DNA Replication & Molecular Cloning PDF

Theme 1: DNA replication & Molecular cloning ============================================ Lesson 1: Structures of DNA AND RNA & PCR & PRIMERS --------------------------------------------------- **Central Dogma of Molecular Biology**: The central dogma of molecular biology states that information c...

Theme 1: DNA replication & Molecular cloning ============================================ Lesson 1: Structures of DNA AND RNA & PCR & PRIMERS --------------------------------------------------- **Central Dogma of Molecular Biology**: The central dogma of molecular biology states that information can be transferred from nucleic acids (DNA and RNA) to proteins, but not the other way around. \ **Picture labelling:** 1. DNA 2. Nucleotide 3. OH group 4. Phosphate group 5. Transcription 6. RNA 7. Translation 8. Protein 9. Amino acid ### Organization of the Genome in Prokaryotes and Eukaryotes **Prokaryote:** - No nucleus - Circular genomic DNA - DNA is free in the cell - No organelles - Smaller cell - Always has a cell wall - 1 gene codes for 1 protein - Higher gene density **Eukaryote:** - Has a nucleus - Linear genomic DNA - DNA in the nucleus - Has organelles - Larger cell - Only plant cells have a cell wall - 1 gene can code for multiple proteins - Lower gene density ### Structure of DNA & RNA **DNA:** - Right-handed double helix: Major and Minor groove. - Complementary: both strands contain information. - Enables replication and transcription. - Antiparallel (5' and 3' are oriented oppositely). - Nucleotide: phosphate group + sugar group + base. - **Pyrimidine (1 ring):** Cytosine, Thymine, Uracil. - **Purine (2 rings):** Adenine, Guanine. ------------------------------------------------------------------------------ **Nucleobase** **Nucleoside** **Nucleotide** ---------------- ---------------- -------------------------------------------- Adenine (A) Purine Deoxyadenosine\ Deoxyadenosine mono-, di-, or triphosphate Guanine (G) Purine Deoxyguanosine\ Deoxyguanosine mono-, di-, or triphosphate Cytosine (C) Pyrimidine Deoxycytidine\ Deoxycytidine mono-, di-, or triphosphate Thymine (T) Pyrimidine Thymidine\ Thymidine mono-, di-, or triphosphate Uracil (U) Pyrimidine Uridine\ Uridine mono-, di-, or triphosphate ------------------------------------------------------------------------------ **RNA:** - Single-stranded - Shorter chains of nucleotides than DNA - RNA can adopt very complex three-dimensional structures when it contains self-complementary sequences (DNA cannot do this because it is double-stranded). - RNA nucleotides contain ribose instead of deoxyribose (OH group at the 2nd carbon atom). ### DNA Polymerization - Most polymerases require a template. - All require a primer (RNA), made by DNA primase. - All synthesize from 5' to 3'. - Some have exonuclease activity: - 3'→5' proofreading. - 5'→3' removal of primer. **Genome Packaging in Prokaryotes and Eukaryotes** - **Genome:** The entire set of genetic material: DNA & RNA. - **Gene:** A segment of DNA that codes for a piece of protein or functional RNA. ### Histone Proteins and Acetylation - Histone proteins together with DNA form a nucleosome. - Chemical groups such as methyl and acetyl groups can be covalently attached to histone subunits. These chemical modifications affect the strength of the interaction between the histones and the DNA. Acetylcation and methylation of the amino acid lysine are common modifications of histones. - Through acetylation, the charge of histones becomes negative, and the interaction with DNA becomes weaker. - **Histone Deacetylase Complexes (HDAC) - Inhibitors** are used in cancer treatment. Why? - They disrupt the expression of oncogenes or tumor suppressor genes. - 0.1 mm of coiled DNA corresponds to 1 meter of uncoiled DNA. ### PCR (Polymerase Chain Reaction) - PCR is a method to amplify a specific piece of DNA using DNA polymerase. - DNA polymerases require primers. - Design a primer pair on known anchor sequences. - Primers provide enormous specificity. - Synthetic ssDNA oligonucleotides: custom primer pairs can be ordered. #### PCR steps 30 cycles of the following 3 steps: 1. **Denaturation (94 °C):** convert dsDNA to ssDNA (break H-bonds). 2. **Annealing (50 °C -- 60 °C):** primers bind to ssDNA (5' \--\> 3'). - If temperature (T) is too low: primers do not bind specifically (quickly form H-bonds). - If temperature (T) is too high: not enough primers bind (not enough product formed). 3. **Elongation (72 °C):** DNA polymerase begins with NTP + dNTP. (NTPs are building blocks of RNA, dNTP are building blocks of DNA) Strands are made double-stranded again. High temperatures; thermophilic polymerases survive due to derived parts from bacteria that can withstand heat. - First round: new products contain the product + remaining genome. - Repeat several times until enough product is present - Unwanted products remain constant in number. #### Reaction Mix - **Primer pair** targeting both ends. - **DNA** with target sequence. - **Nucleotides (dNTPs)**. - **Nuclease-free water**. - **DNA polymerases**. - **Reaction buffer** (to maintain constant pH) (e.g., MgCl₂). #### PCR Primer Design Guidelines: 1. **Length:** 17-28 nucleotides - Longer → annealing becomes more difficult - Shorter → less specificity 2. **GC content:** 50-60% (percentage of guanine and cytosine) - Higher → melting temperature too high - Lower → less specificity - **Melting temperature (Tm):** The temperature at which a primer dissociates from the template (55°C -- 65°C). The longer the primer, the higher the Tm. Count the number of C-G pairs. - **Annealing temperature (TA):** The temperature at which a primer binds to the template. This is typically about 5°C below the average Tm. - Primers do not dissociate during elongation because DNA polymerase starts building from 50°C. DNA polymerase stabilizes the primer-template complex. - Primers should end with **C or G** at the 3' end to prevent dissociation. #### Reaction Stops - The reaction stops when NTPs run out → analyze reaction products on a gel → if a single band is visible → sequence it. ### How to prepare primers? **Example:** You want to prepare a forward and reverse primer for this sequence. How do you do that?\ 5' **GCTGAATCGA** \-\-\-\-\-\-\-\-\-\-\-\-\-\-\--**GGGTACCGTA** 3' Forward primer: **GCTGAATCGA -\>** copy & paste 1st part. Reverse primer: **TACGGTACCC -\>** based on 2nd part: Rewrite 2nd part of sequence in reversed form:\ **GGGTACCGTA -\>** ATGCCATGGG\ Write complementary sequence for reverse sequence:\ ATGCCATGGG -\> TACGGTACCC -\> answer ### Cystic fibrosis **CFTR Protein**: The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride (Cl⁻) transporter in epithelial cell membranes. It regulates the transport of sodium (Na⁺) and water (H₂O), crucial for keeping the lung mucus hydrated to trap and expel bacteria. **Mutated CFTR Protein**: CFTR mutations reduce Cl⁻ export, leading to increased Na⁺ reabsorption and mucus dehydration, which causes lung infections. These mutations also affect other organs, such as the pancreas and sweat glands. Cystic fibrosis is inherited in an autosomal recessive manner, with over 2500 known mutations. Lesson 2: Origins of replication, dna polymerases ------------------------------------------------- ### Replication Bubble in prokaryotes - **Initiator proteins** recognize the **Origin of Replication (ORI)** and bind to it, leading to the unwinding of the double helix and the activation of **primase activity**, resulting in the formation of a **replication bubble**. - The replication bubble: replication is usually **bi-directional**. Both DNA polymerases meet opposite the ORI. The replication stops **Exonuclease Activity** In addition to adding nucleotides to a strand, exonuclease activity allows the removal of nucleotides. ### Different DNA Polymerases #### DNA Polymerase (DNApol) - Most require a **template**. - All require a **primer** (RNA), which is synthesized by **DNA primase**. - All synthesize in the **5' to 3'** direction. - Some have exonuclease activity: - **3' to 5' proofreading**. - **5' to 3' removal** of the primer. #### DNA Replication by DNA Polymerase - **Template-dependent**. - **Semiconservative**: one old strand and one new strand. - **Triphosphate nucleotides** serve as substrates. - Bonding occurs at the **3' end** (OH group). - Two phosphate groups are cleaved off, releasing energy, while one phosphate remains incorporated. - Approximately **1000 nucleotides per second** in prokaryotes. - Approximately **50 nucleotides per second** in eukaryotes. ### DNA Synthesis by DNA Polymerase - **DNA polymerase** reads in the **3' to 5'** direction and builds in the **5' to 3'** direction. - **Leading strand:** 3' to 5', so it can be read and synthesized easily. - **Lagging strand:** 5' to 3', which must be read and synthesized in fragments.  ### Okazaki Fragments - **Okazaki fragments:** newly synthesized pieces of DNA along with primers. ### Key Enzymes in DNA Replication - **Helicase:** unwinds the DNA double helix. - **Exonuclease Activity of DNA Polymerase (DNApol):** - Removes the **5' to 3'** primer. - Performs **3' to 5' proofreading** (repairing any mistakes). - **Ligase:** seals the backbone of the DNA strands. ### Synthesis Direction - DNA polymerase builds in the **5' to 3'** direction. If it were to build in the opposite direction, proofreading would block further elongation since there would be no triphosphate (energy) available for the addition of nucleotides. ### Important nucleotide repeats in prokaryotes - **5x 9 nucleotide repeats:** This is a sequence consisting of a repetition of 9 nucleotides where the initiator complex binds (specifically, dnaA initiator proteins). - **3x 13 nucleotide repeats:** This region is rich in A-T pairs, making it easier to break open due to the torsional stress that occurs after the binding of the dnaA complex. This is because A-T pairs have fewer hydrogen bonds compared to C-G pairs. A close-up of a dna test Description automatically generated ### Origin of replication in prokaryotes - **ORI activation:** The ORI can be activated only once per cell division. - **Fully methylated ORI:** Activated → replication can occur. - **Hemimethylated ORI:** Inactive → replication cannot occur (as long as the DNA is hemimethylated, an inhibitor protein is bound to the ORI). - After complete methylation of the adenines in the GATC sequences by a DNA methylase, the ORI can be reused. ### Initiation in prokaryotes - **DnaA:** Initiator protein - Binds to a specific sequence → induces torsional stress → DNA opens at A-T rich sequences. - **DnaB:** Helicase - Binds to DnaA with the help of DnaC. - **DnaC:** Helicase loading protein - Blocks the helicase until it is correctly placed on the ORI. - **DnaG:** Primase - Places an RNA primer on single-stranded DNA (ssDNA). ### Eukaryotic Origin of Replication (ORI) - **ORC Binding Site:** - A binding site for the **Origin Recognition Complex (ORC)**. - **Unwinding Region:** - A sequence that is rich in **A-T** base pairs. - **Abf1 Binding Site:** - A binding site for proteins that help attract the ORC. ### Initiation in eukaryotes - The **ORC** is bound to an ORI throughout the entire cell cycle, except during the replication of the origin itself. Only a **non-phosphorylated ORC** can form a **prereplicative complex**. - **Is the ORI activated?** → Replication forks are initiated → The ORC becomes phosphorylated. - **Dephosphorylation of ORC** occurs only at the end of mitosis.  ### Helicases in pro- and eukaryotes - Break hydrogen bonds between the bases. - DNA becomes single-stranded and is available for DNA polymerase. - Consumes energy (ATP). - Can process up to **1000 base pairs** per second. ### Digestion and Ligation - **Digestion:** The process of breaking down the whole into smaller pieces. - **Ligation:** Ligase restores the phosphodiester bonds in the backbone. ### Priming Before replication starts, priming of nucleotides is necessary by a primase (a special type of DNA polymerase). This process occurs in both prokaryotes and eukaryotes. Subsequently, another polymerase is responsible for the elongation of the new strand. **Prokaryotes:** - **DNA Polymerase I** - primer removal. - **DNA Polymerase III**. - **RNA Primer**. - Okazaki fragments of **2000 base pairs**. **Eukaryotes:** - **DNA Polymerase α** -- priming. - **DNA Polymerase δ**. - **RNA-DNA Primer**. - Okazaki fragments of **200 base pairs**. ### cDNA (Complementary DNA) #### Synthesis of cDNA: 1. Isolate (m)RNA (recognized by the **poly-A tail**). 2. Treat with **DNase** to eliminate genomic DNA. 3. Convert RNA into cDNA using: - **Oligo dT** (primers). - **Reverse Transcriptase** (of viral origin). - **RNase H**. - **DNA Polymerase I**. ### Cloning steps: 1. Isolation of genomic DNA/RNA 2. PCR of the gene of interest 3. Digestion of plasmid 4. Ligation 5. Transformation 6. Culturing bacteria 7. Plasmid isolation 8. Validation ### Cloning Vectors - **Cloning Vector:** - **Restriction Nuclease:** Cuts the plasmid. - **DNA Fragment to be Cloned:** - **DNA Ligase:** Joins the DNA fragment into the vector. - **Recombinant DNA:** ### Restriction Enzymes & Replication - **Bacterial Strains:** Have unique restriction enzymes. - **Diversity:** More than 3000 different types known. - **Commercial Availability:** Over 600 are commercially available. - **Naming:** Refers to the original strain (e.g., EcoRI from *E. coli*). - **Unique Combination of:** 1. **Recognition Sequence:** Palindromic sequence of 4-8 nucleotides. 2. **Cutting Style:** Either blunt or sticky ends. ### Types of restriction enzyme cutting style: A screenshot of a computer Description automatically generated ### Compatible overhangs: Fragments with the same overhang can be connected to each other  ### Multiple Cloning Site (MCS) - **Definition:** A DNA segment with multiple unique restriction enzyme sites. - **Purpose:** Enables insertion of foreign DNA into a cloning vector. - **Usage:** Researchers cut vectors at MCS sites to facilitate cloning. ### Ligation Ligase restores the phosphodiester bond in the backbone.  Lesson 3: Primer deletion & topoisomerases & telomeres ------------------------------------------------------ ### Primer deletion in prokaryotes - DNA polymerase III only has 3\'→5\' exonuclease activity (proofreading) and therefore cannot remove primers. - DNA polymerase I takes over replication and can remove the RNA primer (it has 5\'→3\' exonuclease activity). - DNA polymerase I fills the gap with DNA nucleotides. - DNA ligase seals the backbone (between the 3\'-OH and the 5\'-P) ### Primer deletion in eukaryotes - DNA polymerase δ only has 3\'→5\' exonuclease activity (proofreading) and therefore cannot remove primers. - RNAse H removes the primer, leaving one nucleotide behind (which is removed by FEN1). - DNA polymerase δ fills the gap with DNA nucleotides. - DNA ligase seals the backbone (between the 3\'-OH and the 5\'-P). ### Topoisomerases (Supercoiling) DNA topoisomerases are enzymes that catalyze changes in the topological state of DNA. - **Topoisomerase I:** - Does not require ATP. - Cuts one DNA strand. - Allows the other strand to pass through the break, then seals it. - **Topoisomerase II:** - Requires ATP. - Cuts both DNA strands. - Holds the ends in place while allowing another helix to pass through, then seals the break. ### Telomeres & Telomerase A telomere is **a region of repetitive DNA sequences at the end of a chromosome. Telomerase** prevents telomere shortening. The telomere shortening is caused by a failure to completely replicate the ends of linear DNA molecules. #### Telomeres: - Found at the ends of linear DNA; consist of non-coding tandem repeats. - Fold back and hybridize with themselves to form a T-loop (three strands, two paired). - Vulnerable to nucleases; protected by specific proteins. #### Telomere Shortening: - Not enough space to add the Okazaki primer. - -Not enough space to replace the Okazaki RNA primer with DNA. Shortly: Limits for primer binding and RNA replacement with DNA. #### Solution via Telomerase: - Human Telomerase Reverse Transcriptase (hTERT) has RNA as a template for extension. - Gradually adds single-stranded tandem repeats, allowing primer binding and DNA polymerase activity. - Nucleases remove primers from the 5\' end, leaving a 3\' single-stranded tail for a new T-loop. ### The Hayflick limit The limit on cell replication imposed by the shortening of telomeres with each division #### Presence: - Found in stem cells, germ cells, tumor cells, and embryonic cells. ### (Heat Shock) Transformation 1. **Preparation:** Use competent E. coli that easily uptake plasmids. 2. **Mixing:** Combine bacteria with plasmids, cool on ice, heat shock at 42 ºC for 45 seconds, then cool again. 3. **Recovery:** Let bacteria recover in a small amount of medium at 37 ºC. 4. **Plating:** Spread bacteria on agar plates and incubate overnight at 37 ºC. #### E. coli as a Replication Machine - **Growth Medium:** E. coli can grow in both liquid and solid media. - **Colony Formation:** On solid media, E. coli cells remain stationary but will divide, forming colonies (clones). #### Practical Application: - Transform the bacteria and plate them on solid media. - Pick several colonies and grow them in separate liquid cultures. #### Plasmid Isolation - **Host Cells:** Use host cells sensitive to antibiotics. - **Vectors:** Employ vectors containing antibiotic resistance genes. - **Cultivation:** Grow transformed cells in the presence of antibiotics. - **Survival:** Only cells with the vector will survive. #### Validation of Recombinant Plasmid - **Restriction Enzyme Digestion:** Isolated recombinant plasmids are cut again with restriction enzymes. - **Fragment Analysis:** Analyze the size of the resulting fragments using gel electrophoresis. ### The bacterial replication fork  Theme 2: Transcription & mRNA expression analysis ================================================= Lesson 4:Polymerases & operons ------------------------------ ### General Gene Expression Regulation Gene expression refers to the process of activating specific genes to manifest in the phenotype. This involves the conversion of DNA into RNA, which is then translated into an amino acid sequence, forming a protein. ### DNA Polymerase vs. RNA Polymerase #### DNA Polymerase: - Requires a template. - Needs a primer (RNA) produced by DNA primase. - Synthesizes DNA in the 5' to 3' direction. - Some have exonuclease activity for: - **3' to 5' proofreading** - **5' to 3' primer removal** #### RNA Polymerase: - - **Prokaryotes:** One type of RNA polymerase. - **Eukaryotes:** Three distinct types of RNA polymerases. - Synthesizes RNA in the 5' to 3' direction, using DNA as a template. - - Can break open DNA strands independently and does not require a primer. - Can remove incorrectly incorporated nucleotides. - **Transcription initiation:** Determined by the orientation of the promoter. - **Transcription synthesis:** Continuously occurs in the 5' to 3' direction. **\ ** ### Prokaryotic Transcription Process - **No mRNA processing:** mRNA is ready for translation immediately after transcription. #### Initiation Steps: 1. The σ-factor associates with RNA polymerase. 2. The σ-factor recognizes the consensus sequence in the -35 box of the promoter. 3. The holoenzyme breaks the hydrogen bonds between the strands in the AT-rich -10 box. #### Elongation: 1. After several attempts, the holoenzyme leaves the promoter region. 2. The σ-factor dissociates from the RNA polymerase complex. 3. RNA polymerase synthesizes an RNA molecule based on the DNA nucleotide sequence. #### Termination: 1. The termination sequence on the DNA template contains a palindromic sequence followed by a stretch of adenines. 2. The complementary sequence in the RNA transcript hybridizes with itself, weakening the interaction with the template DNA. 3. The interaction between the adenine stretch on the DNA template and the uracils on the RNA transcript is weak enough to cause the dissociation of the DNA-RNA-RNA polymerase complex. A close-up of a logo Description automatically generated #### Operon Structure: - An operon often contains one or more operators. - **Operator:** A DNA sequence near the promoter where repressor or activator proteins bind to regulate RNA polymerase\'s affinity for the promoter. #### σ-Factor Functionality: - The affinity of the σ-factor for the promoter depends on the specific promoter sequence of a gene. - There are \"strong\" and \"weak\" promoters, which can differ in binding strength for RNA polymerase by up to a factor of 1000. - Under varying conditions, an alternative σ-factor can be expressed, altering its affinity for promoter sequences. This can convert a weak promoter into a strong one and vice versa. ### Differences in Gene Expression Between Eukaryotes and Prokaryotes #### Eukaryotes: - Each gene has its own promoter. - Typically, one mRNA (monocistronic) is produced per protein, meaning no operons are present. #### Prokaryotes: - - Sets of functionally related genes are arranged sequentially under the control of a single promoter and operator, forming an operon. - Produces one mRNA (polycistronic) that contains multiple open reading frames - (ORFs), each with its own ribosome binding site (RBS), allowing for the synthesis of multiple proteins. ### Variation of operons and σ-factors - The affinity of the σ-factor for the promoter depends on the specific promoter sequence of a gene. - There are \"strong\" and \"weak\" promoters, depending on the binding strength of RNA polymerase (which can vary by a factor of 1000). - Depending on the conditions, an alternative σ-factor can be expressed with a different affinity for promoter sequences. A weak promoter may now become strong, and vice versa.  ### Regulation of the Tryptophan Operon in Prokaryotes **Tryptophan Operon:** - Encodes five enzymes responsible for synthesizing tryptophan from simpler molecules when tryptophan concentration is low. - The promoter contains an operator (a part of the operon near the promoter). - Tryptophan binds to a repressor, activating it. - The activated repressor binds to the operator, preventing RNA polymerase from binding to the promoter. - Consequently, no mRNA is produced, leading to a lack of enzymes necessary for tryptophan biosynthesis. ### Regulation of the Lac Operon in Prokaryotes **Lac Operon:** - Comprises three genes (LacZ, LacY, LacA) involved in lactose uptake and metabolism. - Activation occurs only in the presence of lactose and when glucose levels are low, utilizing two mechanisms: 1. **Positive Regulation (CAP):** - Low glucose levels result in high cAMP concentrations. - cAMP binds to CAP (Catabolite Activator Protein). - CAP binds to the CAP binding site, facilitating RNA polymerase binding and transcription. 2. **Negative Regulation (Repressor):** - Lactose must be present. - Allolactose (an isomer of lactose) binds to the repressor. - This binding induces a conformational change, causing the repressor to release from the operator, allowing RNA polymerase to transcribe the genes. Inducible expression of lac promotor: **Natural situation**: in *E. coli*: Due to the presence of lactose, the isomeric form allolactose is also present, which induces the expression of the cloned gene. **In the lab:** IPTG (isopropyl-beta-D-thiogalactopyranoside) is used. It also binds to the repressor (acts as an inducer), but it is not broken down by LacZ into galactose and glucose. d Lesson 5: Transcription in Eukaryotes ------------------------------------- Transcription in eukaryotes has 3 stages: initiation, elongation, termination. ### Polymerases - Prokaryotes have only one type of RNA polymerase, while eukaryotes have 3 (RNA polymerase 1, II, and III)\ \  - Eukaryotic polymerases need several transcription factors to start, and prokaryotes need only one (sigma (σ) factor) RNA polymerases I, II, and III each have their own class of promoters. Each promoter class consists of multiple consensus sequences (elements). These consensus sequences are recognized by general transcription factors. General transcription factors are proteins that help RNA polymerases bind to the promoter and activate transcription. **General transcription factors (TFIIs) for RNA polymerase II:** 1. TFIID (composed of TBP and TAFs) forms a platform for the other TFIIs: **TBP (TATA-binding protein) binds to the TATA box. -- no test material** **TAFs (TBP-associated factors) stabilize this interaction.** 2. TFIIH: phosphorylates the C-terminal domain (CTD) of RNA polymerase II → it causes a conformational change→ RNA polymerase II to detach from the TFIIs platform. TFIIH also functions as a helicase, unwinding the DNA for transcription. After promoter clearance, most transcription factors remain on the promoter, allowing for quick binding of new RNA polymerase II. ### Pre-mRNA processing **Eukaryotic mRNA:** - 5' capping - Splicing - 3' polyadenylation **Prokaryotic processing:**\ \ - no processing **5\' Capping proteins** - A methylated G is attached via a 5\'-5\' linkage. - This cap is added as soon as the mRNA emerges from RNA polymerase II. **Functions of the 5\' cap** - Distinguishes mRNA from other non-coding RNAs. - Facilitates mRNA export from the nucleus. - Regulates translation. - Protects mRNA from degradation. **Splicing proteins** - Remove introns(intervening) and splice exons(expressed sequence) together **Alternative splicing:** Different introns are removed in different cell types, resulting in different protein isoforms. \+ One gene can produce multiple proteins. ### mRNA splicing - Consensus regions are found on introns. - snRNPs (small nuclear ribonucleoproteins) bind (snRNA + proteins = U proteins). - A loop is formed with adenosine at its center. - A spliceosome forms, removing introns and splicing exons together. - Catalysis is carried out by snRNAs (ribozymes). - Exon-exon junctions are marked by exon-junction complexes. **Exon Skipping:** Exon can be skipped (intentionally or unintentionally) during the splicing process. **Cryptic Splice Site:** Cryptic splice sites may resemble true splice sites and be used in error.\ \ **Alternative splicing in Cystic Fibrosis** Exon skipping/frameshift → Less functional CFTR with unstable mRNA + truncated and / or malfunctioning proteins **Export of mRNA from the nucleus to the cytoplasm** Only properly formed mRNA can exit the nucleus (marked by proteins such as CBC, poly-A-binding proteins, and EJC) and they receive a nuclear export receptor. Junk RNA is destroyed in the by **exosome** (protein complex) If RNA is defective (due to premature stop codons from mutations or incorrect intron splicing), it is destroyed by the nonsense-mediated decay (NMD) system. **3' polyadenylation** 1. Cleavage factors are attached to the CTD of RNA polymerase II. 2. Cleavage factors recognize the polyadenylation signal (AAUAAA) on mRNA and bind to it. 3. Cleavage factors cut the mRNA at the recognition sequence. 4. Poly-A polymerase attaches \~250 A's to the 3\' end of the mRNA. 5. The cut mRNA becomes unstable and disintegrates. 6. Termination of transcription **Specific transcription factors:** - Known as transcription regulators. - Activate individual genes. - Co-activators and co-repressors bind nearby them ### Gene Control Region: Promoter: General transcription factors. Cis-regulatory elements: Specific transcription factors. ### Mediator complex The mediator complex integrates upstream and downstream regulatory signals → it allows RNA polymerase II to start. ### Eukaryotic promotors A close-up of a table Description automatically generated Lesson 6: Epigenetics & Sanger sequencing ----------------------------------------- ### DNA methylation & DNA acetylation Acetylation and methylation are 2 competing processes that regulate gene expression. They are reversible(deacetylation and demethylation) and the occur primarily on lysine residues on histone tails. There are 4 4 forms of acetylated/ methylated lysines: 1. Acetyl lysine 2. Monomethyl lysine 3. Dimethyl lysine 4. Trimethyl lysine **DNA methylation** - Methylation is the addition of methyl group(CH3) to a molecule. - In general, DNA methylation represses transcription(gene silencing) , and loss of methylation is associated with gene activation. **DNA acetylation** - Acetylation is the addition of acetyl group (CH3CO-) to a molecule. - This process is regulated by two classes of enzymes:\ - Histone acetyltransferases (HATs)\ - Histone deacetylases (HDACs) - Acetylation is accompanied by loss of positive charge, decreasing its attraction to DNA → DNA becomes more accessible - Hyperacetylated chromatin is transcriptionally active, and hypoacetylated chromatin is silent. ### Histone modifications Histone modifications examples: methylation, acetylation, phosphorylation, ubiquitylation, and SUMOylation ### Cis-regulatory elements **Cis-regulatory elements** Silencers: Inhibit transcription. Enhancers: Activate transcription. ### Chromatin density Chromatin density is lowest in the nuclear interior and highest in the heterochromatin near the nuclear lamina. 1\. **Heterochromatin:** a highly condensed form.\ There are 2 main types of heterochromatin: \- **Constitutive heterochromatin**: compact, contains genes that do not need to be expressed or cannot be expressed; usually located in regions like centromeres and telomeres. \- **Facultative heterochromatin:** can switch between compact and more relaxed state; location can vary between different cell types 2\. **Euchromatin:** less condensed form, makes up about 90 -- 92% of human genome ### **Sanger sequencing** Sanger sequencing is determining of the order of DNA. #### Requirements: - Labeled primer - Target fragment in ssDNA - DNA polymerase - Reaction mixtures:\ - dNTP (A+T+C+G in excess)\ - ddNTP (labeled) (A + T+C or G in limited amounts). Once incorporated, the molecule is complete, and polymerase cannot extent the DNA molecule further. #### Reaction: → Denaturation → Annealing → Adding of reaction mixtures (4 dNTPs + 4x ddNTP + DNA polymerase) → Elongation → Denaturation of dsDNA (double-stranded DNA) → Electrophoresis of ssDNA (PAGE) → Dry gel and autoradiography #### Reaction Info: - Synthesis of complementary chains stops at random A T, G or C - With sufficient template DNA, all nucleotides are included - As a result, a mix of different lengths is created #### Primer design: ##### Universal primers - First primer matches a part of the vector DNA - No sequence information needed about the fragment - Can start from the 5' or 3' end of the insert - After sequencing, you gain information on the first \~750 bp ##### Internal primers - Design an internal primer on the last base pair - Repeat sequencing for longer fragments #### Disadvantages of Sanger sequencing - Limited sequence length (max \~750 bp) - Requires a known sequence for primer design - Very labor-intensive for entire genes or multiple genes **DNA polymerase in Sanger sequencing** Lesson 7: --------- **rRNA Synthesis in Eukaryotes and Prokaryotes** Ribosomes consist of: - RNA - Proteins **Prokaryote:** - Pre-rRNA → cleaving rRNA. - Smaller ribosome. **Eukaryote:** - Undergoes chemical modifications through small nucleolar RNAs (snoRNAs) + small nucleolar ribonucleoproteins (snoRNPs): snoRNAs attach to rRNA and facilitate modifications and cleaving. - Uracil → pseudouridine (isomer). - 2\'-O-methylated ribose. - Pre-rRNA → cleaving rRNA. - Larger ribosome (about 3 times larger than prokaryote). **tRNA Synthesis in Eukaryotes and Prokaryotes:** - 20 different genes for tRNAs (20 amino acids). - Pre-tRNA: - Cleaving. - Intron splicing. - Adding CAA-3\' end. - Chemical modifications (in addition to A, C, G, and U, also \'D\', \'Ψ\', and T\'s: 50 different tRNA modifications known). - Internal base pairing. - Adopting 3D conformation. - Binding to the corresponding amino acid. - Reads the mRNA in triplets. - Has a cloverleaf structure. - Ribosome helps tRNA to the correct position on the mRNA. The ribosome moves 3\' → 5\' over mRNA. - Aminoacyl tRNA synthetase: binds an empty tRNA with an amino acid. Each amino acid has a different aminoacyl tRNA synthetase. Some recognize multiple tRNAs (isoaccepting tRNAs). **Peptide Bonds:** Growth from N → C. - Linked by peptide bonds (peptide bond). - tRNA releases from the growing chain via peptidyl transferase (ribozymes (RNA with catalytic activity), part of the large ribosomal subunit). - 20 amino acids → 48 tRNAs through isoaccepting tRNAs → 68 coding codons + 3 stop codons through \'wobbling\' (multiple tRNAs can fit on different codons). **Wobbling:** - Inosine (deaminated guanine) in the anticodon of tRNAs allows base pairing with A, C, and U. - G-U wobble: G-C can also be G-U. - A-U base pairing can also be G-U. → 2 tRNAs for 4 codons. rRNA provides structure, correctly places tRNA on mRNA, and catalyzes the formation of covalent peptide bonds. - Positions for tRNAs: - E: exit tRNA. - P: tRNA with the growing polypeptide chain. - A: tRNA with an amino acid that still needs to bind. - Functions of Subunits: - **Small subunit:** placement of the correct tRNA on the codon of mRNA. - **Large subunit:** formation of peptide bonds between amino acids (contains peptidyl transferase activity). **Function of Nucleolus:** - Multiple genes on multiple chromosomes lead to a lot of rRNA synthesis. - The nucleolus appears as a dark area in the nucleus due to extensive rRNA transcription. *mRNA Stability:* Initiation Processes of mRNA in Eukaryotes and Prokaryotes **Prokaryote:** mRNA in bacteria is unstable (t1/2 \< a few minutes). RNAse - Endo/Exonucleases (3' → 5'). Degradosome - Complex of proteins to degrade prokaryotic RNA. If a (terminal) w is present, exonucleases cannot access the RNA. The hairpin must first be removed by an endonuclease. **Eukaryote:** mRNA in eukaryotic cells is relatively stable (t1/2 = 30 min). Exosome - Protein complex in the nucleus for cleaning up leftover RNA, such as introns. Instability elements - Unstable regions in mRNA where endonucleases have access. Deadenylation-dependent mRNA decay -- 3' → 5' degradation of the polyA tail in the cytoplasm. This is followed by 3' → 5' degradation of the mRNA and/or 5' decapping and 5' → 3' degradation of the mRNA. Nonsense-mediated mRNA decay (NMD) -- Errors in splicing and frameshifts are detected, leading to mRNA degradation. **RNA Interference** - Once mRNA enters the cytoplasm, degradation of the polyA tail begins through deadenylase (3' → 5'). - Deadenylase also binds to the 5' cap, followed by decapping. - The unprotected mRNA is further degraded by exonucleases. - Stability of mRNA depends on the length of the polyA tail. - Competition between polyA shortening and decapping versus translation. **NMD** - An mRNA surveillance system. Degradation of mRNAs with a stop codon that does not occur at the end of the coding region (due to mutation or incorrect splicing).  **Quantitative Real-Time PCR (qPCR)** Live measurement of PCR: back-calculating to the starting material. - Fluorescent labeling with probes or SYBR green. - Measuring expression: converting mRNA to cDNA → amplifying cDNA → fluorescence increases per cycle, measuring per cycle → measuring change and back-calculating to the start. Halfway through the cycle, a threshold is set to measure samples 1 and 2. If one has 9 and the other has 13: 10, 11, 12, 13 = 24 = 16. → Sample 1 contains 16 times more starting material than Sample 2. CT value: cycling threshold, the cycle number at which the signal exceeds the threshold. **Taqman Probe** - Has a fluorescent group at the 5\' end and a quencher at the 3\' end. - Hybridizes with the PCR target fragment → 5\'→3\' exonuclease activity of Taq polymerase cleaves the Taqman probe → the fluorescent group and the quencher are separated → fluorescence is emitted. **SYBR Green** - Intercalating: binds only to dsDNA. - Fluoresces only when bound to dsDNA. - Requires a thermocycler with a laser and detector. - **Disadvantage:** not specific for target DNA. By-products, primer dimers, etc., are also bound and thus measured. **Determining the Initial Concentration of Target DNA:\ **Difference in cycles that yield the same amount of material on the curve and back-calculating using the formula: 2\^Cycles. **Melting Curve** - Check whether the correct fragment is being amplified using a melting curve. - Measure fluorescence. When amplifying one specific product, there is a loss of fluorescence. **S-Curve:** - Begins low (the amount is too low to be measured). - The curve that starts to rise first contains the most starting material. - The curve levels off at the end: dNTPs are depleted. Thema 3: Translation & Reverse genetics ======================================= Lesson 8: --------- **Naming of the Translated and Untranslated Regions of mRNA**\ **Translated Region = Open Reading Frame (ORF):**\ The area between the start and stop codons that is translated (via translation) into a protein.\ **Untranslated Regions:** - 5' UTR (Untranslated Region) - 3' UTR (Untranslated Region) ### The Global Structure of Proteins #### Primary - Sequence of amino acids - Linear #### Secondary - α-helices - β-sheets - N-H and C=O in the backbone form hydrogen bonds → makes the backbone visible. #### Tertiary - R-groups interact with each other: non-covalent and covalent bonds. - Domains: functional areas within the tertiary structure (e.g., DNA-binding, activator, ligand-binding (receptor), catalytic (enzyme) domain). #### Quaternary - Multiple polypeptides connected together (multi-subunit protein). - Homo-dimer, -trimer, -tetramer = multiple identical polypeptides connected together. - Hetero-dimer, -trimer, -tetramer = multiple different polypeptides connected together. ### Co-Translational Protein Folding: During translation, proteins spontaneously fold into their secondary and tertiary structures.\ **Chaperone Proteins:** Assist in folding and recognize incorrectly folded proteins. - Heat shock proteins (HSP) assist in the folding of proteins at elevated temperatures and ensure that expression is upregulated. - **Chaperonin (Hsp60):** - Helps misfolded proteins to fold correctly. The protein enters a capsule (hydrophobic environment) → the protein has a chance to unfold and refold properly. - **Hsp70 Chaperone:** - Binds to the hydrophobic parts of the polypeptide during translation → prevents folding → waits until the rest of the protein has folded → Hsp70 releases → continues protein folding. - If the protein still does not fold correctly → degradation of the protein. ### Genetic Code ### Anticodon on tRNA - Anticodon tRNA binding with codon on mRNA - tRNA binds amino acid **Reading Frame:** - There are 3 possible reading frames on mRNA, and 2x3 possible reading frames on DNA. **tRNA as an Adaptor and Wobbling:**\ Wobbling is the incorrect placement of a nucleotide, which can result in a different amino acid.\ The adaptor is the scheme by which the amino acids should be read. **Structure of tRNA:**\ tRNA has the structure of a cloverleaf. This structure arises due to existing hydrogen bonds.\ It has at least two important components: an anticodon and a binding site for one of the amino acids. ### Structure of Ribosome: - rRNA provides structure, correctly places tRNA on mRNA, and catalyzes activity to form covalent peptide bonds. - Sites for tRNAs: - E: exit tRNA. - P: tRNA with growing polypeptide chain. - A: tRNA with an amino acid that is yet to be linked. - Functions of subunits: - Small subunit: placement of the correct tRNA on the codon of mRNA. - Large subunit: formation of peptide bonds between amino acids (contains peptidyl transferase activity). - Protein synthesis begins at the N-terminus. #### Initiation ##### Prokaryote: 1. Binding of the small ribosomal subunit + initiation factors (IFs) to the ribosome binding site (RBS) (located 3-10 nucleotides before the start codon).\ The RBS is complementary to the 3' end of 16S rRNA. The small ribosomal subunit can (along with IFs) use 16S rRNA to occupy the correct position on the mRNA, in the proper orientation. 2. Binding of initiator tRNA + IFs\ The AUG start codon is recognized by the initiator tRNA + formyl-methionine (methionine with a blocked N-group → synthesis can only occur in one direction). 3. Binding of the large ribosomal subunit\ The large ribosomal subunit is loaded after the initiator factors are released → the ribosome is ready for the elongation phase. ##### Eukaryote: 1. Formation of the initiation complex: The initiator tRNA associates with the small ribosomal subunit. 2. Association of the initiation complex with the 5' cap. 3. The initiation complex scans for the Kozak sequence (with start codon): The initiation complex moves from 5' to 3' over mRNA until tRNA binds to AUG. 4. The Kozak sequence is the part where the tRNA is bound to the AUG (start codon). 5. Binding of the large ribosomal subunit. ### Elongation 1. Coupling of the second aminoacyl tRNA: After positioning the ribosome with the initiator tRNA at the start codon, a second aminoacyl tRNA binds to the codon that is available in the A-pocket. 2. Formation of a peptide bond between amino acids: The peptide chain is released from the tRNA in the P-pocket and is linked to the amino acid on the tRNA in the A-pocket using peptidyl transferase (a ribozyme, part of the large ribosomal subunit). A diagram of a protein Description automatically generated ### Termination 1. The ribosome encounters one of the three stop codons. 2. A release factor (RF) binds in the A pocket (there are no tRNAs for stop codons). 3. Peptidyl transferase catalyzes the addition of H2O instead of an amino acid to the polypeptide chain. 4. Translation is blocked, and RFs release. The complete polypeptide is released and is then folded and possibly modified (see next lesson). The ribosome dissociates into two subunits → reused for other translations. 5. mRNA can be reused or degraded. ### Translational Factors \ **Polyribosomes:**\ It is a cluster of ribosomes that simultaneously read the same mRNA but are working on different parts of the mRNA. Lesson 9 & 10: -------------- ### Structure and Folding of Proteins **Amino Acids:**\ An amino acid consists of a hydrogen atom (H), an amine group (NH₂), a carboxyl group (COOH), and a variable side chain (R group). Only the side chain changes between amino acids. There are 20 different amino acids, each represented by a letter. Each amino acid is encoded by three nucleotides. **Covalent and Non-covalent Bonds:**\ Proteins fold through covalent (actual bonds between atoms) and non-covalent (attractions) interactions between amino acids, determining the protein's structure. - **Covalent bonds:** Strong bonds between atoms. - **Non-covalent bonds:** Weaker forces like hydrogen bonds and ionic interactions. **Folding:** - **Polar amino acids:** Found on the outside of the protein, allowing it to dissolve in the cytoplasm or extracellular matrix. - **Non-polar amino acids:** Buried inside the protein, away from water. **Functional Domains** Proteins consist of one or more functional domains, which are: - Regions of 40 to 350 amino acids that fold independently into a 3D structure. - Typically conserved across species and protein families. A dna model with multiple strands Description automatically generated with medium confidence ### Protein Modifications ( mono- & polycistronic) **Monocistronic Protein Synthesis (Eukaryotes):** - Occurs in eukaryotic cells. - mRNA contains **one gene**. - Translates into **one protein**. - Has a single **translation start** and **termination point**. - Undergoes **post-translational modifications**. **Polycistronic Protein Synthesis (Prokaryotes):** - Occurs in prokaryotic cells. - mRNA contains **multiple genes**. - Has multiple **translation and termination points**. - **No post-translational modifications**. - Translates into **multiple proteins**. Unfolded proteins can form aggregates, which may lead to diseases. ### RNA Interference (RNAi) - **RNA interference (RNAi):** Process where RNA molecules influence mRNA expression. ### miRNA (microRNA): - Regulates expression of **own genes**. - A separate gene codes for miRNA, which hybridizes with mRNA. - **DICER** enzyme recognizes double-stranded RNA (dsRNA) and cuts it into miRNA. - **RISC** complex (with proteins and miRNA) searches for complementary mRNA sequences. - **Full base pairing**: mRNA is cleaved. - **Partial base pairing**: Translation is suppressed. - miRNA can be transported to other cells via **vesicles**. ### siRNA (small interfering RNA): - Provides **protection against foreign RNA**. - **DICER** cuts dsRNA into siRNAs. - RISC removes one strand of siRNA and uses the other to scan for matching mRNA. - **Match found**: RISC attacks and degrades mRNA or silences it. **Reverse Genetics:** - Manipulating a gene to observe changes in the **phenotype**, revealing the gene\'s function. ### Creating a Knockout Mouse **Gene Knockout:** Inactivation of a specific gene in a mouse to study its function. 1. **Gene-targeting vector** is inserted into **embryonic stem cells**. 2. **Homologous recombination** replaces the original gene with a sequence containing an antibiotic resistance gene. 3. **Selection and proliferation** of manipulated stem cells are done using antibiotics. 4. The manipulated stem cells are **injected into a blastocyst**, which grows in a surrogate mother. 5. The offspring carrying the mutated gene in their **germline** are crossed to produce a knockout mouse line. ### Knockdown Using siRNA - **Knockdown:** Reduces gene expression at the **RNA level** by degrading mRNA. - **RNAi** (RNA interference): Short RNA fragments (siRNA or miRNA) can silence gene expression by binding to matching mRNA sequences (gene silencing). - This can be **temporary and reversible**, or **long-term and even heritable**. - **Synthetic siRNA** or **shRNA** (short hairpin RNA) can be used to mimic this gene silencing effect. ### Reportergen & DNA Footprinting - **Reportergen**: A gene inserted in the same frame as the gene of interest to assess gene expression regulation in different cells. - **DNA Footprinting**: Technique used to identify specific protein-DNA interactions. The bound protein protects the DNA from DNase digestion, and the uncut regions on a gel indicate the binding location. ### Protein Degradation - **Targeting Proteins for Degradation**: - Misfolded or abnormal proteins are degraded to prevent unwanted activity and aggregation. - **Proteins no longer needed** (e.g., cyclins during cell division) are selectively degraded. - **Proteasome**: Large protein complexes in the cytoplasm and nucleus responsible for protein degradation. - **Proteins marked for degradation** are tagged with ubiquitin via specific enzymes like ubiquitin ligases. - Ligases recognize **exposed hydrophobic regions** of misfolded proteins. ### Protein Aggregation - **Protein Misfolding**: Mutations may lead to improper folding, exposing hydrophobic regions. - Misfolded proteins can form **amyloid fibrils** with stacked β-sheets, leading to **resistance to degradation**, which can result in cell damage and death A grey symbol with white text Description automatically generated ### Protein-Protein Interactions - **Protein Complexes**: Most proteins function within larger protein complexes via non-covalent bonds. Examples include: - Transcription initiation complex - Nuclear pore complex - Spliceosome ### Use of Reporter Genes - Reporter genes encode proteins whose expression is easy to detect. Examples include: - GFP (Green Fluorescent Protein) - Luciferase - Antibiotic resistance genes ### Applications of Reporter Genes - Visualization of proteins and cells - Measuring promoter activity - Selecting bacteria or cells that have taken up recombinant DNA ### Bioluminescence Imaging with a Luciferase Reporter Gene #### Application: - Used to make **proteins and cells** visible, for example, for the **localization and quantification** of tumor cells in vivo. #### Approach: - Introduce an **extra gene** for luciferase expression in tumor cells. - The reporter gene includes its **own promoter and start codon**, allowing tracking of the location and activity of the tumor cells. ### Promoter Assays #### Application: - Used to measure the **activity of a promoter**. #### Approach: - Clone the reporter gene, such as **luciferase or GFP**, as a replacement for the original gene behind the promoter. - The reporter gene contains its **own start codon**, enabling direct measurement of promoter activity. Thema 4: Mutagenesis & Identification of mutations ================================================== Lesson 11: ---------- ### Types of Mutations, Mutation Rates, and Mutagens 1. **Point Mutation:** Change in nucleotide 2. **Silent:** The last nucleotide in the codon changes, but the amino acid does not change. 3. **Missense:** A nucleotide in the codon changes, resulting in a different amino acid. 4. **Nonsense:** A nucleotide changes, causing the codon to become a stop codon, which shortens the protein. 5. **Read Through:** A nucleotide changes, and the stop codon changes, leading to an extended protein. #### Deletions and Insertions (in exon): - 1 or 2 nucleotides removed or added, causing a frameshift. - 3 nucleotides or multiple codons lead to a shortened or extended protein, which is less severe. #### Mutations in Introns - Can lead to splicing errors. #### Replication Errors - Tautomers. - SNP: Variation of 1 nucleotide within a population. Arises as a point mutation, but the error is not corrected. - **Slippage:** DNA polymerase has difficulty reading repeat DNA sequences. These sequences can become longer than they actually are during replication (no repair). #### Chemical Changes - **Base Analog:** 5-bromouracil can bind with A and G due to easy tautomerization (can form more hydrogen bonds, thus becoming a different base opposite it). - **Depurination:** Loss of A or G leads to the loss of a nucleotide pair. - **Deamination:** Loss of an NH2 group. C deamination leads to U, and after replication, U pairs with A, so U=A and G=C in new strands. - **Intercalation:** A substance that binds within DNA (e.g., Ethidium Bromide). #### Physical Changes - **UV Radiation:** Adjacent thymines bond to each other, leading to one thymine and causing a frameshift. This causes covalent bonds between pyrimidines (C and especially T). - **Heat:** Can cause depurination. - **Ionizing Radiation** - **Radioactive Sources** #### Mutagenesis: - **Unicellular:** Changes are passed on to the offspring. - **Multicellular:** Changes in germ cells are passed on to the offspring. ### (Post-) replicative DNA Repair #### Proofreading: - 3'→5' exonuclease activity (removal of incorrectly incorporated nucleotides by DNA polymerase). #### Mismatch Repair - **Strand Directed Mismatch Repair:** MutS recognizes the mismatch, MutL looks for \'nicks\' (often in the new strand, distinguishing between the old and new strand) → MutL triggers the degradation of the nicked strand. In prokaryotes, MutH distinguishes between old and new strands by methylated A on the old strand. #### Homologous Double-Strand Break Repair - More accurate. Aligns with sister chromatids (less heterozygous, mother and father alleles). Nucleases create a 3'-overhang → the overhang is complementary to the sister chromatid sequence → hydrogen bonds are formed, acting as a primer → DNA polymerase synthesizes further as the repair system → hydrogen bonds are broken → DNA polymerase continues synthesizing → ligation → complete. ### Pre-replicative DNA Repair #### Base Excision Repair - Repairs deaminated base → recognized by DNA glycosylase → incorrect base is removed → backbone is removed → DNA polymerase fills the gap. This is inefficient. #### Nucleotide Excision Repair - Pyrimidine dimer is distorted by, for example, UV radiation → excision nuclease removes a stretch of nucleotides → DNA polymerase fills the gap again. #### Emergency (SOS) Repair - Emergency repair in case of massive damage: DNA is randomly glued together = quick, no proofreading, and inaccurate. Cells become senescent (stop dividing) or undergo apoptosis (if the damage is too extensive). #### Non-Homologous End-Joining - Joins DNA fragments together: quick & dirty. Ends are recognized by Ku proteins → marked and protected → joined together even if nucleotides may have been trimmed by nucleases → \'end joining ligases\'. ### Southern Blotting Transfer of DNA from gel to membrane. - DNA fragments from the gel are transferred to nitrocellulose, a membrane with a banding pattern identical to the gel. - An accessible print is made of the original banding pattern. - The paper is incubated with a labeled probe → unbound probes are washed away. - Detection of DNA sequences. #### Labeling with Probes Incorporating labeled nucleotides: - Denature DNA → binding of hexanucleotides (primers consisting of 6 random nucleotides).\ → Incorporation of modified nucleotides by DNA polymerase. - Incorporation of many labeled nucleotides → easy to detect. - Incorporation into the genome or plasmid can be recorded. #### Hybridization with DNA Probes: - Single-stranded probes that are complementary to the target sequence. - Linked to a chemical group and can be detected by fluorescently labeled antibodies. #### FISH (Fluorescent in situ hybridization): - Localizing positions of genes on chromosomes. - Different probes are labeled with different color labels. - Each probe shows 2 binding sites per chromosome because the chromosomes are duplicating/copying the genes. By using different probes, sometimes a mixture of two fused chromosomes can be observed. - Applied for genome diagnostics based on karyotypes. Theme 5: The applications of Molecular biology and techniques ============================================================= Lesson 12: ---------- ### Next-Gen Sequencing All platforms work differently, which affects their reliability, speed, and cost of use. Well-known platforms include Illumina and Oxford Nanopore. **Protocol:** 1. Fragment DNA 2. Ligate fragments to adapter sequences 3. Immobilize DNA fragments 4. Clonally expand fragments ### Illumina (by synthesis): 1. Add fluorescently labeled nucleotides: a. Each of the four types of dNTPs is labeled with a different fluorescent group, and the 3' OH group is blocked. b. In each cycle, the polymerase incorporates only one nucleotide because a second nucleotide cannot be added without a 3' OH group. 2. Imaging 3. Cleave the label and wash (making the OH group available again): a. The fluorescent label is cleaved off. b. The 3' OH group is made available again, allowing for another nucleotide to be incorporated in the next cycle. ### Oxford Nanopore The DNA strand is pulled through a pore, causing a change in the resistance of the membrane, which results in peaks at different times. This reflects a change in physical properties. ### Assembly of Large Genomes: - A genome is sequenced multiple times (sequence depth). - It is fragmented in various ways (shotgun method), using restriction enzymes or through sonication (random). - **Known Genome:** Overlapping sequences are aligned against a reference genome (resequencing). - **New Genome:** Assembly of sequences based on overlap and genomic markers (SNPs, genes, and restriction sites). - Overlapping ends are identified using software (In Silico). - This yields sequence contigs: assembled puzzle pieces. - The genome is reconstructed based on contigs. #### Problems Due to Low Sequencing Depth: - Some regions of a genome are sequenced more often than others. - The more frequently a region is sequenced, the higher the reliability. - Regions sequenced very little (low sequencing depth) result in unreliability. #### Problems Due to Tandem Repeats: - The length of these repeats can never be accurately determined. #### Problems Due to Genome-Wide Repeats: - Incorrect linking of areas on different (parts of) chromosomes. ### Site-Directed Mutagenesis - Mutations can be mimicked by mutating genes in expression vectors. - Primers are designed so that the mutation can be introduced using PCR. - The ends of the linear PCR product are ligated together using ligase. ### Gene Editing with CRISPR/Cas9: Gene editing - Precisely adjusting/correcting genes in living cells. #### Applications: - **Medical:** After detecting hereditary mutations (NGS), is correction possible? - **Biotechnology:** Crop improvement, protein engineering, etc. (VL5 BMR). - **Research:** Disease models, studying gene function, etc. #### Difference with Classical Recombinant DNA Techniques: Diagram of a cell division Description automatically generated #### Four systems: - Meganucleases - Zinc Finger Nucleases - Transcription Activator-Like Effector Nucleases (TALEN) - Clustered Regularly Interspaced Short Palindromic Repeats system (CRISPR/Cas9) #### Principle: 1. A specialized nuclease (molecular scissors) cuts DNA at a specific location. 2. A double-strand break occurs. 3. Repair by the cell, potentially in the presence of a 'template' with the desired modification. #### Possible DNA Manipulations: - Gene inactivation - Insertion - Deletion - Modification - Replacement - Gene induction - Multiple manipulations within a single cell **CRISPR/Cas9:** CRISPR - Clustered Regularly Interspaced Palindromic Repeats: An inherited library of DNA fragments from phages that a bacterium (or an ancestor) has been exposed to. Cas - CRISPR associated genes system. #### CRISPR/Cas9 as a Bacterial 'Immune System' **\ ** Prokaryotic defense against phages via: - Restriction enzymes: unspecific. - CRISPR-Cas: specific ('memory'). #### CRISPR/Cas9 as an Editing Tool: - Of all Cas proteins, Cas9 is the most useful nuclease. - Cas9 is guided by guide RNA (gRNA). - The gRNA sequence can be customized to direct Cas9 to a specific site to make double-strand cuts. #### CRISPR/Cas9 Double Strand Breaks: - **Non-Homologous End Joining (NHEJ):** - Fast repair by ligating loose ends (without telomeres!) using Ku proteins. - Inaccurate, with loss of nucleotides. - Gene damaged → usable for making a gene knock-out. - **Homologous Recombination:** - Accurate, no loss of nucleotides. - Using an unbroken sister chromatid (template) works only after DNA replication (S/G2 phase). ### NHEJ or Homologous Recombination? - Only a few cells are in S/G1 phase. If you provide dsDNA (donor DNA) during repair, that is used as a template → large insertions of desired sequences are possible. - If no template is provided → NHEJ → small random insertions/deletions → damage to the gene A diagram of a dna molecule Description automatically generated Lesson 13: ----------