Seminario 11 Técnicas de Biología Molecular aplicadas a Entidades Monogénicas - Sebastián Giusti

Name: Seminario 11 Técnicas de Biología Molecular aplicadas a Entidades Monogénicas - Sebastián Giusti
Uploaded: 2025-04-27T21:46:49.000Z
Duration: 2 h 32 min 53 s

Introduction to Molecular Biology Techniques

Overview of Class Objectives

The class will analyze various molecular biology techniques used in medical genetics to detect different allelic variants in the genome.

The first part focuses on PCR (Polymerase Chain Reaction) techniques, while the second part covers DNA sequencing methods.

Emphasis will be placed on understanding the molecular fundamentals and clinical contexts where these techniques are necessary.

Understanding PCR Fundamentals

Historical Context and Significance

PCR was developed by biochemist Kary Mullis in the 1980s, earning him a Nobel Prize in Chemistry in 1993.

This technique allows for the amplification of specific DNA segments from complex mixtures, enabling targeted analysis.

Key Components of PCR

Essential elements include genomic DNA as a template, Taq polymerase (a heat-resistant enzyme), dNTPs (deoxynucleotide triphosphates), and primers.

Primers serve dual functions: they stabilize the reaction and determine which DNA segment is amplified during PCR.

Mechanism of Action in PCR

Requirements for Polymerization

Taq polymerase requires a buffer solution to maintain pH stability and divalent cations like magnesium as cofactors for activity.

dNTP substrates must be present in excess to ensure rapid and effective amplification during reactions.

Limitations of Polymerases

All DNA polymerases, including Taq, cannot initiate synthesis de novo; they require a pre-existing single-stranded template with a free 3' hydroxyl group for nucleotide incorporation.

Steps Involved in PCR Cycles

Cycle Breakdown

Denaturation: The reaction is heated to approximately 95°C to separate double-stranded DNA into single strands, making them available for amplification.

Annealing: Temperature is lowered (50–70°C) allowing primers to bind to their complementary sequences on the single-stranded templates.

Extension: Taq polymerase synthesizes new strands by adding nucleotides complementary to the template strand.

This structured approach ensures precise amplification of target regions within the genome through repeated cycles of denaturation, annealing, and extension.

These notes provide an organized overview of key concepts discussed regarding molecular biology techniques relevant to medical genetics, particularly focusing on PCR's principles and mechanisms.

PCR Process and Visualization Techniques

Steps in PCR Amplification

The initial step involves lowering the temperature to allow primers to hybridize with DNA strands, which is crucial for the amplification process.

The second phase occurs at 72°C, the optimal temperature for Taq polymerase activity, leading to effective elongation and polymerization of DNA fragments.

Repeating these cycles results in exponential amplification; each cycle doubles the amount of target DNA, leading to billions of copies after approximately 30 cycles.

This exponential growth means that the original genomic DNA becomes negligible compared to the amplified product by the end of PCR.

Visualization of Amplified Products

Gel electrophoresis is commonly used to visualize PCR products; it separates charged nucleic acid molecules based on size when subjected to an electric field.

Nucleic acids are negatively charged due to phosphate groups, causing them to migrate towards a positive electrode during electrophoresis through a porous gel matrix.

Smaller DNA fragments move more easily through this matrix than larger ones, allowing for size-based separation during electrophoresis.

Staining and Detection Methods

Ethidium bromide is often used as a fluorescent intercalating agent that binds between base pairs in DNA; it fluoresces under UV light, enabling visualization of separated bands in gels.

Different lanes in a gel can contain various samples or molecular weight markers, aiding in estimating the sizes of amplified products by comparison with known standards.

Alternative Electrophoresis Techniques

Capillary electrophoresis serves as an alternative method where separation occurs within thin capillary tubes instead of traditional gels but follows similar principles regarding charge and size separation.

In capillary systems, fluorescence detection occurs at specific points along the capillary, producing digital readouts (electropherograms) that represent peaks corresponding to different sized DNA fragments.

Interpretation of Results

Understanding PCR in Medical Genetics

Overview of PCR Technique

The discussion begins with an example of a PCR reaction that produces two distinct amplification products, represented as a series of two peaks.

Components and Applications of PCR

After reviewing the fundamentals and reagents involved in basic PCR reactions, the focus shifts to its applications in medical genetics, particularly for detecting specific allelic variants.

Hypothesis-Driven Approach

It is emphasized that PCR is a hypothesis-driven technique; its clinical application relies on the assumption that specific allelic variants are being sought based on clinical context.

Case Study: Cystic Fibrosis

A case study on cystic fibrosis illustrates how symptoms can lead to suspicion of this genetic condition, which is caused by pathogenic variants in the CFTR gene.

The most common mutation associated with cystic fibrosis is ΔF508, resulting from a three-nucleotide deletion affecting phenylalanine coding.

Clinical Relevance of Allelic Variants

In patients showing signs compatible with cystic fibrosis, it may be beneficial to test for the presence or absence of the ΔF508 deletion in the CFTR gene.

Designing Primers for Specific Mutations

Conventional PCR can effectively detect insertions or deletions guided by clinical hypotheses. Primers are designed flanking the region where mutations may occur.

Amplification Products Comparison

Two allelic variants are compared: one without and one with the deletion. Both will yield amplicons due to primer binding but differ slightly in size due to nucleotide differences.

Gel Electrophoresis Analysis

Polyacrylamide gels can be used to distinguish between these small size differences during electrophoresis, allowing for effective analysis of amplification results.

Family Case Study Analysis

A family case study examines individuals showing clinical signs of cystic fibrosis. Individual four has a unique amplification product larger than individual two's product.

Interpretation of Results Based on Genomic Structure

The diploid nature of human genomes means each individual has two alleles per gene. The differing sizes suggest individual four carries both non-deletion alleles while individual two carries both deletion alleles.

Amplification Products and Allelic Variants

Understanding Amplification in PCR

The amplification products from two allelic variants used as templates during PCR generated the same amplification product.

Individuals 1 and 3 exhibited two distinct amplification products, indicating one variant has a deletion while the other does not, suggesting heterozygosity.

PCR can detect insertions and deletions due to size differences in amplicons that are separated by gel electrophoresis.

Detecting Substitution Mutations

To identify allelic variants caused by substitution mutations, specific variations of PCR must be employed, such as allele-specific PCR.

The design of primers is crucial; one primer must bind specifically to one allelic variant at its 3' hydroxyl end for successful amplification.

If designed correctly, only one allelic variant will amplify during the PCR reaction based on whether there is a proper match with the template.

Analyzing Patient Samples

In testing five patients, those with amplification products (patients 1, 2, and 5) likely possess at least one CG variant in their genome.

Patients 3 and 4 showed no CG variants initially but later revealed AT variants upon further testing with different primers.

Genetic Configurations

Individual 1 likely carries both CG and AT alleles due to positive results from both types of PCR amplifications.

A question is posed regarding individual 5's genetic configuration based on their unique amplification results.

PCR RFLP Technique for Variant Detection

Introduction to RFLP

Another technique called PCR RFLP (Restriction Fragment Length Polymorphism) detects substitution mutations affecting restriction sites within DNA sequences.

Mechanism of RFLP

This method utilizes restriction enzymes that cleave DNA at specific recognition sequences to differentiate between allelic variants.

The designer must ensure that the mutation affects a restriction site recognized by an enzyme; if it does not cut due to a mutation, this indicates a difference between the alleles.

Practical Application of RFLP

PCR Techniques and Applications

Overview of PCR Amplification

The process begins with designing primers that flank the restriction site where the variant is located, allowing amplification of both allelic variants without affecting polymorphic regions.

Both alleles are amplified through PCR, resulting in products of the same size before any further processing.

Restriction Enzyme Treatment

After amplification, the PCR products are treated with a restriction enzyme; only one fragment will be cut based on whether it contains the restriction site affected by the allelic variant.

This results in a polymorphism in fragment length, which can be analyzed using electrophoresis to separate DNA fragments of different sizes.

Case Study: Genetic Variants Analysis

In analyzing five patients' genetic constitution, two variants (AT and TA) were identified. The AT variant retains the restriction site while TA does not.

For example, patient four exhibited a large amplification product indicating both parental alleles were TA type, while patient one showed smaller fragments indicative of AT type variants.

Complex Interpretations

Patients three and five presented three amplification products; they had one TA allele (not cut by the enzyme) and one AT allele (cut), leading to varying fragment sizes.

Applications of Multiplex PCR

Contextual Use in Forensic Genetics

Multiplex PCR is utilized in forensic genetics to determine biological relationships between individuals rather than for pathological contexts.

Microsatellites as Genetic Markers

Short tandem repeats (STRs), or microsatellites, serve as highly polymorphic markers that differ among individuals at specific chromosomal locations due to variations inherited from parents.

Enhancing Fidelity in Analyses

To improve accuracy in paternity testing, multiple microsatellites are analyzed simultaneously. The FBI standardized this approach by examining 13 specific sequences across various chromosomes since 1997.

Technical Aspects of Multiplex PCR

Unlike traditional PCR that uses a single pair of primers for amplification, multiplex PCR employs multiple primer pairs designed for different microsatellite regions within a single reaction tube.

Complexity and Efficiency

Fluorescent Amplification in PCR

Overview of Fluorophores in PCR

The use of fluorophores allows for the visualization of amplification products, with different colors emitted by molecules linked to primers. This enables differentiation among various PCR products based on color.

A multiplex PCR approach is employed due to the complexity of reactions involving at least 13 distinct amplification products, utilizing the same fluorophores for genes with amplicons of varying sizes.

Detection and Analysis Techniques

Multiplex PCR is typically combined with capillary electrophoresis to detect different products, where fluorescence from various colored fluorophores indicates specific primer pairs used.

An example electroferogram illustrates multiple peaks corresponding to different amplification products, where peak height reflects fluorescence intensity and horizontal separation indicates amplicon size.

Detailed Signal Separation

Digital analysis allows for the separation of signals from different colored channels, facilitating a more detailed examination of amplification results. For instance, blue and green fluorophore channels can be analyzed separately.

Specific design of primers helps identify regions yielding amplification products from particular microsatellites; two peaks indicate variations in repeat numbers (e.g., 12 vs 13 repeats).

Implications for Genetic Analysis

The presence or absence of peaks can indicate allele variants; a single peak suggests identical maternal and paternal alleles in terms of repeat number. This information aids in assessing biological relationships between individuals.

By combining data from multiple primer pairs associated with different fluorophores, one can determine exact repeat counts across tandem repeat regions and evaluate potential biological affiliations among subjects.

Triplet Prime PCR: A Diagnostic Tool

Introduction to TPPCR

Triplet prime PCR (TPPCR) is introduced as a technique used for diagnosing genetic diseases caused by triplet expansions, which are characterized by non-classical monogenic disorders that will be discussed further in future seminars.

Characteristics of Triplet Expansion Diseases

Normal allelic variants exhibit a range between 5 to 50 trinucleotide repetitions; however, some individuals may carry premutation alleles with higher repetition counts without showing symptoms but have increased risk for offspring developing related conditions.

Challenges with Conventional PCR

Conventional PCR struggles to detect triplet expansions due to difficulties amplifying regions containing multiple repeats effectively; this often leads to incomplete or erroneous amplification results when targeting expanded alleles like those found in the FMR1 gene's UTR region.

Limitations Highlighted

Understanding TP PCR and Its Applications

Overview of Allelic Variants and Amplification Challenges

The presence of an expanded allelic variant can lead to no amplification product, masking results in gel electrophoresis where only the non-expanded variant is visible.

Southern blotting was previously used to address these issues but is now largely outdated; variations of PCR have emerged as solutions.

TP PCR Technique

The modified PCR technique employs three primers: one forward primer flanking the amplification region, a second complementary to repeat regions, and a third reverse primer.

The unique feature of the repeat-binding primer allows it to bind at various locations within the repeat region, generating diverse amplification products.

Amplification Products and Analysis

This setup leads to multiple simultaneous reactions, producing amplicons of varying lengths based on primer interactions.

TP PCR enables estimation of expanded allelic variants through two methodologies: assessing complete expansion product amplification and analyzing fragment sizes from different regions.

Results Interpretation via Capillary Electrophoresis

Results are typically analyzed using capillary electrophoresis, visualized in electroferograms showing peak heights (fluorescence intensity) against product size variation.

Different length ranges allow classification into normal, premutation, or fully mutated allelic variants.

Case Studies: Normal vs. Expanded Alleles

In normal alleles with trinucleotide repeats, strong amplification peaks indicate successful binding by both flanking primers; additional peaks arise from the repeat-binding primer's activity.

Premutated individuals show broader peak series due to increased fragment diversity; however, overall signal strength diminishes due to lower efficiency in amplifying larger fragments.

Comparison with Fully Expanded Alleles

Individuals with fully expanded alleles exhibit a wider range of molecular weights in their peak patterns; however, the height of peaks corresponding to full-length products is reduced due to lower production efficiency.

Conclusion on TP PCR Utility

TP PCR serves as a specialized adaptation for detecting triplet expansion variants effectively.

Next Steps: DNA Sequencing Techniques

Introduction to DNA Sequencing Methods

Sanger Sequencing and Its Evolution

Introduction to Sanger Sequencing

The discussion begins with an overview of the Sanger sequencing method, developed by Frederik Sanger, who won the Nobel Prize in Chemistry in 1980 for this innovation. This method was pivotal in producing the first human genome sequence as part of the Human Genome Project.

High-Throughput Sequencing Developments

Following Sanger's work, advancements over the last 15 to 20 years have led to high-throughput sequencing methodologies, also known as next-generation sequencing (NGS). These methods build upon Sanger's foundational techniques.

Molecular Mechanisms of Nucleic Acid Polymerization

Understanding nucleic acid polymerization is crucial for grasping Sanger's methodology. DNA polymerase uses a single-stranded DNA template but cannot initiate synthesis de novo; it requires a pre-existing complementary strand with a free 3' hydroxyl group.

Role of Dideoxynucleotides in Sequencing

Sanger introduced dideoxynucleotides (ddNTPs), which lack a hydroxyl group at the 3' position. When incorporated into a growing DNA strand during polymerization, they terminate further extension because no additional nucleotides can be added.

Experimental Setup for Sequencing Reaction

In an experimental setup, standard deoxynucleotides and small amounts of ddNTPs are mixed with DNA polymerase and a single-stranded DNA template. Each ddNTP is labeled with different fluorophores to allow identification during sequencing.

Primer Design and Polymerization Process

A chemically attached known sequence at one end of the target DNA allows researchers to design a complementary primer that initiates polymerization. This primer binds to the known region, enabling amplification of the unknown sequence through PCR-like processes.

Parallel Reactions and Random Incorporation

The reaction occurs millions of times simultaneously within a tube, leading to various outcomes where most molecules undergo complete polymerization while some randomly incorporate ddNTPs at specific positions, thus terminating their growth prematurely.

Polymerization and DNA Sequencing Techniques

Overview of Polymerization Process

The polymerization process results in a complex mixture of fully polymerized molecules, with some containing variations at different positions.

Millions of molecules participate in the polymerization, leading to statistical representation across all positions, ensuring diverse outcomes.

Fragment Length Variation

Molecules that experience interruptions during polymerization will have varying lengths, differing by one nucleotide.

Electrophoresis is utilized to separate DNA fragments based on size; this can be done using porous matrices like agarose gels or capillaries.

Identifying Nucleotides through Electrophoresis

Each fragment emits fluorescent light corresponding to the nucleotide at its end, allowing for sequence reconstruction based on emitted signals.

Results are visualized as electropherograms showing peaks of fluorescence that indicate differences in nucleotide length among fragments.

Allelic Variants Detection

Different individuals may show distinct nucleotides at specific positions; for example, one may have adenine while another has guanine.

An individual displaying two simultaneous peaks indicates heterozygosity at that position due to differing alleles from each parent.

Limitations and Applications of Sanger Sequencing

Sanger sequencing was widely used until 2000 but only sequences one type of molecule per experiment, making it costly and limited in scope.

This method is typically applied when there’s a clinical reason to suspect relevant variants within a specific gene or exon.

High-throughput Sequencing Methods

In contrast to Sanger sequencing, high-throughput methods can sequence millions of molecules simultaneously and are more cost-effective for whole-genome analysis.

Genetic Variants and Their Implications in Medical Genetics

Understanding Genetic Variants

The challenge in medical genetics lies in identifying whether genetic variations from the human reference genome are causative of diseases or simply benign polymorphisms reflecting population diversity.

Techniques for Genome Sequencing

Advanced sequencing techniques allow not only whole genome sequencing but also targeted sequencing of specific genomic subsets, such as exonic regions, which focus on coding variations.

Molecular Diagnosis Scenarios

In diagnosing monogenic disorders, clinical signs may suggest a specific gene's involvement. A strong clinical hypothesis can guide the choice of diagnostic techniques.

If there is a clear clinical indication of a genetic disorder, molecular diagnosis can be pursued differently than when no strong hypothesis exists, necessitating unbiased high-throughput sequencing methods.

Hypothesis Testing with Genetic Variants

When using high-throughput sequencing, variants differing from the reference genome may be identified. These findings can lead to hypotheses about their potential causal role in disease if corroborated by functional studies or similar cases.

Targeted Approaches Based on Clinical Hypotheses

If a specific gene is suspected based on symptoms, one might check for known mutations before resorting to broader techniques like Sanger sequencing to identify any relevant variants.

In cases where frequent mutations are documented for a gene, more focused techniques such as PCR can be employed to detect specific alleles rather than broad screening methods.

Specific Techniques for Variant Detection

Depending on mutation types (insertions/deletions vs. substitutions), different PCR methodologies are applicable; conventional PCR works well for size differences while allele-specific PCR or RFLP is suited for substitution mutations.

For conditions involving triplet repeat expansions, specialized techniques like Triple Prime PCR (TPPCR) are preferred over traditional Southern blotting due to implementation challenges and declining usage.

Applications in Forensic Genetics