separate fragments are analyzed to read the DNA base sequence.

Electrophoresis allows us to visualize DNA fragments according to their size, which facilitates the interpretation of genetic sequences. The use of this technique has been essential for advances in genomics, such as the sequencing of the human genome.

References:

Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual (3.ª ed.). Cold Spring Harbor Laboratory Press.

Mullis, K., & Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 155, 335–350. https://doi.org/10.1016/0076-6879(87)55023-6

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DNA profiling and DNA sequencing are fundamental techniques in genetics that allow analyzing and understanding the genetic information contained in the DNA. Detailed explanations of each are presented below, based on recognized medical sources.

DNA profiling:

DNA profiling, also known as genetic fingerprint, is a process that identifies unique characteristics of a person's DNA. This technique is used in various areas, such as:

• Forensic identification: Comparing DNA samples found at crime scenes with those of suspects or databases.

• Paternity detection: Establishing biological relationships between parents and children.

• Diagnosis of genetic diseases: Identifying specific mutations associated with certain medical conditions.

The process involves extracting DNA from a biological sample (such as blood or saliva), amplifying specific regions using PCR, and analyzing the resulting patterns to create a unique profile.

DNA sequencing:

DNA sequencing determines the exact order of nucleotide bases (adenine, thymine, cytosine, and guanine) in a DNA molecule. This information is crucial for:

• Genetic research: Understanding the function of specific genes and their relationship with diseases.

• Personalized medicine: Adapting treatments based on individual genetic variations.

• Diagnosis of diseases: Identifying genetic mutations responsible for hereditary disorders.

There are several sequencing techniques, with Sanger sequencing and new generation sequencing (NGS) being the most common. The choice of method depends on factors such as the scope of the analysis and the available resources.

References in APA format:

Mayo Clinic. (2020). Genetic testing. Mayo Clinic. Recuperado el 3 de marzo de 2025, de https://www.mayoclinic.org/tests-procedures/genetic-testing/about/pac-20385080

National Human Genome Research Institute (NHGRI). (2020, 1 de junio). DNA sequencing. U.S. Department of Health and Human Services. Recuperado el 3 de marzo de 2025, de https://www.genomeweb.com/genetic-testing/dna-sequencing

National Institutes of Health (NIH). (2020, 8 de septiembre). Genetic testing and its uses. National Institutes of Health. Recuperado el 3 de marzo de 2025, de https://www.nih.gov/news-events/news-releases/what-genetic-testing-and-why-it-important

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What are plasmids?

A plasmid is a small molecule of DNA found in cells, especially in bacteria, and that is separated from chromosomal DNA. The plasmids contain additional genetic information, but are not essential for the survival of the cell. Plasmids often carry genes that can be useful, such as those that allow bacteria to resist antibiotics.

In summary:

• Plasmids are extra pieces of DNA that can be transferred between bacteria.

• They are independent of the main DNA of the cell (chromosomal DNA).

• They are essential for genetic manipulation and biotechnology.

How are plasmids handled?

Plasmid manipulation is a key process in biotechnology, since it allows genes to be inserted or modified within cells. I explain the process step by step.

Step 1: Plasmid insulation

1. Plasmid extraction: The first step is to obtain the plasmid from a bacterial cell. This is done through a process called plasmid extraction or plasmid preparation. In this process, the bacterial cell is broken (using chemicals or enzymes) and the plasmid is separated from the chromosomal DNA.

Step 2: Cut the plasmid with restriction enzymes

2. Use of constraint enzymes: To insert a gene into the plasmid, we need to cut the DNA in specific places. This is achieved through the use of restriction enzymes, which are proteins that cut DNA into very specific sequences. This process leaves "sticky ends" in the DNA where a new DNA fragment can be inserted.

Step 3: Insertion of the gene of interest

3. Gene insertion: Once the plasmid has been cut, the next step is to insert the gene of interest (the gene you want to study or use) into the plasmid. For this, an enzyme called DNA ligase is used, which binds the inserted DNA fragment with the plasmid, closing the structure and forming a new recombinant plasmid.

Step 4: Introduction of the plasmid in a cell (Transformation)

4. Transformation: The next step is to introduce the recombinant plasmid into a bacterial cell (usually E. coli). This process is called transformation. There are several ways to do it, such as:

• Electroporation: An electric field is used to make the cell membranes become more permeable and allow the entry of the plasmid.

• Thermal shock method: The bacteria is exposed to temperature changes so that it acquires plasmid.

Step 5: Selection of transformed cells

5. Selection of bacteria with the plasmid: Not all bacterial cells will have the recombinant plasmid, so those that contain it must be selected. This is done using a marker, such as an antibiotic resistance gene, that is present in the plasmid. By cultivating bacteria in a medium that contains antibiotics, only the bacteria that have acquired the plasmid survive.

Step 6: Expansion and analysis

6. Growth of transformed bacteria: Bacteria containing the recombinant plasmid are grown in large quantities to produce copies of the plasmid and the gene of interest.

7. Plasmid analysis: Then, scientists can study or purify the inserted gene to investigate its function, produce proteins or even modify organisms.

Summary of the plasmid handling process:

1. Isolation of the plasmid of a bacterial cell.

2. Cutting the plasmid with restriction enzymes.

3. Insertion of the desired gene using a ligase DNA.

4. Transformation of bacterial cells with recombinant plasmid.

5. Selection of bacteria that have the plasmid.

6. Growth and analysis of transformed bacteria.

What are plasmids used for?

Plasmids have many applications in biotechnology, such as:

• Production of recombinant proteins: Like human insulin in bacteria.

• Genetic research: As the study of the function of specific genes.

• Creation of genetically modified organisms (GMOs): To improve cultures

Sources:

• National Institute of General Medical Sciences (NIGMS). (2020). Plasmids. Retrieved from https://www.nigms.nih.gov/

• Mayo Clinic. (2020). Genetic engineering and biotechnology. Retrieved from https://www.mayoclinic.org/

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Genetic modification in bacteria is a process in which the DNA of a bacterium is altered to introduce specific genes or modify its function. This process is used to study genes, produce proteins, or create genetically modified organisms (GMOs).

Key steps:

1. Selection of the bacteria: A bacterium, such as E. coli, is chosen to be modified.

2. Insertion of a gene: Using techniques such as gene cloning, a new gene is introduced (for example, an antibiotic resistance gene or a gene that produces a protein of interest).

3. Transformation: Modified DNA is introduced into the bacterial cell by methods such as electrophoresis or thermal shock.

4. Selection: Bacteria that have integrated the new gene are selected using markers, such as antibiotic resistance.

5. Culture and analysis: Modified bacteria are cultivated to produce the desired proteins or perform genetic research.

This process is essential in biotechnology to produce medicines, industrial enzymes, and study diseases.

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CRISPR/Cas9 is a genetic editing tool that allows precise changes to the DNA of living cells. "CRISPR" refers to DNA sequences that act as a kind of genetic "memory", while "Cas9" is an enzyme that cuts DNA in a specific location. The system works like a "molecular scissors", allowing scientists to eliminate, aggregate or modify genes with great precision. This technology has revolutionized molecular biology and has potential applications in medical treatments, agriculture and genetic research.

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Genetic modification in plants is a technique that allows the DNA of plants to be altered so that they develop specific characteristics that they do not have naturally. This is achieved by introducing, eliminating or modifying genes within the genome of the plant.

How is genetic modification carried out in plants?

1. Identification of the gene of interest: The gene that confers the desired characteristic is selected, such as pest resistance or drought tolerance.

2. Introduction of the gene into the plant: Methods such as the bacterium Agrobacterium tumefaciens or bioballistics (gene gun) are used to insert the gene into the plant's DNA.

3. Culture and selection: Modified cells are cultivated under special conditions that allow identifying and selecting those that have successfully incorporated the gene of interest.

For example, through genetic modification, tastier tomatoes have been developed without reducing their size. Researchers inactivated certain genes that limited the production of sugars during maturation, increasing fructose and glucose levels by 30%, without affecting the size or quantity of the harvest.

It is important to note that genetically modified plants can offer benefits such as greater resistance to diseases, better adaptation to adverse environmental conditions and nutritional improvements. However, its use also generates debates about food security and environmental impact.

Explanation with a video: https://youtu.be/o8Yw1NMmc_Y?si=CIufUJBAcP1VwMET

Summary: The video "Genetically modified plants" addresses the use of genetically modified crops in the country, highlighting their adoption as one of the highest in relation to other agricultural technologies. Although no specific details about the characteristics or benefits of these crops are provided in the available fragment, their relevance in modern agricultural production is emphasized.

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Genetic modification in animals involves altering their genetic material by inserting, modifying or eliminating specific DNA sequences. This process is carried out to study gene functions, model human diseases or improve specific characteristics in animal species.

Genetic modification procedure in animals:

1. Identification and isolation of the gene of interest: The gene that you want to introduce, modify or eliminate in the animal's genome is selected.

2. Vector preparation: The gene of interest is inserted into a vector, which can be a modified virus or a small DNA molecule, designed to transport the gene inside the animal's cells.

3. Introduction of the vector in animal cells:

• Microinjection: The vector is introduced directly into the nucleus of individual cells, such as developing embryos.

• Embryonic stem cell transfer: Embryonic stem cells are modified in the laboratory and then implanted in embryos, allowing modifications to be transmitted to future generations.

4. Selection and breeding of modified animals: Animals that correctly integrate and express the modified gene are identified and bred to establish stable lines of genetically modified animals.

Techniques used:

• CRISPR/Cas9: A genetic editing tool that allows you to make precise cuts in the DNA, facilitating the insertion, elimination or modification of specific genes.

• Nuclear transfer: It consists of transferring the nucleus of a somatic cell to an egg without a nucleus, creating an embryo with the desired genetic material.

These techniques have allowed significant advances in biomedical research, the production of animal models for human diseases and the improvement of specific characteristics in animals of agricultural interest

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Genetic modification in humans refers to the deliberate alteration of human DNA with the aim of correcting genetic defects, treating diseases or potentially improving certain physical or cognitive characteristics. This practice, which is part of the broader field of genetic engineering, has advanced significantly in recent decades, offering innovative therapeutic possibilities, but also raising important ethical and social debates.

Genetic modification techniques in humans

1. Somatic gene therapy: It consists of the introduction, elimination or modification of genes in somatic cells (all cells of the body except the germ cells) to treat diseases in a specific individual. These changes are not transmitted to future generations.

2. Germ genetic editing: It involves the modification of germ cells (eggs, sperm or embryos), which results in changes that can be inherited for generations. This practice is highly controversial due to its ethical implications and the possibility of unwanted effects on offspring.

Applications and advances

• Treatment of hereditary diseases: Gene therapy has shown potential in the treatment of genetic disorders such as cystic fibrosis, hemophilia and certain forms of anemia. For example, advances have been made in the use of CRISPR-Cas9 to correct specific mutations in genes that cause hereditary diseases.

• Fight against cancer: Genetic modification is used to design therapies that allow immune system cells to recognize and attack cancer cells more effectively. An example is CAR-T cell therapy, where the patient's T cells are genetically modified to express specific receptors against cancer.

• Treatment of viral infections: Gene therapies are being developed to treat chronic infections, such as HIV, by modifying genes in immune cells to make them resistant to the virus.

Ethical and social considerations

Genetic modification in humans raises deep ethical debates, especially in relation to germinal line editing. Concerns include:

• Safety: Risks of off-target effects or unwanted modifications in the genome, which could cause mutations or new diseases.

• Consent: Difficulties in obtaining the informed consent of future generations that will carry the genetic modifications.

• Equity: Possibility that these technologies are available only to certain groups, increasing social inequalities.

• Definition of normality: Questions about what constitutes a "normal" or "desirable" characteristic and the risks of promoting a culture of genetic perfection.

A case that exemplifies these dilemmas is that of the Chinese geneticist He Jiankui, who in 2018 announced the birth of twins whose genomes had been edited to give them resistance to HIV. This act was widely condemned by the international scientific community due to the ethical implications and the lack of global consensus on the germ edition.

Regulations and future prospects

Worldwide, human genetic editing is subject to strict regulations. For example, the U.S. National Institutes of Health. The U.S. does not fund studies on germinal editing in humans. The World Health Organization has issued recommendations to guide the research and application of these technologies, focusing on safety, efficacy and ethical considerations.

For the future, genetic modification in humans offers significant promises for the treatment of diseases and health improvement. However, it is essential to carefully address the associated ethical, legal and social aspects, ensuring that their implementation is responsible and benefits humanity as a whole.

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A genetically modified organism (GMO) is one whose genetic material has been altered by genetic engineering techniques to introduce, eliminate or modify specific genes. This allows the body to acquire characteristics or abilities that it did not naturally possess. GMOs can be plants, animals or microorganisms.

Common applications of GMOs:

• Agriculture: Crops resistant to pests, diseases or adverse environmental conditions, as well as plants with nutritional improvements.

• Medicine: Production of medicines, vaccines and gene therapies using modified organisms.

• Industry: Microorganisms that synthesize chemical compounds used in the manufacture of various products.

The creation and use of GMOs are regulated by country-specific laws and guidelines, aimed at ensuring their safety and effectiveness.

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Human genome editing is an emerging field that offers opportunities to treat and prevent genetic diseases. The World Health Organization (WHO) has established guidelines to guide its ethical and safe application.

In gene therapy, viral vectors are used to deliver therapeutic genetic material to the patient's cells. These vectors, such as retroviruses and adenoviruses, have been modified to be safe and effective in gene transfer. Their design and selection are fundamental to the success of gene therapies.

Genetic modification in animals has been studied due to its scientific and ethical implications. Research has shown that modifying genes can influence the expression of other genes, affecting phenotypes and diseases. These findings are essential to understand genetic complexity and its potential applications.

In addition, the production of laboratory-grown meat and the use of genetically modified animals for food consumption have been topics of debate. These technologies raise questions about their safety, ethics and social acceptance, reflected in academic and cultural discussions.

The polymerase chain reaction (PCR) is a fundamental technique in molecular biology that allows the amplification of specific DNA sequences. This methodology is essential for diagnostics, genetic research and forensic tests, highlighting its importance in multiple scientific fields.

References

• Organización Mundial de la Salud. (s.f.). Edición del genoma humano. Recuperado de https://www.who.int/health-topics/human-genome-editing#tab=tab_1

• Nadeau, J. H. (2001). Modifier genes in mice and humans. Nature Reviews Genetics, 2(3), 165–174. Recuperado de https://citeseerx.ist.psu.edu/document?repid=rep1&type=pdf&doi=fbe41d0139ccb2b07c101fc9ce43fc36639ac35e

• McHugh, S. (2010). Real artificial: Tissue-cultured meat, genetically modified farm animals, and fictions. Configurations, 18(2), 181–197. Recuperado de https://dune.une.edu/cgi/viewcontent.cgi?article=1003&context=eng_facpubs

• National Center for Biotechnology Information. (2013). PCR: Polymerase Chain Reaction. NCBI Bookshelf. Recuperado de https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102308/

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Gene delivery methods:

Electroporator: Use electricity to make cells membranes permeable for genes to enter.

Microinjection: Use a needle to inject genetic material into another cell.

Biological viruses: No harmful viruses, insert cells the gene desired.

Chemical polymer and liposome: Molecule with desired gene and insert into the cells.

Gene gun: Loaded with gold particles covered with particles of the gene and yet inserted.

Gene modification in humans

If genetic engineering is done on somatic cells, the only injected area is the contaminated.
On the contrary, the germline one will inject the whole body cells.