Methods in MBG 2 ÇALIŞMA (PDF)

METHODS IN MBG 2 ÇALIŞMA DERS NOTLARI: ➔ Cells or tissues of interest must be DNA Extraction: isolated, and their membranes must...

METHODS IN MBG 2 ÇALIŞMA DERS NOTLARI: ➔ Cells or tissues of interest must be DNA Extraction: isolated, and their membranes must be disrupted to release the DNA. This The goal of DNA extraction is to obtain DNA in a is typically done using an "Extraction relatively purified form, which can then be used in Buffer." downstream applications such as PCR, sequencing, or enzymatic digestion. Components of the Extraction Buffer: Basic Protocol for DNA Extraction: 1. EDTA (Ethylenediaminetetraacetic Acid): Chelates Mg²⁺ ions, (cofactor)which are 1. Preparation of a Cell Extract crucial for maintaining the structure of cell 2. Purification of DNA from the Cell components and enzymes like DNAse. Extract 3. Collecting DNA 4. Measurement of Purity and DNA Concentration 2. SDS (Sodium Dodecyl Sulfate): Aids in the disruption of cell membranes by solubilizing the lipid components. -It is amphiphilic, which allows it to denature proteins by disrupting hydrophobic interactions and 1. Preparation of a Cell Extract hydrogen bonds. a. Sample Collection and Preparation 2. Purification of DNA from the Cell Extract b. Cell Lysis: The cell extract contains not only DNA but also significant amounts of proteins and RNA, Detergents: Used to disrupt the lipid which need to be removed to pure DNA. bilayer of the cell membrane. Denaturants: Employed to release Denaturation results in: chromosomal DNA and denature proteins. Additional Enzymes: Required for Decreased protein solubility specific cell types: Loss of biological activity Gram-positive bacteria: Improved digestion by proteases Lysozyme to break down the Release of chromosomal DNA from cell wall. nucleoprotein complexes. Yeasts: Lyticase to disrupt the cell wall. Plant cells: May require cellulase pre-treatment for cell wall disruption. Protein Removal: 3)Selective DNA Binding: Proteinase K Enzyme: Commonly Using specific binding used to degrade proteins in the cell agents to selectively extract. bind and separate DNA from other cellular RNA Removal: components. Ribonuclease (RNase A) Enzyme: Advantages: Used to rapidly degrade RNA molecules into their ribonucleotide 1. Speed and subunits. convenience 2. No organic solvents Additional Purification Methods: 3. DNA purification column containing a 1)Organic Extraction Method:(Phase silica membrane Separation) 4. Amenable to automation/miniaturization Deproteinization: Typically involves adding phenol. Support Materials : ○ Principle: DNA is polar (negatively charged) and Silica therefore insoluble in organic Anion-exchange resin solvents, which precipitates 5. Collecting DNA proteins but leaves nucleic acids in the Role of Salt in DNA Precipitation: aqueous phase. Neutralization of DNA Charges: Phase Separation: The Salts like sodium acetate neutralize aqueous solution the negative charges on the DNA’s containing nucleic acids phosphate backbone, reducing its is carefully removed solubility in water. using a pipette, leaving ○ Mechanism: Sodium ions behind the organic phase (Na⁺) neutralize the negative that contains the phosphate groups (PO₃⁻), denatured proteins. making the DNA less hydrophilic and more likely to 2) Salting Out: precipitate out of solution. At high salt concentration, proteins are Ethanol Precipitation: dehydrated, lose solubility and precipitate. Common Method: Ethanol is used to Precipitated proteins, leaving DNA in precipitate DNA, especially in the solution(in the supernatant.) presence of salt and at low Usually sodium chloride, potassium acetate temperatures (below -20°C). or ammonium acetate are used. ○ Process: After precipitation, the DNA can be collected by centrifugation and dissolved in a small volume of water or TE buffer (Tris-EDTA). Role of Ethanol: Quantification of Extracted DNA Sample: The electrostatic attraction between the Na+ OD260 of sample X dilution factor X 50 ions in solution and the PO3- ions are dictated μg/ml (1 OD) = μg/ml DNA by Coulomb’s Law, which is affected by the dielectric constant of the solution. PS:50 μg/ml of DNA = 1 OD (optical density) Dielectric Constant: Ethanol has a Example; If 5 μl of extracted DNA in lower dielectric constant than water, 1000μl (1ml) gives an OD260 = 0.14 which reduces the solubility of DNA in the solution and facilitates its ➔ Dilution factor = 1000 / 5 = 200 precipitation. ➔ 0.14 X 200 X 50 = 1400 μg/ml 6. Measurement of Purity and DNA Prokaryotic DNA Isolation: Concentration DNA Sources: Quantification by Spectrophotometry: Chromosomal DNA Absorbance at 260 Extrachromosomal DNA (plasmids, nm (A260): Used to bacteriophages) determine DNA concentration, as Plasmids: DNA absorbs UV light at this Small, circular, wavelength. double-stranded, extrachromosomal DNA (distinct from chromosomal DNA.) They can replicate itself independently from the host's chromosomal DNA. Naturally exist in bacteria; provide functions like antibiotic resistance.But also exist naturally in archaea and eukaryotes such as yeast and plants All natural plasmids contain an origin Purity Ratios: of replication. -Pure DNA has an OD260/OD280 ratio of ~1.8 Origin of Replication: This is because proteins absorb maximum 1. Controls the host range (which UV light at A280. Ratio of less than 1.8 is species the plasmid can replicate in). indicative of protein contamination. 2. Regulates the copy number of the plasmid (how many copies of the -Pure DNA & RNA has an OD260/OD230 plasmid exist within a cell). ratio of ~2.0-2.2 3. Common examples are genes that confer antibiotic resistance, allowing phenolic contamination which hamper the the host cell to survive in the presence downstream enzymatic activity) of specific antibiotics -Pure RNA has an OD260/OD280 ratio of ~2.0 Low ratios could be caused by protein or phenol contamination. Plasmid elements : 1. Origin of Replication (ORI): DNA sequence which allows initiation of replication within a plasmid by recruiting (devreye sokarak) replication machinery proteins. 2. Antibiotic Resistance Gene: Allows for selection of plasmid-containing bacteria. 3. Multiple Cloning Site (MCS): Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. -In expression plasmids, the MCS is often downstream from a promoter. ! Bacteriophages are not plasmids. 4. Insert: The DNA sequence, such as a Bacteriophages are viruses. gene or promoter, cloned into the MCS for further study. Prokaryotic Chromosomal DNA Isolation: 5. Promoter Region: Controls gene expression and the production of 1. Cell growth recombinant proteins.. 2. Harvesting Cells: Centrifugation 6. Selectable Marker: Used to select cells that have taken up the plasmid. 3. Weakening Cell Wall &Cell Wall Lysis: ! The antibiotic resistance gene allows a. Physical methods: for selection in bacteria. However, Freeze/thaw, homogenization, many plasmids also have selectable ultrasonication. markers for use b. Chemical methods: Use of in other cell lysozyme, EDTA(chelation), types. and SDS.(Cell Wall Lysis) 4. RNA and Protein Removal: 7. Primer a. RNA: RNase enzyme. Binding Site: A b. Proteins: Proteases short c. Rest of proteins and single-stranded oligopeptides: DNA sequence Phenol-chloroform extraction. used as an 5. DNA Precipitation: Using alcohol. initiation point for PCR DNA is precipitated using typically amplification or isopropanol or ethanol. sequencing of the primers.. Salt Function: Neutralizes the - charges on the PO3- in the DNA backbone, allowing the DNA to come together. Alcohol Role: Allows Na+2 from the salt to interact with the PO3-. This interaction neutralizes the - , causing the DNA to aggregate and precipitate out of the solution.  6. Further Purification: a. Dialysis of proteins b. CsCl density gradient centrifugation(Caesium 4. Neutralization:(pH: 5.5) chloride) c. chromatography The solution is neutralized with potassium acetate (CH3CO2K) or Extrachromosomal DNA Isolation: another acidic buffer. This allows the plasmid DNA to renature into its By removing chromosomal DNA: The plasmid double-stranded form. preparation becomes purer. It's important to be gentle because vigorous Current extrachromosomal DNA isolations use mixing or vortexing will shear the gDNA alkali and the anionic detergent SDS= (genomik) producing shorter stretches that can Alkaline Lysis Method re-anneal and contaminate. Alkaline Lysis Method: 5. Centrifugation: This method exploits the difference in size The mixture is centrifuged, and the and structure between plasmid and precipitated chromosomal DNA and chromosomal DNA. debris are pelleted at the bottom. The plasmid DNA remains in the Key Steps: supernatant. 1. Cell Growth and Harvesting: 6. Plasmid Purification: 2. Re-suspension (pH: 8.0): The pellet is May be further purified by additional then re-suspended in a solution containing steps such as ethanol precipitation Tris, EDTA, glucose and RNase A. or using silica column. 3. Cell Lysis (pH: 12-12.8): Eukaryotic DNA Isolation Cells are then treated with an alkaline Sources: Blood, semen, saliva, urine, hair, solution, typically containing (NaOH) bone, tissue, etc. and detergent (SDS). NaOH Function: Breaks down the cell Methods: wall and disrupts hydrogen bonds between DNA bases, converting 1. Organic Extraction: (phenol-chloroform) (dsDNA) into (ssDNA) in a process 2. Non-Organic Extraction: Proteinase K called denaturation. and salting out.) Plasmid DNA is able to renature 3. Chelex Extraction: (ion exchange resin) when the conditions are neutralized. 4. Silica-Based Extraction: (silica exchange In contrast, chromosomal DNA does resin) not. SDS Function: Denatures most Organic Extraction Buffer: cellular proteins, Phenol: Usually equilibrated with a aiding in their buffer (e.g., TE) and contains 0.1% separation hydroxyquinoline and 0.2% from plasmid β-mercaptoethanol. DNA later in the process.  Hydroxyquinoline: Acts as an antioxidant and RNA Isolation gives phenol a yellow color for easier phase identification. RNA used for: Gene expression analysis, gene cloning, cDNA library construction. β-mercaptoethanol: Helps denature proteins by breaking disulfide bonds between cysteine RNA is especially labile (unstable) for several residues and removes tannins and reasons.WHY? polyphenols during plant DNA extraction. 1. 2'-Hydroxyl Group: RNA has a Chloroform: Typically used in a 24:1 reactive 2'-OH group, making it prone (v/v) mixture with isoamyl alcohol, (YATKIN) to hydrolysis and strand which prevents foaming. cleavage. 2. Single-Stranded Structure ! Phenol/Chloroform/Isoamyl Alcohol 3. Susceptibility to RNases (hassas) Ratio: 25:24:1. RNases MUST be eliminated or inactivated ! DNA, remains in the water phase and BEFORE isolation. does not dissolve in phenol (a less polar solvent) due to its PO3- charged Lab Glassware: phosphate backbone. Wash with Diethylpyrocarbonate Steps: (DEPC): DEPC inactivates RNases by modifying histidine residues, ○ Cell Lysis preventing RNA degradation. ○ Protein Digestion: with Proteinase K. DEPC-treated water (RNase-free): ○ DNA Extraction: Phenol-chloroform ○ Prepare by treating water with ○ DNA Precipitation: Using ethanol and salt. 0.1% v/v DEPC for at least 2 ○ DNA Resuspension and Storage: In TE hours at 37°C. buffer, stored at 4°C or -20°C. ○ Autoclave the solution for at least 15 minutes to inactivate Concentrating DNA (Alcohol Precipitation): any remaining traces of DEPC. Isopropanol: Used instead of ethanol to precipitate DNA (1 volume of Total RNA Composition: isopropanol vs. 2 volumes of ethanol). 2. Ribosomal RNA (rRNA): Makes up Advantage: Requires a smaller volume to 80-90% of the total RNA in a sample. centrifuge. 3. Messenger RNA (mRNA): Accounts for 2.5-5% of total RNA, representing Disadvantage: Less volatile, harder to the transcribed genes that guide remove, and more easily co-precipitates salts protein synthesis. like sodium chloride with DNA. RNA Isolation Methods: Resuspension and Storage of DNA: Organic RNA Extraction: TE Buffer: Typically 10 mM Tris-HCl (pH 8.0) with 1 mM or 0.1 mM EDTA. 1. Lyse/homogenize cells. 2. Add phenol:chloroform Storage: !Prevent depurination, and EDTA alcohol:Mix with lysed sample and chelates Mg²⁺, inactivating DNases. centrifuge. Can be stored at 4°C for extended periods; for long-term storage, -20°C is preferred.  This separates the sample into two NOT: phases: Relative Positions of Different DNA Forms Organic phase (bottom) on a Tris-Acetate Agarose Gel contains solvents. Aqueous phase (top) contains total RNA. Debris layer (interface-ara faz) holds cellular debris and genomic DNA. 3. Transfer RNA to a clean tube. 4. Precipitate RNA: Use ethanol to precipitate RNA, wash, and resuspend in water. Affinity Purification of RNA: 1. Lyse cells and spin PS: The nicked DNA 2. Apply lysate to a glass membrane is the slowest due to column: the floppy, relaxed ○ Nucleic acids bind to the shape, allowing us to glass membrane. distinguish between ○ Cellular contaminants wash DNA forms. through the column. ○ DNase treatment is used to remove DNA contaminants. 3. Wash with alcohol: This helps remove remaining contaminants. 4. Elute RNA: Apply water to release DNA Migration on Agarose Gel with purified RNA from the membrane for Restriction Enzyme Treatment collection. Isolation of PolyA (Messenger) RNA: PolyA tail: Found in 6% of bacterial mRNA and 1.1% of mammalian mRNA. Oligo(dT) probes: Bind to the polyA tail and selectively purify mRNA. Elution: mRNA is released from the oligo(dT) matrix using water or low-salt buffer.  Quality of Isolated DNA: ÖZET: Simple method for quality check: 1. Prepare Acrylamide Gel: ○ Mix acrylamide solution (e.g., 6-8% for 1. Mix DNA with a loading buffer. DNA). 2. Load into agarose gel. ○ Add APS (ammonium persulfate) and 3. Analyze under UV light. TEMED to initiate polymerization. ○ Pour the solution into the casting tray, Quality Control of Nucleic Acids: insert comb, and let the gel solidify. 2. Prepare DNA Samples: Agarose Gel Electrophoresis is used to 3. Load Samples: analyze DNA and RNA quality. 4. Run Gel Electrophoresis: ○ Fill the tank with a running buffer. Staining methods: ○ Place the gel into the tank. ○ Run the gel at 80–120 V for 1-2 hours, Ethidium Bromide: toxic, mutagenic. depending on fragment size. SYBR Green I: less harmful, doesn't need 5. Stain the Gel: UV for visualization. ○ Soak the gel in ethidium bromide or SYBR Safe for 10–20 minutes. Matrix Materials ○ Rinse the gel with water. 6. Visualize DNA: 1. Agarose ○ Place the gel on a UV transilluminator. 2. Polyacrylamide 7. Quantify DNA: 3. Starch (potato) ○ Compare the intensity of your DNA bands 4. Agarose-polyacrylamide with the ladder bands. ○ Use quantification software if available to measure the DNA concentration. 8. USING DNA!: ○ You could cut out the band of DNA using a razor blade or scalpel. ○ Then you put the gel piece in a tube and run it through the gel extraction kit. NOT: Gel Electrophoresis Purpose Uses electromotive force to separate molecules The concentration of agarose determines the Separation based on size, charge, and size of molecules that can be separated. shape Lower concentration = larger molecules separation. Why Perform Electrophoresis on dsDNA? Agarose Gel Loading Dye Recipes: To separate fragments To determine fragment sizes and presence/amount of DNA  Factors Affecting DNA Mobility in Agarose TBE buffer: Gel Electrophoresis ○ Higher buffering capacity, making it more DNA size stable for extended electrophoresis. Agarose concentration ○ Slower DNA migration due to lower conductivity. ↑[agarose] → ↓pore size ○ Ideal for resolving smaller DNA fragments ( Tm Repeats (VNTR) PCR: Targets areas = 62°C, Annealing Temp = 57°C of the genome exhibiting length variation. PCR Types:: 7. Asymmetric PCR: Preferentially amplifies one strand of the target 1. Conventional (Qualitative) PCR: DNA. Routine PCR applications. 8. Nested PCR: Increases specificity 2. RT-PCR (Reverse Transcription of DNA amplification with two sets of PCR): Used to reverse-transcribe and primers in successive reactions. amplify RNA to cDNA. Using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity. Uses: Detection of pathogens that occur with very few amount.  9. Quantitative PCR(qPCR ) / Nomenclature used in qPCR :(Key Terms) Realtime : Measures the specific amount of target DNA (or RNA) in a Baseline: PCR cycles where fluorescent sample. signals are present but undetectable. ΔRn: The change in fluorescence per Enables reliable detection and measurement cycle, plotted against cycle number. of products during each cycle of the PCR Threshold: A set fluorescence level; process. signals above this are considered real. Ct (Threshold Cycle): The cycle number Uses fluorescent dyes (e.g., Sybr Green) or when fluorescence exceeds the threshold; fluorescent DNA probes (e.g., TaqMan) to it indicates the amount of target DNA. measure the amount of amplified product as the amplification progresses.Measures the Ct Value: Inversely proportional to target progress of DNA amplification in real-time by nucleic acid amount: detecting the release of fluorescent signals during amplification.A computer monitors the ➔ Lower Ct = more target nucleic acid rate of fluorescent "flashes" in experimental (below 29 cycles). PCR reactions compared to a control reaction. ➔ Higher Ct = less target nucleic acid (above 38 cycles). 10. Hot-start PCR: Performed by heating reaction components to DNA melting temperature before adding polymerase. 11. Touchdown PCR: Annealing temperature is gradually decreased in later cycles. 12. Assembly PCR (Polymerase Cycling Assembly, PCA): Synthesizes long DNA structures by Applications: assembling multiple DNA pieces. 13. Colony PCR: Screens bacterial Relative and absolute quantification of colonies directly by PCR, often for gene expression. screening DNA vector constructs. Validation of DNA microarray results. 14. Digital PCR: Amplifies thousands Variation analysis, including SNP of samples simultaneously, each in discovery and validation. separate droplets in an emulsion. Counting bacterial, viral, or fungal loads, 15. Suicide PCR: Ensures specificity among other uses. by using primers only once, often used in paleogenetics to avoid false positives. 16. COLD-PCR (Co-amplification at Lower Denaturation Temperature-PCR): Enriches variant alleles from a mixture of wildtype and mutation-containing DNA. Notes:  Ders NOTU 8: Mechanism and Types of Cuts: Restriction Endonucleases :Restriction Blunt Ends: Produced endonucleases (REs) are enzymes that cut by enzymes cutting both DNA at specific recognition sequences DNA strands at the same (restriction sites). These enzymes, found in location. Blunt-ended bacteria, act as molecular scissors. fragments can be joined to any other blunt-ended → precisely-located sites so that a small set DNA of homogeneous fragments are produced. Importance: For DNA sequencing, it’s essential to cut DNA into smaller, precise Sticky Ends: fragments. Random Overhanging cuts by other single-stranded ends enzymes aren’t created by some suitable for this enzymes. These "sticky" purpose, which is why ends can form base REs are critical. pairs with complementary sequences, making them highly useful in DNA cloning. Enzyme Activity: Recognition and Palindromes: Palindromes: These sequences enable REs to cut DNA symmetrically on both strands. Recognition Sites: Most REs recognize palindromic sequences in DNA. ! Why don’t bacteria destroy their own DNA with their restriction enzymes?, RE Categories: To protect their own DNA, bacteria add methyl Isoschizomers: groups to certain nucleotides within their own Enzymes that recognize DNA at the same recognition sites. and cut the same sequence. Types of REs: Neoschizomers: Recognize the same Type I: Recognizes specific sequence but cut at sequences but cuts at variable different sites. distances, less useful for precise Isocaudomers: manipulations. Recognize slightly different sequences Type II: Cuts directly at recognition but produce the same ends. sites, revolutionizing DNA manipulation and cloning techniques..  Reaction Conditions: Genetic Engineering Applications: The activity of REs depends on factors Recombinant DNA: Sticky ends allow like pH, temperature, salt DNA fragments to base pair, and DNA concentration, and ion presence. ligases permanently join them, Example: The enzyme XbaI is resulting in recombinant DNA incubated at 37°C (optimal) and later molecules. inactivated by heating at 65°C. Key Contributions: REs enabled the production of therapeutic proteins like NOT: An Enzymatic Unit (U) is defined as human insulin and factor VIII, the amount of enzyme required to digest 1 revolutionizing biotechnology. mg of DNA under optimal conditions. Techniques and Tools: Typical RE Reaction Traditional Cloning: Stanley Cohen 20 μl reaction için: and others used REs to move DNA fragments between organisms, leading 1. 10 μl DNA (~1 μg total) to the development of plasmid vectors 2. 7 μl water for cloning. 3. 2 μl 10X reaction buffer 4. 1 μl RE 10 U/μl (10x for complete digestion) Incubate 1 h at appropriate temperature. Double Digestion: A technique where 2 REs are used simultaneously to cut DNA at multiple sites, often performed in compatible buffers to avoid enzyme interference. Caution: some enzymes display *star activity* in certain buffers which causes them DNA Mapping: In the 1970s, Daniel to digest the DNA at sites other than the Nathans' restriction mapping of DNA standard recognition site. initiated the comparison of genomes, aiding the detection of SNPs and ★ If a DNA molecule contains several genetic diversity. recognition sites for a RE, it is possible that certain sites are cleaved but not others. 10. Advanced Applications: ★ These incompletely cleaved fragments of DNA are called partial digests (partials). CRISPR and Artificial Restriction ★ Partials can arise if Enzymes: REs, including an insufficient amount CRISPR-Cas9, now allow of enzyme is used or programmable cutting of DNA at the reaction is stopped desired sequences, facilitating gene after a short time. editing. This has significant ★ Reactions containing implications for gene therapy and partials may also synthetic biology. contain some molecules that have been completely cleaved. Restriction Enzyme (RE) Mapping: Compare the patterns between: 1. Isolate the DNA: The single-enzyme digests (which cut ○ Start by extracting the DNA the DNA at one site). that you want to map. The double-enzyme digests (which cut 2. Choose Restriction Enzymes: the DNA at two or more sites). ○ Select appropriate restriction enzymes (e.g., BamHI, PstI, HindIII) that recognize and cut 8. Construct a Map: specific DNA sequences. ○ Using the fragment sizes from 3. Digest the DNA: the gel, calculate where the ○ Set up reactions where the restriction enzymes cut on the DNA is cut with: DNA. Each enzyme individually ○ Match fragment sizes to (e.g., BamHI alone, HindIII create a restriction map, alone). showing the relative positions A combination of enzymes of enzyme cut sites along the (e.g., BamHI + HindIII DNA. together). 9. Confirm the Map: Leave one sample ○ Ensure the sum of the undigested as a control. fragment sizes matches the 4. Perform Gel Electrophoresis: total length of the DNA. ○ Load the digested DNA ○ Repeat the process with samples onto an agarose gel. additional enzymes if needed ○ Apply an electric field to to refine the map. separate DNA fragments based on size. The smaller Genetic engineering: the fragment, the farther it moves. DNA fragments can join through base pairing 5. Stain and Visualize the Gel: between sticky ends, and DNA ligase forms ○ Stain the gel using a covalent bonds to create recombinant DNA DNA-binding dye (like (rDNA). ethidium bromide) and visualize the bands under UV Recombinant DNA technology revolutionized light. Each band represents a genetics and biotechnology, enabling the DNA fragment of a specific production of therapeutic proteins like size. human insulin and factor VIII. 6. Determine Fragment Sizes: ○ Compare the bands on the gel Applications Utilizing Restriction Enzymes to a DNA ladder (molecular weight marker) to estimate Traditional Cloning: the size of each DNA fragment. Restriction enzymes, combined with DNA 7. Analyze Results: ligases, allow for "cut-and-paste" workflows, ○ For each digestion, identify moving DNA fragments between organisms. how many fragments were produced and their sizes. Stanley Cohen's work with plasmid vectors in E. coli led to the making of cloning vectors used for recombinant protein production. Restriction enzymes are used for post-cloning verification..  DNA Mapping: before your gene. Mark a terminator after the ○ Daniel Nathans pioneered gene to show where it stops. restriction mapping in the 1970s to study SV40 DNA. 5. Place any promoters or terminators. ○ Restriction mapping helps detect SNPs and Indels for If your plasmid genetic disorder identification, includes a genetic diversity studies, and promoter parental testing. (where gene Epigenetic Modifications expression In vitro DNA Assembly: starts), draw a ○ Techniques like BioBrick™, small arrow Golden Gate Assembly, and before your Gibson Assembly facilitate gene. Mark a synthetic biology and DNA terminator after library construction. the gene to Gene Editing Technologies: show where it ○ Zinc Finger Nucleases stops. (ZFNs), TALENs, and CRISPR-Cas9 are advanced 6. Label antibiotic resistance genes (if tools for precise gene editing. present). Artificial Restriction Enzymes: ○ Synthetic enzymes like Many plasmids contain genes that give cells CRISPR-Cas9 allow targeted resistance to antibiotics. Draw another arrow DNA cuts at desired for these and label them (e.g., “AmpR” for sequences for genome ampicillin resistance). editing. 7. Show the plasmid size. HOW TO DESIGN A PLASMID MAP? Somewhere around the circle, mention the 1. Decide on important features: total size of the plasmid (e.g., “5.4 kb”), which represents how long the DNA sequence. Origin of replication (ori),Genes of interest, Promoters and terminators: Restriction From electrophoresis: enzyme sites ETC Use the fragment sizes to determine the 2. Position the origin of replication distance between each site.For double (ori). digests, identify where two enzymes cut relative to one another.For example:If enzyme Mark a spot on the circle and label it “ori” (the B cuts within the 3,000 bp fragment and starting point for DNA replication). produces two fragments of 1,500 bp and 1,500 bp, place the enzyme B site halfway through 3. Add your gene of interest. the 3,000 bp fragment. Draw an arrow or box at the correct location on the circle where your gene is located. Label it with the gene name (e.g., “GFP” for green fluorescent protein). 4. Place any promoters or terminators. If your plasmid includes a promoter (where gene expression starts), draw a small arrow DNA Polymorphism Analysis DNA Sequence Polymorphism: ○ Single Nucleotide Human Genome Overview: Polymorphism (SNP): A single base difference between DNA The human genome consists of over 3 billion sequences. base pairs. Approximately 99.9% of the genome is identical between any two Short Tandem Repeat (STR) humans; the remaining 0.1% difference Polymorphisms: translates to about 3 million bases.Despite this high similarity, the genome is more complex STR are and dynamic than previously understood due repeats of to processes like replication, recombination, nucleotide and repair. sequences. Different DNA Polymorphisms: alleles contain different numbers of repeats. DNA polymorphisms are inheritable – TTCTTCTTCTTC - four repeat allele sequence differences present in at – TTCTTCTTCTTCTTC - five repeat least 1–2% of a population. STRs exist in non-coding regions of These can involve changes as small DNA and vary between individuals. as a single base or as large as These sequences can be amplified thousands of bases. and analyzed based on their length. Most polymorphisms are found in non-coding DNA and may not have Detection of STR Polymorphisms: immediate phenotypic effects. DNA polymorphisms are important STR polymorphisms can be detected markers for genetic analysis since through Southern blot analysis and they represent differences between PCR. individuals. Southern blot: STR alleles are If the location of a polymorphic analyzed by fragment size. sequence is known, it can serve as a landmark or marker for locating other genes or genetics regions. Each polymorphic marker has different versions or alleles. Allele: It is an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome.-different versions of same gene Major Types of DNA Polymorphisms: STR alleles can also be analyzed by amplicon (a segment of chromosomal DNA Length Polymorphism: DNA that undergoes amplification and -Short Tandem Repeat contains replicated genetic material.) (STR) Polymorphism: 2–9 size.( pcr) base pairs. -Variable Number Tandem Repeat (VNTR) Polymorphism: 10–100 base pairs. STR Polymorphisms detection by using A haplotype can refer to a combination multiplex PCR of alleles or to a set of single nucleotide polymorphisms (SNPs) Multiple loci are genotyped in the found on the same chromosome. same reaction using multiplex PCR. Amplification of multiple targets in a Chimerism Testing Using STR: single PCR experiment. More than one target sequence can be amplified STR analysis is crucial in monitoring by using multiple primer pairs in a allogeneic bone marrow transplants, reaction mixture. evaluating engraftment by analyzing Multiplex-PCR: It is a special type of the presence of donor chimerism. the PCR used for detection of multiple pathogens or targets by using Multiple primers sets each one targets a particular pathogen/target. Uses: This permits the simultaneous analysis of multiple targets in a single sample. Analysis: PCR products of STR genotypes can be analyzed using gel or capillary electrophoresis. - Birden fazla genotipe sahip hücrenin Capillary electrophoresis: Allows veya dokunun bir arada canlılıklarını precise sizing of DNA fragments using devam ettirebilmeleri durumu kimera standards, achieving single-base olarak tanımlanmaktadır. resolution. Allelic ladders are standards representing all alleles observed in a population. Notlar: Y-STR Polymorphisms: STRs on the Y chromosome are inherited as a block without recombination, representing paternal inheritance. In other words, STR on the Y chromosome are inherited paternally as a haplotype. A haplotype is a set of DNA variations, or polymorphisms, that tend to be inherited together. Ders Notu 10: Nucleic Acid, Blotting, Labeling and Detection: Labeling, and Detection Probe Design, Production, and Applications A probe is a single-stranded nucleic acid (DNA or RNA) with a strong affinity to a specific target sequence. The hybrid( target-probe) sequences must be complementary but not necessarily exactly. Used in various blotting and in situ techniques to detect nucleic acid sequences. General stages of the probe preparation, labeling and detection: Types of Label: Probe Types 1. Radioactive Labels: ○ Use isotopes (e.g., 32P, 35S) for detection. ○ Autoradiography or Geiger-Muller counters are used for detection. 2. Non-radioactive Labels: ○ Safer, more stable, efficient, 1. Gene Probes (200–600 bases): and allow in situ detection ○ Generated via cloning or (e.g., Biotin,Enzymes, PCR. Chemiluminescence, ○ Offer greater specificity due to Fluorescence chemicals, longer sequences. Digoxigenin DIG). 2. Oligonucleotide Probes (15–50 bases): Labeling Methods ○ Target specific sequences within genes. 1. Nick Translation: ○ Uses DNase I and E. coli DNA Shorter probes lack specificity; longer probes polymerase I to add take more time for hybridization. nucleotides at nicked DNA sites. Probe Design Considerations: GC content of 40–60% is recommended. Avoid sequences with complementary regions that form hairpin structures. Avoid sequences containing long stretches (more than four) of a single base The targeted sequence should be from unique regions.  ○ Nick translation reaction involves E. ○ During PCR amplification of coli DNA pol I adding nucleotides to the probe, DIG–dUTP is the 3'-OH created by DNase I's incorporated into the DNA nicking activity. strands, with a reduced ○ Simultaneously, the 5' to 3' amount of dTTP in the dNTP exonuclease activity of DNA mixture. polymerase I removes nucleotides ○ PCR–DIG labeling from the 5' side of the nick. incorporates more DIG ○ For radioactive labeling of DNA, an moieties along the DNA [α32P]dNTP is used as the strands compared to precursor nucleotide. random-primed DIG labeling. ○ For nonradioactive labeling, a 4. Photobiotin Labeling: digoxigenin or biotin moiety ○ Photobiotin labeling is a attached to a dNTP analog is used. chemical reaction, not enzymatic. ○ Biotin and DIG can be attached to a nitrophenyl 2. Random-Primed Labeling: azide group, which, when exposed to UV or strong Random-primed visible light, converts to a labeling of DNA highly reactive nitrene. fragments ○ Nitrene forms stable covalent (double- or bonds with DNA and RNA single-stranded) during photobiotin labeling. is an alternative ○ Materials used in photobiotin to nick translation labeling are more stable and for producing less expensive compared to uniformly labeled the enzymes required for nick probes. translation or oligonucleotide labeling 3. DIG-PCR Labeling: ○ Digoxigenin (DIG) is a steroid found in the flowers and leaves of Digitalis (foxglove) plants, forming glycosides when attached to sugars. ○ DIG is a hapten, a small molecule with high antigenicity, used in molecular biology like other haptens such as 2,4-Dinitrophenol, biotin, and fluorescein. ○ A hapten can bind to a specific antibody but lacks inherent antigenicity. Probe Formats 1.Solid Support Formats: Solid supports include nitrocellulose or nylon membranes, latex or magnetic beads, or microtiter plates.  Types of filters/membranes include: Applications of Probes in Medical ○ Nitrocellulose Research ○ Nylon ○ Positively charged nylon 1. Detection of pathogenic (Hybond) microorganisms. ○ PVDF (hydrophobic 2. Detection of nucleic acid sequence polyvinylidene difluoride) changes. Nitrocellulose membranes are 3. Detection of tandem repeat commonly used due to their low sequences. background signals. Nitrocellulose membranes can bind Nucleic Acid Hybridization large amounts of DNA but become brittle and release DNA during Binding of 2 strands (probe and hybridization. sample or template) known as Positively charged nylon HYBRIDIZATION.Hybridization is membranes are recommended for used to form double-stranded nucleic optimal signal-to-noise ratio when acid molecules. using the DIG system. Applications include Southern Activated cellulose membranes are blotting, DNA chips, FISH. more difficult to prepare but can be Factors affecting hybridization include reused, as DNA is irreversibly bound. ionic strength, base composition, Membranes vary in properties such as and mismatched base pairs. binding capacity, tensile strength, mode of nucleic acid attachment, and Nucleic Acid Hybridization Assays: lower size limits for nucleic acid retention. Labeled nucleic acid (probe) Target: A complex mixture of In Solution Format: unlabeled nucleic acid molecules. Key principle: Base complementarity Both probe and target are in solution, with a high degree of similarity maximizing movement and reaction between the probe and the target. chances, leading to faster results. Factors Affecting Tmof Nucleic Acid In Situ Format:(in original place) Hybrids: Probe solution is added to fixed Destabilizing agents: Examples tissues, sections, or smears, which are include formamide and urea. then examined under a microscope. Ionic strength: Affects hybrid stability. The probe label (e.g., fluorescent Base composition: G/C content and marker) reveals a visible change if the presence of repetitive DNA influence target sequence is present and Tm. hybridization occurs. Mismatched base pairs: Lower Sensitivity may be low if the target stability. nucleic acid amount is low. Duplex length: Longer sequences This method is used for gene result in a higher Tm. mapping on chromosomes and detecting microorganisms in Equations for Calculating Tm for Different specimens. Hybrids: DNA-DNA hybrids DNA-RNA hybrids RNA-RNA hybrids Oligonucleotide probes Components of a Typical Hybridization Solution: High salt solution: Either SSC (Saline-Sodium Citrate) or SSPE (Saline-Sodium Phosphate-EDTA). Blocking agent: Examples include Denhardt’s solution, salmon sperm DNA, or yeast tRNA. SDS: Sodium dodecyl sulfate used for denaturing proteins. BLOTTING Blotting is a technique used to identify and characterize macromolecules such as DNA, 2. Northern Blot (RNA detection): RNA, or proteins. ○ RNA separated, transferred, and hybridized to identify 1. Electrophoretic Separation gene expression. 2. Transfer & Immobilization 3. Probe Binding Procedure: 4. Visualization 1. RNA Separation: RNA (total or Blotting Techniques mRNA) is separated by gel electrophoresis. 1. Southern Blot (DNA detection): 2. Transfer: RNA is transferred to a ○ DNA fragments separated via nitrocellulose membrane. electrophoresis, transferred 3. Hybridization: Incubate with a to a membrane, and single-stranded DNA probe that pairs hybridized with labeled with complementary RNA sequences probes. to form a double-stranded RNA-DNA ○ Used for identifying DNA molecule. rearrangements and gene 4. Detection: The probe is either structures.(paralogs and radioactive or has an enzyme (e.g., orthologs),Construction of alkaline phosphatase or horseradish restriction maps peroxidase) for visualization. Procedure: DNA Separation: by gel electrophoresis. Transfer & Immobilization: Denatured DNA is transferred from the gel to a membrane (nylon or nitrocellulose) by placing the membrane on top of the gel and using capillary action with absorbent materials. DNA is then fixed to the membrane by heating or UV light. Hybridization: Add labeled probes in excess to the membrane, which hybridize with 3. Western Blot (Protein detection): complementary ○ Detects specific proteins from DNA sequences. a mixture via antibodies. Additional Blotting Techniques: 1. Dot and Slot Blot: ○ DNA/RNA spotted directly onto filters without prior electrophoresis. 2. In Situ Hybridization: ○ Detects target nucleic acid in Notlar: intact (non damaged) cells or tissues using radioactive or fluorescent probes. Fluorescent In Situ Hybridization (FISH) Uses fluorescent probes to bind to chromosome sequences. Fluorescence microscopy used for detecting probe binding. DNA Chips & Microarrays Analyze gene information using DNA probes placed on glass microspots. DNA chips are valuable for clinical diagnostics, particularly for diseases like cancer. Ders notu 11 Biochips and Microarrays (Technology II) Molecular Diagnostics: A diagnostic testing Biochips: Arrays of biomolecules method used to understand the molecular immobilized on a surface for molecular mechanisms of an individual's disease. biology use. Microarray: DNA microarrays allow Role in Personalized Medicine: for rapid gene sequencing and analysis. Early Detection: Helps in the early Spot sizes are typically smaller detection of diseases. than 20 mm in diameter. Selection of Appropriate Treatment: Used for analyzing gene Ensures the chosen treatments are safe expression changes in normal and effective. and diseased cells. Therapeutic Integration: Integrates It is comprised of DNA probes molecular diagnostics with treatment. formatted on a microscale Monitoring and Prognosis: Monitors (biochips) plus the instruments the therapy and helps in determining the needed to handle samples prognosis. (automated robotics), read the reporter molecules (scanners), Molecular Diagnostic Technologies and analyze the data (bioinformatic tools). Genetic Testing: Molecular diagnostics are vital for genetic testing and screening large populations. Key Technologies in Personalized Medicine: SNP Genotyping: Used for detecting single nucleotide polymorphisms, key markers in disease. Microarrays (DNA chips): Tools for rapid sequencing and gene analysis. DNA Sequencing (Technology I) Clinical Applications: DNA sequencing was initially used only for research purposes but has now become a GeneChip (Affymetrix): Widely used routine tool in molecular diagnostics. for DNA analysis. AmpliChip CYP450 (Roche): 1. cost lowered Analyzes gene variants important for 2. speed is increased drug metabolism. 3. can read millions of DNA sequences simultaneously Applications in Personalized Medicine: HIV Resistance HCV Genotyping Genetic Diseases Molecular Cytogenetics (Technology III) Omics Technologies Fluorescent In Situ Hybridization ○ Genomics: Study of genes and (FISH): A technique that detects their functions. chromosomal changes using ○ Proteomics: Study of proteins. fluorescent probes. ○ Metabonomics: Study of metabolic Advances: molecules. Probes: New, high-quality ○ Transcriptomics: Study of mRNA. probes allow detailed analysis. ○ Glycomics: Study of Cytogenetic Applications: carbohydrates in cells. Helps understand ○ Lipomics: Study of cellular lipids. disease-related chromosomal changes and their impact on SNP Genotyping (Technology IV) treatment. Robust, high-throughput, and cost-effective. Cytomics Techniques Used: Study of Molecular Phenotypes of Single Cells: Investigates multiple Sequencing: High specificity and biochemical properties of selectivity. heterogeneous cell systems. Restriction fragment length Techniques include: polymorphism, RFLP and TaqMan Assays: Flow Cytometry: Commonly used genotyping methods. Cell sorting based DNA Microarrays: Another frequent on fluorescence. tool for genotyping. Laser Scanning Clinical Applications: Cytometry: High-content SNPs relate to disease susceptibility , screening for drug responses,markers to segregate bioimaging. individuals with different levels of response to treatment in clinical. An alternative approach to SNP genotyping is haplotyping  Haplotyping: Refers to a set of DNA Applications in Point-of-Care Diagnostics: variants along a single chromosome (poc) that tend to be inherited together. They tend to be inherited together Potential for self-diagnostics and bedside because they are close to each other applications. on the chromosome, and recombinations between these Gene Expression Profiling (Technology VI) variants are rare. Importance: Knowledge of gene expression in healthy vs. diseased tissues helps identify proteins for normal functioning and the ones involved in diseases. Technologies: 1. DNA Microarrays: for gene expression. 2. Single-Cell Gene Expression: Provides insights into disease mechanisms at the cellular level. ! Single cell isolation methods include flow cytometry cell sorting and laser capture microdissection. ! Use of the nucleus as the substrate for parallel gene analysis can provide a platform for the fusion of genomics and cell biology and it is termed “cellular genomics.” This technique takes a snapshot of genes International HapMap Project: A public that are switched on in a single cell. catalog of genetic variants (SNPs) common in human populations. 3. Alternative Splicing: A gene is first transcribed into a pre-mRNA, which is a copy Purpose: of the genomic DNA containing intronic regions destined to be removed during Helps in large-scale genetic studies. pre-mRNA processing (RNA splicing), as Supports personalized medicine by linking well as exonic sequences that are retained genetic variants with disease and drug within the mature mRNA. response. Deregulation of Splicing: Leads to disease as a result of signaling Nanodiagnostics (Technology V) cascades or changes within spliceosome machinery. Nanobiotechnology: Focuses on developing Techniques: nanodevices for molecular diagnostics. ○ EXPRESSION PROFILING Improves sensitivity and precision in by microarrays(SPLICE diagnostics. JUNCTION MICROARRAYS, EXON ARRAYS, TILED Quantum Dots: Widely used in GENOMIC ARRAYS): nanotechnology-based diagnostics. They allow rapid, label-free, and highly specific detection Used to study the impact of RNA splicing on of DNA and protein interactions. gene expression and disease pathways. LAB NOTLARI: Steps: Pipetting with Micropipettes 1. Cutting the tissues in small pieces 2. Grind in a mortar and pestle with liquid Each micropipette has a specific range (e.g., nitrogen and CTAB: 0.2-10 µL, 10-100 µL). 3. Collect the homogeneous mixture in a tube 4. Incubate at 60°C for 30 minutes: 5. Centrifuge the sample&Collect the supernatant: 6. Add chilled ethanol 7. Centrifuge the tube & Collect the pellets and remove supernatant 8. Add 70% ethanol 9. Centrifuge the tube& To the pellet, add Solution Preparation TE buffer 50x TAE Buffer (pH 8.0): Plasmid DNA Isolation from E. coli ○ ITris-base, Glacial Acetic Acid, and EDTA. Alkaline Lysis Method: Lysis Buffer for Plasmid Extraction: ○ Glucose, Tris-HCl, and EDTA. Differential denaturation is used to separate Alkaline SDS: Prepared with NaOH chromosomal and plasmid DNA. and SDS. Potassium Acetate (5M): Potassium Steps: Acetate ,Glacial Acetic Acid,Dissolved in dH₂O. 1. Overnight Culture TE Buffer: Used for DNA dissolution, 2. Centrifuge composed of Tris-HCl and EDTA. 3. Resuspend Pellet: Resuspend the bacterial pellet in 100 µL lysis buffer CTAB Method for DNA Isolation from Plant and add 4 µL RNase 4. Alkaline Lysis Challenges in Plant DNA Isolation: 5. Neutralization: Add potassium ○ Differences in metabolites and acetate biomolecules make DNA 6. Centrifuge isolation from plants complex. 7. DNA Precipitation: Add ethanol ○ CTAB 8. Wash Pellet: Add ethanol, centrifuge (Cetyltrimethylammonium 9. Resuspend: Resuspend the DNA Bromide) is used to separate pellet in 40 µL TE buffer polysaccharides and polyphenols, which complicate the isolation process. Extraction Buffer Components: ○ CTAB: Disrupts membranes. ○ β-mercaptoethanol: Denatures proteins and removes polyphenols. ○ EDTA: Chelates magnesium ions. ○ NaCl: Aids in DNA precipitation. ○ Tris