Chapter 9. DNA Technology
Higher category: 【Biology】 Biology Index
2. DNA library
3. PCR
8. DNA-protein interaction studies
9. Protein-protein interaction studies
10. Gene editing
a. Nucleic Acid (DNA, RNA) Extraction and Purification Experiment
b. Western Blotting Experiment
c. The Romanov Dynasty and Forensic Science
1. DNA recombination
⑴ 1st. Target gene generation from mRNA (for eukaryotic DNA)
① mRNA is reverse transcribed to synthesize cDNA (complementary DNA to mRNA).
② RT-PCR: Add DNA primers to the mRNA solution and treat with reverse transcriptase (RT) once.
○ Oligo dT primer: Enables the synthesis of complete cDNA by using the poly A tail, which is present in eukaryotic mRNA, as a template.
○ Random primer (usually hexomer): Can form a variety of cDNA pools.
③ RT-PCR: Amplify the amount of cDNA like a normal PCR.
⑵ 2nd. Recombinant plasmid
① Restriction enzyme: A kind of endonuclease. A scissors that cuts DNA.
○ Restriction enzymes are produced in bacteria to target and degrade foreign DNA.
○ Their own DNA, which could be targeted, is protected by methylation.
○ Blunt ends: Ends that cannot rejoin after being cut by a restriction enzyme.
○ Sticky ends: Ends that can re-form complementary hydrogen bonds after being cut by a restriction enzyme.
○ Restriction enzymes that form sticky ends are used for DNA recombination.
○ Palindrome recognition: When reading from 5’ to 3’, the sequences on the sense strand and the antisense strand are identical.
○ The above example is a sticky end.
○ Examples of restriction enzymes
○ AgeI: 5’-A▼CCGGT-3’
○ AluI: 5’-AG▼CT-3’
○ BamHI: 5’-G▼GATCC-3’
○ BglII: 5’-A▼GATCT-3’
○ DpnI: 5’-G metA ▼TC-3’
○ DpnII: 5’-▼GATC-3’
○ EcoRI: 5’-G▼AATTC-3’
○ FspEI: 5’-CmC(N)12▼-3’
○ HindIII: 5’-A▼AGCTT-3’
○ HpaII: Cuts at 5’-C▼CGG-3’, but not at 5’-CmC▼GG-3’ where mC represents methylated cytosine.
○ LpnPI: 5’-CmCDG(N)10▼-3’
○ McrBC: 5’-PumC(N40-3000)PumC-3’
○ MspI: 5’-C▼CGG-3’, 5’-C▼mCGG-3’ where mC represents methylated cytosine.
○ MspJI: 5’-mCNNR(n)9▼-3’
○ MSTII: 5’-CCTN▼AGG-3’, where is a nucleotide sequence with n palindromic structures.
○ NcoI: 5’-C▼CATGG-3’
○ PstI: 5’-CTGCA▼G-3’
○ SalI: 5’-G▼TCGAC-3’
○ Sau3AI: 5’-▼GATC-3’
○ SmaI: 5’-GGG▼CCC-3’, forming blunt ends.
○ XbaI: 5’-T▼CTAGA-3’
○ Recombinant sequence by BamHI is also cleaved by Sau3AI. The opposite holds true only in specific cases.
○ The fragment by BglII and the fragment by BamHⅠ bind to each other and are not cut again by restriction enzymes.
○ When linear DNA is cut once by a restriction enzyme, it becomes two fragments, whereas circular DNA, when cut once by a restriction enzyme, becomes a single fragment.
○ Restriction mapping
② 2nd-1st. Cut the target DNA and the plasmid DNA with the same restriction enzyme to form the same sticky ends.
○ 2nd-1st-1st. To prevent self-ligation when treating with only one restriction enzyme, treat with alkaline phosphatase (e.g., Calf Intestinal Phosphatase (CIP)).
○ 2nd-1st-2nd. Provide directionality to plasmid DNA binding to target DNA by treating with two or more types of restriction enzymes.
③ 2nd-2nd. Insert the truncated gene and plasmid into one test tube → Complementary sticky ends bind to each other.
④ 2nd-3rd. Create a recombinant plasmid by ligating with DNA ligase.
⑶ 3rd. Transformation: Insert the recombinant gene into the host cell (bacteria) to induce transformation.
① Transformation process
○ 3rd-1st. CaCl2: Ca2+ binds to phospholipids and stabilizes cell membranes.
○ 3rd-2nd. Thermal Shock 42 ℃: A temporary pore is formed in the cell membrane, allowing the recombinant plasmid to be inserted into the host.
② Host: Escherichia coli, yeast, insect cells, etc.
○ To obtain the product of a recombinant gene, it must be put into a host cell with metabolic function.
○ Condition 1. First generation period should be short.
○ Condition 2. Easy to cultivate and grow well on cheap media.
○ Condition 3. Not pathogenic.
○ Condition 4. Antibiotic intolerance for screening process.
③ Vector: Plasmid or bacteriophage DNA that carries and replicates recombinant DNA.
④ Cloning vector: A vector used for gene replication.
○ Four conditions: Origin of replication (ori), promoter, restriction enzyme recognition sequence, selection marker (antibiotic resistance gene)
○ Additional condition: Small size, higy copy number
○ Cloning site
⑤ Expression vector: Vectors for expressing foreign genes in other hosts with different expression systems.
○ Prokaryotic host condition: Promoter, SD sequence, 70S ribosomal binding site, transcription terminator. Note that cloning gene is cDNA.
○ Proteins that process tertiary structures (e.g., disulfide bonds) are also required.
○ Eukaryotic host condition: Promoter, poly A sequence
○ Example: When a promoter is attached after the activator gene of the lac operon.
⑥ Vector examples
○ Example 1. pBR322 plasmid
○ 4361 bp. ori is present.
○ It has recognition sequences for restriction enzymes such as EcoRI, BamHI, and HindIII.
○ ampr: The ampicillin resistance gene from plasmid pBR322, recognized by PstI.
○ tetr: The tetracycline resistance gene from plasmid pBR322, recognized by HindIII, BamHI, and SalI.
○ Recombinant strain: Select a tetracycline-sensitive, ampicillin-resistant strain.
○ Example 2. pUC19 plasmid
○ 2686 bp. ori is present.
○ Has restriction enzyme recognition sequences such as ApaLI.
○ lacZ: Produces a blue-colored product using X-gal as a substrate.
○ ampr
○ Example 3. Ti plasmid
○ Agrobacterium tumefaciens (Rhizobium radiobacter): Pathogens of plant crown gall (benign tumor).
Figure 1. Rhizobium radiobacter attached to carrot cells
○ Ti plasmid: Rhizobium radiobacter plasmid, including T-DNA.
○ T-DNA: 20 kb of DNA encodes opines and auxin·cytokinin synthase (forming crown gall).
○ Opine: Nutrient sources of Rhizobium radiobacter.
○ Process: Agrobacterium tumefaciens infects the plant → vir (virulence DNA, 25 bp) cleaves both ends of T-DNA and randomly inserts it into the plant nuclear chromosome.
○ DNA recombination: Inserting target DNA into the T-DNA region after injuring the plant and infecting it with recombinant Rhizobium radiobacter.
○ The recombinant Agrobacterium tumefaciens does not synthesize auxin or cytokinin, preventing the formation of crown gall tumors.
○ Example 4. pBHA
○ Example 5. pBIC-A
○ Example 6. Gene library vector: BAC Library, YAC Library
○ Example 7. Bacterial plasmid: F plasmid, R plasmid (antibiotic resistance gene inserted). Approximately 1-10 kb.
○ Example 8. Virus vector
○ Genetic information is inserted into bacterial DNA through bacterial infection, proliferation, lysis, release and cleavage.
○ λ phage (about 25 kb) is mainly used.
○ Sometimes retrovirus is used.
○ Example 9. Cosmid: Introduces the phage cos end using the in vitro packaging method, approximately 50 kb in size.
○ Example 10. pOXA-48 plasmid
○ Synthesizes carbapenemase to give resistance to carbapenem, a beta-lactam antibiotic
○ Like this, Beta-lactamidase gene creates beta-lactam ring to confer penicillin resistance.
⑷ 4th. Screening: Screening for cells or populations with recombinant genes
① Antibiotic resistance gene (selection based on proper insertion of recombinant plasmid)
○ Example: Ampicillin resistance gene, tetracycline resistance gene
○ 1st. Replica formation: After incubating the entire colony, imprint the entire culture plate onto filter paper.
○ 2nd. Add antibiotics to a portion of the colonies imprinted on the filter paper.
○ 3rd. Colonies that die due to the antibiotic indicate that they were not transformed.
○ 4th. Select colonies corresponding to those that survived the antibiotic on the filter paper.
② Antibiotic resistance genes (selection according to recombination success)
○ 1st. Replica formation: After incubating the entire colony, imprint the entire culture plate onto filter paper.
○ 2nd. Add antibiotics to a portion of the colonies imprinted on the filter paper.
○ 3rd. Colonies that died due to the antibiotic indicate that the restriction enzyme cut the antibiotic resistance gene.
○ 4th. Select colonies corresponding to those that died due to the antibiotic on the filter paper.
③ X-gal (selection according to recombination success)
○ Cloning gene is inserted into lac Z gene.
○ 1st. X-gal is originally white, but becomes blue due to lac Z.
○ 2nd. If a colony treated with lacZ-bearing plasmid appears white on X-gal, it indicates successful transformation.
④ Reporter Gene
○ Use genes that are not expressed in the host cell.
○ Examples: eGFP, tdTomato.
⑤ Southern blotting
⑸ 5th. Cloning amplification: Large quantities of product are obtained by mass fermentation and purification.
① Reason for using antibiotics in large-scale culture with antibiotic-resistant plasmids (non-selective purpose)
○ 1st. Plasmids within the bacterial strain are randomly distributed to daughter cells during cell division.
○ 2nd. As division continues, some cells end up without any plasmids.
○ 3rd. Cells containing plasmids consume more energy to replicate them, leading to a difference in growth rate compared to plasmid-free cells.
○ 4th. Ultimately, plasmid-free cells become the majority.”
② Inclusion Body
○ Since the gene introduced during transformation is not originally present in the host, the produced protein may not fold properly and aggregates.
⑹ Examples
① Hormones and bioactive substances: Growth hormone, insulin, secretin, etc.
② Therapeutic and diagnostic reagents: Hepatitis B vaccine, diagnosis of HIV infection, interferon, neoendorphin, analgesic, blood coagulation factor, etc.
③ Agriculture: Golden rice, herbicide-tolerant crops, pest-tolerant crops
④ Ti plasmid transgenic neofunctional crop: Herbicide tolerance, anti-anthrax crops (seed potatoes, etc.)
⑤ Recombinant strain development strategy
○ Feedback / antibiotics resistant mutant
○ Auxotrophic mutant
○ Overexpression mutant
2. DNA library
⑴ Vector for DNA library
① Bacterial Artificial Chromosome (BAC)
○ Advantage: No cross occurs.
○ Disadvantage: Must be adapted to the prokaryotic expression system.
○ Used in human genome projects.
○ It is still used today.
② Yeast Artificial Chromosome (YAC; pYAC3)
○ Artificial chromosome into yeast (~1 Mb), contains ARS, and inserts DNA between the centromere and telomere.
○ Advantage: Suitable for producing human proteins as it utilizes a eukaryotic system.
○ Disadvantage: Crossover occurs during prophase I of meiosis.
③ Virus genome library
○ Disadvantage: Small capacity. Difficult to control breeding.
⑵ Required DNA elements
① Centromere: A GC-rich sequence that attaches to the spindle fibers during cell division, ensuring proper distribution of chromosomes to each daughter cell.
② Telomere: A nucleotide sequence located at the end of a chromosome that prevents chromosome shortening due to cell division.
③ Origin of replication: The site where DNA replication begins.
④ Selection marker: A specific DNA sequence used to confirm the proper insertion of artificial chromosomes into cells.
⑤ Others: Promoter, gene expression regulatory mechanisms.
⑶ Types
① gDNA Library
○ 1st. Precipitate histones by treating with phenol.
○ 2nd. Nucleic acids are present in the genomic DNA supernatant.
○ 3rd. Separate the supernatant → Trichloroacetic acid → Nucleic acid precipitate.
○ 4th. After digesting gDNA with a restriction enzyme, clone all fragments and insert them into a BAC vector.
○ 5th. Selectively amplify Escherichia coli containing the target DNA.
○ 6th. Used in intron research.
○ Triton X-100 is amphipathic and does not denature the DNA structure.
○ SDS-PAGE denatures the DNA structure.
② cDNA Library
○ 1st. Affinity chromatography: Attach oligo-dT (poly T) to beads.
○ 2nd. Reverse transcribe the separated mRNA using reverse transcriptase.
○ 3rd. Treat with a low concentration of RNase: Functions as a primer.
○ 4th. Treat with DNA polymerase I.
○ 5th. Treat with ligase.
○ 6th. Treat with restriction enzyme.
○ 7th. Insert into a vector.
○ 8th. Cloning amplification.
○ 9th. Used in the study of gene expression.
③ Library Screening
○ Transfer library clones onto a filter and activate with a probe to identify the locations of clones containing the same sequence as the probe.
3. PCR
⑴ Overview
① Definition: A DNA amplification technique based on the principles of DNA replication.
② Developed by Kerry Mullis in 1983; he was awarded the Nobel Prize in Chemistry in 1993.
⑵ Sample: DNA sample, DNA primer (2 types), dNTP, Taq polymerase, Buffer (pH stabilization), MgCl2 (DNA stabilization)
① DNA Primer
○ Condition 1: GC content ＞ 50%
○ Example: 10 out of 18 bases (= 18/2 + 1) are G or C
○ Example: 11 out of 20 bases (= 20/2 + 1) are G or C
○ Condition 2: The 5’ end should end with AT, and the 3’ end should end with GC
○ Reason: Prevents hairpin formation due to self-ligation
○ Example: 5’-ATGCCTATGCG-3’
○ Example: 5’-ATGCAGGCTAAT-3’ can cause self-ligation
○ Condition 3: The 3’ end should have 2 to 3 overlapping Gs or Cs
○ Reason: A=T bonds are double bonds, whereas G≡C bonds are triple bonds, making GC-rich sequences more stable
○ Example: 5’-ATGCCTATGCG-3’
② dNTP
○ Note that NTPs are the building blocks for RNA polymerization.
○ Reason why an E. coli colony can be used instead of a DNA template in colony PCR: Due to heat-induced denaturation.
○ In other words, the E. coli colony is broken down under denaturation, providing the necessary materials for DNA polymerization.
③ Taq polymerase
○ Taq polymerase is derived from the archaea Thermus aquaticus, which inhabits high-temperature hot springs.
○ It was first discovered in the 1960s in Yellowstone National Park.
○ Taq polymerase has an optimal polymerization temperature of 55°C and denatures at 92–95°C.
○ After completing polymerization to the 3’ end, Taq polymerase adds an extra A nucleotide.
○ Since it does not require a template, it can be applied to recombinant DNA with blunt-ended sequences.
○ Taq polymerase lacks 3’ → 5’ proofreading ability, resulting in an error rate of 1/104.
○ In contrast, typical DNA polymerases have a replication error rate of 1/107.
④ MgCl2
○ DNA has a negatively charged phosphate backbone, leading to structural instability → Na+ or Mg2+ can stabilize DNA.
⑤ IPP (Inorganic Pyrophosphatase)
○ Enhances the binding of NTPs.
⑶ Process
① 1st. The heating step to separate the two DNA strands. 94 ℃. 30-60 seconds.
② 2nd. Annealing: As the mixture cools, the primers bind to the DNA template. 50-55 ℃. 1 minute.
③ 3rd. DNA elongation: Polymerase starts synthesis at primer. 72 ℃. 1 minute per 1000 bp.
④ 4th. Repeating steps ① to ③ theoretically results in the exponential amplification of the DNA sample by a factor of 2.
⑤ 5th. DNA ligase action (40 ℃) and stabilization (4 ℃).
⑥ 6th. Switch to E. coli cloning amplification after a few cycles: In reality, the amount of artificial nucleotides is insufficient, producing much less DNA.
⑷ Tm vs annealing temperature
① DNA Melting Curve: A graphical representation of the degree of DNA denaturation as a function of temperature.
○ It shows the denaturation level of linear double-stranded DNA at different temperatures using absorbance at 260 nm (A260).
○ A260 value comparison: Double-stranded DNA ＜ Single-stranded DNA ＜ Fragmented nucleic acids
○ Reason: Less base stacking leads to lower shielding and higher absorbance.
② Tm (Melting Temperature): The temperature at which 50% of the DNA remains double-stranded, while the other 50% has denatured into single strands.
○ Generally proportional to (A + T) × 2 + (G + C) × 4.
○ Tm value is independent of repetitive sequences.
○ The Tm of dsRNA is about 15 ℃ higher than that of dsDNA with the same sequence due to the -OH group in RNA stabilizing dsRNA.
③ Annealing
○ The annealing temperature is typically set 5–10°C below Tm.
○ If the annealing temperature is too high, hybridization is inefficient; if too low, mis-hybridization increases.
○ The presence of metal ions affects the optimal annealing temperature.
⑸ Ct (Threshold Cycle)
① Definition: The cycle at which the signal exceeds the threshold value.
② Ct and copy number have an inverse correlation.
⑹ Limitation: The nucleotide sequences at both ends of the template DNA must be known.
① Assumption: If there is a known sequence in the middle and the template DNA has blunt ends, this limitation can be overcome.
② Solution 1. Inverse-PCR
○ 1st. Form circular DNA from the template DNA using DNA ligase.
○ 2nd. Generate linear DNA by cutting within the known sequence using a restriction enzyme.
○ 3rd. PCR is possible because we know sequence of both ends.
③ Solution 2. Anchored-PCR
○ 1st. Attach a new primer to the template DNA using DNA ligase and amplify one half.
○ 2nd. The other half goes through the same process.
④ Solution 3. Oligonucleotide-directed mutagenesis
○ Case 1: Replicating the template DNA using a primer with only a few mismatched bases.
○ Case 2: Replicating the template DNA using a primer with an added nucleotide sequence that induces a hairpin structure in the middle.
○ Case 3: Replicating the template DNA using a primer with a removed nucleotide sequence to allow the template DNA to form a hairpin structure.
⑺ Types
① RT-PCR: Includes the process of synthesizing cDNA from RNA using reverse transcriptase.
② Real-time PCR (qPCR, quantitative PCR): Allows real-time monitoring of amplified DNA quantity.
○ Initially, the given nucleotide fragment does not fluoresce because the distance between the fluorescent reporter and quencher is too short.
○ During polymerization in PCR, the fluorescent reporter and quencher separate, allowing fluorescence to appear.
○ TaqMan qPCR: A TaqMan probe (containing a fluorophore and quencher) binds to the target DNA and is cleaved during DNA synthesis, generating fluorescence.
③ Multiplex PCR: Amplifies multiple types of DNA samples within a single PCR reaction.
④ Allele-Specific PCR (AS-PCR): A method for detecting allele-specific variations using PCR.
4. DNA fingerprinting
⑴ Overview
① Objective: Paternity confirmation, prediction of genetic diseases, forensic perpetrator identification.
② Using the fact that no two individuals, except for identical twins, are genetically identical.
⑵ Electrophoresis: Allows for the separation of substances by size and the estimation of their quantity.
① Stationary phase, mobile phase
○ Stationary phase (compression gel, stacking gel)
○ Higher gel density results in slower transport of DNA samples or proteins.
○ Reason for high gel density: To keep the moving speed constant regardless of DNA sample or protein size.
○ Mobile phase (running gel)
○ Low gel density allows rapid movement of DNA samples or proteins.
○ Gel density is 6-15%.
② Nucleic acid electrophoresis: Agarose gel
③ Protein electrophoresis: Polyacrylamide gel + Bisacrylamide gel, SDS-PAGE, TEMED
○ Material 1. Acrylamide: Forms polymers in the gel.
○ Material 2. RIPA Buffer: Isotonic solution. Used as a buffer for cell lysis.
○ Material 3. Loading Buffer or Laemmli Sample Buffer: A buffer that breaks disulfide bonds.
○ Does not contain methanol but includes SDS-PAGE.
○ SDS-PAGE: Used for more efficient separation. Applied in denaturing gels.
○ SDS-PAGE consists of SDS (an anionic surfactant) and β-ME (β-mercaptoethanol).
○ SDS: Removes ionic bonds and hydrophobic interactions. Equalizes the charge density per unit mass of proteins.
○ β-ME: Eliminates disulfide bonds.
○ When SDS-PAGE binds to each amino acid, the repulsion between negative charges results in all proteins having the same charge density, forming a secondary structure.
○ DTT: An additive that breaks disulfide bonds between cysteine residues.
○ Material 4. Transfer Buffer: Used for transferring from gel to membrane.
○ Contains methanol but does not include SDS-PAGE.
○ Native-PAGE: Used in non-denaturing gels.
○ Methanol: Functions to cool the heat generated during the transfer process.
○ Material 5. Ammonium Persulfate (APS): Acts as an enzyme that forms cross-links between polyacrylamide and bis-acrylamide gels.
○ Material 6. TEMED (N,N,N’,N’-tetramethylethylenediamine): Stabilizes APS and helps the gel solidify quickly.
④ Typically the top is the anode, the bottom is the cathode.
⑤ Moving distance = Δt × (a × log M + b), a < 0, M: molecular weight
○ When treated with special substances such as SDS, molecular weight is linearly proportional to size, resulting in greater resistance in the mobile phase and a shorter migration distance.
○ Molecular weight has the greatest influence on migration distance.
○ Migration distance is proportional to time (Δt).
○ DNA ladder (size marker): Provides migration distances based on size, allowing estimation of the size of unknown substances.
○ Supercoiled DNA experiences less resistance in electrophoresis.
⑥ Type 1. General electrophoresis
○ Nucleic acids and proteins with smaller molecular weights migrate further.
○ **In protein electrophoresis, SDS-PAGE causes proteins to migrate from the (-) electrode to the (+) electrode.
⑦ Type 2. Isoelectric electrophoresis: Electrophoresis using isoelectric points, which are unique values of proteins.
○ Electrophoresis with a pH gradient moves proteins to the point where the total charge is zero (isoelectric point).
○ After electrophoresis, proteins are stained using protein dyes such as Coomassie Blue or silver nitrate.
Figure 2. Isoelectric electrophoresis
⑧ Type 3. 2D electrophoresis
○ O’Farrel law is common.
○ X-axis: Separation based on charge through isoelectric electrophoresis in the presence of urea or electrophoresis under non-denaturing conditions.
○ In other words, separation occurs according to the isoelectric point.
○ Urea: Removes all R-R interactions except disulfide bonds.
○ Y-axis: Separation based on molecular weight through electrophoresis in the presence of SDS.
○ More than 1,000 types of proteins can be separated.
○ Application 1. Separate both the X-axis and Y-axis using the same method, but add a specific substance during Y-axis separation → Substances deviating from the diagonal indicate interaction with the added substance.
○ Application 2. For tRNA and small RNA molecules: Use 10% polyacrylamide gel for 1D and 20% for 2D → Effectively separates various types of RNA.
⑶ Fingerprinting Method 1. RFLP (restriction fragment length polymorphism): Paternity testing, genetic disease analysis
① Process: Target genes.
○ 1st. DNA isolation from tissue.
○ 2nd. PCR: Amplify the amount of DNA.
○ 3rd. Cutting DNA into fragments using restriction enzymes.
○ 4th. Generate DNA fragments of different sizes with the same restriction enzyme recognition sites.
○ 5th. Electrophoresis: Separates fragments based on size differences for visualization.
② Cause: Single nucleotide polymorphism (SNP)
○ Defition: Difference between people due to one base being different by point mutation.
○ In humans, approximately one SNP appears per 1,000 base pairs (bp).
○ SNPs can occur in both exons and introns.
○ If an SNP occurs in a restriction enzyme recognition site, RFLP may occur.
○ Difference between mutation and polymorphism: If it occurs in less than 1% of the entire population, it is considered a mutation; otherwise, it is classified as polymorphism.
○ Number of SNPs: As of 2017, there are approximately 10,000 SNPs.
Figure 3. Number of SNPs
③ Limitation: Since SNPs do not necessarily occur at restriction enzyme recognition sites, RFLP variations among individuals are not significant.
○ Difficult to use for criminal identification and similar applications.
⑷ Fingerprinting Method 2. Tandem repeat: Used for suspect identification. A more powerful genetic fingerprinting method than RFLP, targeting repeat sequences.
① VNTR (variable number tandem repeat): 15-100 repetitions
○ Principle: The number of repeats in repeat sequences varies among individuals, resulting in fragments of different sizes.
○ Cause: Uneven crossover of homologous chromosome
○ 1st. DNA fragments are separated and amplified based on length using electrophoresis.
○ 2nd. Separated DNA fragments are transferred to filter paper.
○ 3rd. After transferring to filter paper, a chemical treatment (alkaline) is applied to break hydrogen bonds → Results in single-stranded DNA.
○ 4th. Compare the location of the VNTR fragments with a VNTR probe labeled with radioactive material.
○ 5th. Band appears when exposing filter paper to X-ray film.
② STR (Short Tandem Repeat): Repeats of 2-4 times.
○ Not significantly different from VNTR.
⑸ Fingerprinting Method 3. Other Fingerprinting Methods
① CNV (Copy Number Variation)
② LOH (Loss of Heterozygosity)
③ Genomic Rearrangement
④ Rare Variant
5. Hybridization
⑴ Southern blotting: Nucleic acid probe hybridization to DNA.
① Overview
○ A kind of RFLP.
○ Developed by Edward M. Southern in 1975.
② 1st. Restriction enzyme treatment → PCR amplification → Electrophoresis
○ Electrophoresis: Agarose gel is used.
○ A master plate can be used instead of electrophoresis: Colonies with recombinant plasmids are arranged on a solid medium.
③ 2nd. The gel is shaken in a 0.5 M HCl solution.
○ HCl treatment accelerates the transfer of DNA from the agarose gel to the membrane.
④ 3rd. The gel is shaken in a 1.5 M NaCl, 0.5 M NaOH solution (DNA denaturation).
○ The NaOH solution, sponge, and gel are stacked in order to separate DNA into single strands.
○ The sponge absorbs the NaOH solution and supplies it to the gel.
○ To prevent partial reformation of double-stranded DNA, an additional nucleic acid denaturant is added.
○ Nucleic acid denaturants: Metal ions, formamide, formaldehyde, etc. The form series weakens DNA binding strength.
⑤ 4th. The gel is shaken in a 1.5 M NaCl, 1 mM EDTA, 0.5 M Tris-HCl (pH 7.2) solution.
○ Tris-HCl: Buffer solution.
⑥ 5th. Capillary phenomenon + blotting
○ 5th-1st. Place a positively charged nylon filter and a paper towel on the gel.
○ Nitrocellulose paper can be used instead of nylon filter.
○ 5th-2nd. The NaOH solution that moves into the gel carries single-stranded DNA and travels to the paper towel stack, where it is absorbed.
○ 5th-3rd. Negatively charged single-stranded DNA sticks to nylon membrane.
⑦ 6th. Autoclave: Nylon membrane combined with DNA is treated under high pressure and high temperature to fix the bond.
○ DNA can also bind to the membrane when exposed to ultraviolet (UV) light.
○ In general, sterilization at 121°C for 15 minutes kills most microorganisms.
⑧ 7th. Blocking: A blocking agent is added to enhance clarity.
○ 7th-1st. Identify DNA that is considered insignificant through repeated experiments or electrophoresis for each allele.
○ 7th-2nd. Add salmon sperm DNA fragments to the DNA.
○ Purpose: To prevent probe DNA from non-specifically binding to the membrane during the 8th hybridization process (enhancing clarity).
○ Note: The membrane strongly interacts with DNA or proteins, requiring a blocking process.
⑨ 8th. Hybridization
○ 8th-1st. The nylon membrane with bound DNA is placed in a Seal-a-Meal bag.
○ 8th-2nd. At 62 °C, a radioactively labeled DNA probe is added to the Seal-a-Meal bag.
○ The labeled probe has a complementary sequence to the gene of interest.
○ Probe DNA: 100–500 bp, single-stranded DNA.
○ Probe DNA is typically labeled with the radioactive isotope P32.
○ Probe DNA can also be labeled with dyes such as EtBr or SYBR Green.
○ 8th-3rd. Probes bind to complementary DNA.
○ Through repeated experiments, other DNA considered insignificant is blocked and covered with salmon sperm DNA.
⑩ 9th. Eliminate unhybridized radioactive probes in seal-a-meal bags.
⑪ 10th. Autoradiogram: Irradiation on nylon membrane.
⑫ 11th. Identify the radioactive positions on the electrophoresis gel (or colony positions).
⑬ Housekeeping Gene
○ A gene that is present in all cells and continuously expressed.
○ Used as a control in Southern blotting.
○ Examples: β-actin (ACTB), tubulin, GAPDH, B2M, RPL11, RPL13A, 18S rRNA (most reliable).
⑭ Stringency
○ Conditions that lower stringency: Create an environment where probe binding is easier.
○ Low temperature or high salt concentration facilitates nucleic acid hybridization, leading to lower stringency.
⑵ Northern blotting: Nucleic acid probe hybridization for RNA.
① Very similar to Southern blotting but with the following differences.
② Difference 1. Extraction of samples
○ Southern blotting uses DNA extraction.
○ Northern blotting effectively isolates mRNA from extracts by affinity chromatography using oligo dT.
③ Difference 2. Use of restriction enzyme
○ DNA dealt by Southern blotting is very long and must be used with restriction enzymes.
○ Northern blotting uses RNA, not DNA, so restriction enzymes cannot be used.
④ Difference 3. Type of solution in capillary action
○ Southern blotting uses basic solutions to denature single-stranded DNA.
○ In Northern blotting, RNA is degraded when exposed to an alkaline solution.
○ Northern blotting uses a salt solution to stabilize RNA.
④ Difference 4. Types of probe
○ Southern blotting uses gDNA probe.
○ Northern blotting uses cDNA probe.
⑶ Western blotting: Antibody probe hybridization for proteins.
① Numerous differences with nucleic acid blotting.
② Difference 1. Type of gel
○ Nucleic acid blotting uses agarose gel.
○ Western blotting uses polyacrylamide gel, SDS-PAGE, etc.
③ Difference 2. Electrophoresis voltage condition
○ In nucleic acid blotting, nucleic acid transfers well to electrophoresis, so a low voltage must be applied.
○ In Western blotting, proteins are not well transported by electrophoresis, so high voltage must be applied.
④ Difference 3. Blotting Process
○ Nucleic acid blotting uses capillary phenomenon during blotting.
○ Western blotting uses an electric field during blotting.
⑤ Difference 4. Nucleic acid pretreatment
○ Nucleic acid blotting uses salmon sperm DNA fragments.
○ Western blotting uses casein, skim milk, bovine serum albumin (BSA), etc.
○ Tween: Surfactants such as Tween-20 are used.
○ A brand name developed by the American company Uniqema.
○ A detergent composed of a hydrophilic group (polyethylene glycol) and a hydrophobic hydrocarbon group.
○ Uses: Helps mix water and oil, disrupts cell membranes.
○ The numbers 20, 40, 60, 80 indicate increasing carbon chain length, making the detergent more hydrophobic as the value increases.
○ BSA can also be used for nucleic acid blotting.
⑥ Difference 5. Types of probe
○ Nucleic acid blotting uses DNA probes.
○ Western blotting uses antibody probe.
○ Primary antibody: An antibody that specifically binds to the target protein.
○ To ensure the specificity of the primary antibody, an enzyme extracted from a different species than the target animal must be used.
○ Secondary antibody: An antibody that specifically binds to the primary antibody. It is typically conjugated with an enzyme that breaks down a chromogenic substrate.
○ To ensure the specificity of the secondary antibody, an enzyme extracted from a species different from both the target animal and the primary antibody host must be used.
○ In Western blotting, the primary and secondary antibodies originate from different species.
⑦ Difference 6. Imaging Method
○ Nucleic acid blotting uses EtBr + self-radiation.
○ Western blotting uses the Coomassie Blue staining method.
⑷ DNA chip (microarray)
① Characteristics
○ Simultaneously detects the expression patterns of multiple genes.
○ Each spot contains a single cDNA probe.
○ Allows identification of tissue-specific gene expression using cDNA DNA chips.
○ Microarray data consists of continuous raw data, whereas RNA-seq raw data is count-based.
○ RNA-seq is more robust in detecting genes with extremely low or high expression levels compared to microarray data.
○ Becoming an obsolete technology due to advancements in NGS (Next-Generation Sequencing).
② Process
○ 1st. A cDNA library of human genes is spotted onto a glass slide to create a cDNA chip.
○ 2nd. The cDNA chip is treated with a 1% BSA solution.
○ BSA prevents nonspecific binding between the chip and DNA, allowing only complementary DNA to bind.
○ 3rd. mRNA X (from normal tissue) and mRNA Y (from abnormal tissue, e.g., cancer) are extracted separately.
○ 4th</sup. Oligo-dT is added to X and Y, binding complementarily to poly-A sequences in mRNA.
○ 5th. RT-PCR (Reverse Transcription PCR)
○ X: dNTP + Cy3 (green fluorescent dye)-dTTP.
○ Y: dNTP + Cy5 (red fluorescent dye)-dTTP.
○ Reverse transcription reaction is performed to generate fluorescently labeled cDNA.
○ 6th. 0.1 N NaOH is added to X and Y, incubated at 70°C for 10 minutes, then neutralized with 0.1 N HCl.
○ Template RNA is degraded due to basic condition.
○ 7th. Synthesized cDNA from X and Y is purified and mixed in equal amounts, then hybridized onto the cDNA chip.
○ 8th. The cDNA chip is washed with buffer solution to remove unbound sequences.
○ 9th. Fluorescence intensity of Cy3 and Cy5 is measured and corrected.
○ Black: Neither X nor Y is expressed.
○ Green: Only X is expressed.
○ Red: Only Y is expressed.
○ Yellow: Both X and Y are expressed.
⑸ ISH (in situ hybridization)
① Overview
○ An experimental technique that hybridizes a specific DNA sequence with a chromosome.
○ Used not only to determine the location of specific nucleotide sequences but also to identify the location of RNA.
○ Rapidly diagnoses chromosomal abnormalities but cannot diagnose DNA mutations.
② Type 1. Fluorescence in situ hybridization (FISH)
○ Hybridize a specific nucleotide sequence with a fluorescent probe complementary to the nucleotide sequence, then observe using a fluorescence microscope.
○ 1st. Prepare epithelial tissue sections on slides.
○ 2nd. Treat with RNase, incubate at 37°C for 1 hour, then wash.
○ Probes that bind to DNA may bind to RNA, so there is a process to remove RNA.
○ 3rd. Remove the culture medium, add a 2% formaldehyde solution, and incubate for 15 minutes.
○ This step fixes the cells.
○ 4th. Remove the formaldehyde solution, add a 0.2% Triton X-100 solution, and incubate for 5 minutes.
○ Triton X-100 is a surfactant.
○ 5th. Remove the Triton X-100 solution, add 2 M HCl, then treat with pepsin and incubate at 37°C for 10 minutes.
○ Pepsin functions in acidic conditions, so the pH is adjusted accordingly.
○ Pepsin degrades intracellular proteins, facilitating probe penetration in subsequent steps.
○ 6th. Wash the sample.
○ 7th. Prepare a probe that binds to the centromere.
○ This probe contains biotin-labeled dTTP.
○ 8th. Hybridize the sample from step 4 with the probe from step 5.
○ 9th. Wash the sample.
○ 10th. After sufficiently treating with fluorescently labeled avidin, wash the sample.
○ 11th. Stain with DAPI.
○ DAPI (4’-6-diamidino-2-phenylindole) is a nuclear stain.
○ DAPI strongly binds to AT-rich regions of DNA minor groove.
○ It is also used as an apoptosis marker.
○ Since DAPI is cell-membrane-permeable, it can be used to stain both live and fixed cells.
○ When UV light is applied to DNA bound with DAPI, it emits blue fluorescence.
○ 12th. The centromere region will display avidin fluorescence, while DNA will appear as blue fluorescence.
○ Application 1. RNA ISH (In Situ Hybridization)
○ rRNA molecules are observed in situ rather than being isolated from individual cells.
○ A labeled riboprobe (complementary RNA sequence) hybridizes with the transcript.
○ Visualization is achieved through a probe that binds to the complementary transcript.
○ Application 2. WM ISH (Whole-Mount ISH) (1989)
○ Requires thousands of animals to create an atlas, making it less commonly used after 2010.
③ Type 2. Radioactive ISH: The first ISH method, developed in 1969.
⑹ Sequencing technology: Determines DNA nucleotide sequences.
⑺ Gene-trap screen
6. Gene deletion
⑴ Knock-out mouse: Study the function of specific genes.
Figure 4. Knockout process
① 1st. Knockout induction of gene X in embryonic stem (ES) cells
○ 1st-1st. Construction of a plasmid vector (targeting vector): Includes gene X which is inactivated by the insertion of neor (neomycin-resistant gene), along with a TK (thymidine kinase) gene located at a distance from the gene X.
Figure 5. The structure of plasmid vector
○ 1st-2nd. Insert targeting vector inside ES cell.
○ 1st-3rd. Some ES cells autonomously replace the existing gene X with the inactivated gene X.
○ To enable genetic recombination, the same gene must be included next to gene X.
○ 1st-4th. Selection: Cells are cultured in medium containing G418 (geneticin, neomycin derivative) and gancyclovir.
○ 1st-4th-1st. Untransformed ES Cells: Killed by G418.
○ 1st-4th-2nd. ES cells in which regions other than gene X have been replaced: These cells contain the TK gene, making them susceptible to ganciclovir-induced cell death (TK gene metabolizes ganciclovir into a toxic compound).
○ 1st-4th-3rd. Only ES cells in which only gene X has been replaced survive.
② 2nd. Selection of knockout ES cells.
③ 3rd. Inject knockout ES cells into the embryo.
④ 4th. The germ cells of the chimera mouse consist of both normal germ cells and germ cells derived from knockout ES cells.
⑤ 5th. F1 mice include both (+/+) individuals and (+/-) individuals.
○ Reason: Because the chimera mouse can be considered a heterozygous mutant.
⑥ 6th. Select (+/-) individuals among the F1 mice through genetic testing and perform self-crossing.
○ There are limitations in selection based on mouse fur color.
⑦ 7th. Among the F2 mice, 25% are knockout mice (homozygous mutants) and must be identified through genetic testing.
⑵ Cre-Lox
① The Cre gene encodes recombinase.
② Cre-mediated DNA recombination occurs only between identical lox sequences.
③ If the paired lox sequences are in the same orientation, DNA deletion occurs by Cre; if they are in opposite orientations, DNA inversion occurs by Cre.
⑶ miRNA, siRNA: Gene expression can be suppressed for all genes using RNA interference (RNAi).
7. Nuclear substitution
⑴ GMO (Genetically Modified Organism): Genetically altered substances, food, organisms.
⑵ Methods
① Nuclear Transplantation
○ Utilizes techniques such as microinjection.
○ Applicable to trait transformation and animal cloning.
② Trait transformation without using a vector
○ Can induce trait transformation using gene guns, etc.
○ Example 1. Formation of recombinant plant cells: Particles coated with recombinant genes are “shot” into plant cells.
○ Example 2. Recombinant plant cells → Callus → Whole plant formation (in nutrient medium).
○ Plant cells exhibit totipotency.
○ Example 3. Maintenance and propagation of purebred
○ Example 4. Proliferation of useful plants
○ Example 5. Tissue culture with meristematic regions
○ Example 6. Induction of differentiation
⑶ Examples
① Example 1. Recombinant animals: Mass production of beneficial gene products in animals.
② Example 2. Edible vaccines
③ Example 3. Genetically Modified Food (GM Food)
○ Past: Gene-modified crops obtained by increasing the frequency of specific alleles through selective breeding (artificial selection).
○ Present: Genetic recombination technology → Increased shelf life, production rate (resistance to pests, weeds, diseases, drought, and cold).
○ Example 3-1. Golden rice: Genetically modified to produce beta-carotene (increases nutritional value of rice).
⑷ GMO Debate and Principles of Safety Assessment
8. DNA-protein interaction studies
⑴ Gene footprinting assay (DNA footprinting technology)
Figure 6. Application of gene footprinting
① Genes binding with proteins appear to be missing on the gel electrophoresis.
② It allows the identification of transcription factor binding sites.
⑵ Electrophoretic mobility shift assay (EMSA; gel shift assay, GMSA)
① Confirms the binding between the transcription factor and the promoter.
② 1st. Probe production: Labels DNA with radioactive isotopes.
③ 2nd. Electrophorese the protein-DNA hybrid, followed by radiation sensitivity analysis.
④ 3rd. Result analysis
Figure 7. Example of EMSA results
○ Premise: Since the top is the cathode and the bottom is the anode, DNA will move from top to bottom. (-) refers to the case where only the probe is moved.
○ Interpretation of a: Protein A and Protein B bind to the probe, and A and B bind together with the probe (they could also bind exclusively with each other…).
○ Interpretation of b: Protein C and Protein D bind to the probe, and C and D do not bind to each other.
○ Interpretation of c: Protein E binds to the probe, while protein F does not bind to the probe but binds to protein E.
⑤ Generally, protein means transcription factor and it can be applied to measure transcription activity.
⑶ ChIP (Chromatin Immunoprecipitation)
⑷ South-Western Blotting
Figure 8. South-Western Blotting
① 1st step: Proteins are initially subjected to Western blotting.
② 2nd step: Subsequently, DNA labeled with fluorescence is subjected to Southern blotting.
⑸ Yeast One-Hybrid Assay
⑹ Filter Binding Assay: Only transcription factors bind to the filter.
⑺ DNA Affinity Chromatography
9. Protein-protein interaction studies
⑴ Dual Hybrid System: A method for identifying proteins that interact with protein X.
① 1st. Transcription factor gene is divided into two parts: DNA binding domain and transcription activation domain.
② 2nd. Bait: Protein X + DNA binding domain.
③ 3rd. Prey: Protein of interest for determining binding + transcription activation domain.
④ 4th. When the two hybrid proteins interact and bind, the prey induces the expression of a reporter gene.
○ Example of a reporter gene: GFP protein.
⑵ Yeast Two-Hybrid: Protein-protein interactions are used to identify protein binding relationships.
⑶ Protein separation and purification using GST-tagged fusion proteins.
⑷ Phage Display Method: Used to obtain monoclonal antibodies.
① Objective: To obtain a monoclonal antibody that binds to a specific protein.
② Method for obtaining monoclonal antibodies in vitro.
③ Conventional monoclonal antibodies: B lymphocytes are fused with myeloma cells (hybridoma), becoming stem cells that proliferate and produce a large amount of antibodies.
④ 1st step. By introducing mutagens and altering the base sequence, a vast phage library is created.
⑤ 2nd step. Screen for phages with desired activity and later confirm the nucleotide sequence.
10. Gene editing
⑴ CRISPR-Cas9 Gene Editing Technology
① Overview
○ Gene Scissors: A mechanism of gene cleavage in bacteria (e.g., E. coli) to eliminate viral DNA.
○ CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
○ Derived from DNA fragments of bacteriophages that infected prokaryotic cells.
○ Found in 50% of bacterial genomes and 90% of archaeal genomes.
○ CRISPR arrays consist of repetitive DNA sequences and spacers located between each repetition.
○ Bacteria or archaea record spacers, which are fragments of external DNA such as viruses, and add repeat sequences.
○ Subsequently, the CRISPR-Cas9 system detects and cleaves viral DNA that matches the spacer.
○ Cas9 (CRISPR-associated protein 9)
○ Acquired from organisms like Streptococcus pyogenes. It has two nuclease domains, HNH and RuVC9.
○ dCas9 (nuclease-deficient Cas9, dead Cas9): Unable to cut DNA but capable of binding to DNA through sgRNA.
② Step 1. CRISPR Processing: The following describes the CRISPR processing of the subtype I-C/Dvulg Cas5d system in Bacillus halodurans.
○ 1st. Cas5d recognizes hairpin structures and 3’ single-stranded sequences in the CRISPR repeat region, cleaving pre-crRNA into unit-length fragments.
○ 2nd. Pre-crRNA processing: Further processing of pre-crRNA into smaller crRNAs.
○ 3rd. Cas5d forms a complex with crRNA, Csd1, and Csd2 proteins.
○ 4th. The crRNA portion in this complex detects and removes viral DNA.
Figure 9. CRISPR Processing
③ Step 2. Reaction to External Nucleic Acids
○ 1st. Cas9 creates a bubble in the double-stranded DNA, and the complementary sgRNA (small guide RNA) binds to the target RNA next to the PAM site within that bubble.
○ The bubble is referred to as DNA double-strand breaks (DSBs).
○ sgRNA: Fragments of viral DNA that remain in the genomic DNA of bacteria (e.g., E. coli) surviving viral infection. Also known as Spacer or gRNA (guide RNA).
○ PAM (Protospacer Adjacent Motif) site: A 5’-NGG-3’ nucleotide sequence that distinguishes self from non-self.
Figure 10. Location of sgRNA in dsDNA
○ 2nd. The sgRNA-Cas9 complex cleaves the template DNA, non-template DNA, and sgRNA at the same location.
○ 3rd. Cas9 and sgRNA separate.
○ 4th. The DNA that formed the bubble reanneals through hydrogen bonding.
○ 5th. The cleaved template and non-template DNA undergo DNA repair mechanisms.
○ 5th-1st. Viruses without DNA repair mechanisms are eliminated by the CRISPR/Cas9 mechanism.
○ 5th-2nd. Non-homologous End Joining (NHEJ): Allows for the joining of completely different chromosomes, following the Holliday model.
○ 5th-3rd. Homology Directed Repair (HDR): Effects such as the substitution of specific bases or the addition of repeat sequences occur.
Figure 11. NHEJ and HDR
○ 6th. In genetic recombination, research is conducted to replace the unnecessary parts of VNTR genes with useful DNA.
○ Utilizes homology-directed repair mechanisms.
○ Can be applied not only to DNA but also to RNA, epigenomes, and other single nucleotides for editing.
○ Types of gene editing: OE (overexpression), KD (knock-down).
④ Off-Target Issues
○ Definition: The problem of editing regions other than the target gene when applying the CRISPR/Cas9 system to gene therapy.
○ Streptococcus pyogenes Cas9 (SpCas9) nuclease is commonly used due to its efficiency but has a high off-target ratio.
○ Nuclease mutation
○ PAM sequence modification
○ gRNA truncation
Figure 12. CRISPR/Cas9 Technology Diagram
⑤ Application 1. Perturb-seq
○ sgRNA/dCas9-BFP-KRAB complex → binding to the target promoter → KAP1 complex → heterochromatinization (suppression of gene expression)
○ Perturbation screening can be performed using different guide RNAs.
⑥ Application 2. Gene therapy: editing alleles that cause rare diseases
⑦ Application 3. Temporal sequencing (e.g., Record-seq): recording events within spacers
⑵ Lysogenic viruses: The viruses primarily used in gene therapy are adenoviruses and retroviruses.
⑶ siRNA, miRNA Therapeutics: Current market situation is not favorable
⑷ Nucleic Acid Delivery Systems
① Overview
○ Adopted commercially with great success by Moderna and Pfizer during the COVID-19 pandemic.
○ Since mRNA is unstable in the body, a delivery vehicle is necessary to ensure stability until it reaches the target tissue or cell.
② Type 1. mRNA Drug Delivery Systems
○ Solid Lipid Nanoparticle (SLN): The most popular mRNA delivery vehicle adopted by Moderna and Pfizer.
○ A single SLN of 80-100 nm can encapsulate around 100 mRNA molecules.
○ Examples: ALC-0315 (Pfizer/BioNTech), SM-102 (Moderna), ALC-0159 (Pfizer/BioNTech), PEG-DMG (Moderna).
○ Cationic liposome
○ Polymer and polymer/lipid hybrid particle
○ Micelle
○ Emulsion
③ Type 2. DNA Drug Delivery Systems
④ Type 3. AAV(adeno-associated virus)
○ Utilized as a formulation for treating Alzheimer’s disease.
Figure 13. Comparison between AAV and LNP
⑹ Mega Nuclease
⑺ TALEN (Transcription Activator-Like Effector Nuclease)
⑻ ZFN (Zinc Finger Nuclease)
Input: 2015.07.03 21:57
Modify: 2016.05.25 00:18