Korean, Edit

Chapter 9. DNA Technology

Higher category: 【Biology】 Biology Index 

1. DNA recombination

2. DNA library

3. PCR

4. DNA fingerprinting

5. Hybridization

6. Gene deletion

7. Nuclear substitution

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 1^st. 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 2^nd. 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’-C^mC(N)₁₂▼-3’

○ HindIII: 5’-A▼AGCTT-3’

○ HpaII: Cuts at 5’-C▼CGG-3’, but not at 5’-C^mC▼GG-3’ where ^mC represents methylated cytosine.

○ LpnPI: 5’-C^mCDG(N)₁₀▼-3’

○ McrBC: 5’-Pu^mC(N_40-3000)Pu^mC-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.

○ 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 2^nd-1^st. Cut the target DNA and the plasmid DNA with the same restriction enzyme to form the same sticky ends.

○ 2^nd-1^st-1^st. To prevent self-ligation when treating with only one restriction enzyme, treat with alkaline phosphatase (e.g., Calf Intestinal Phosphatase (CIP)).

○ 2^nd-1^st-2^nd. Provide directionality to plasmid DNA binding to target DNA by treating with two or more types of restriction enzymes.

③ 2^nd-2^nd. Insert the truncated gene and plasmid into one test tube → Complementary sticky ends bind to each other.

④ 2^nd-3^rd. Create a recombinant plasmid by ligating with DNA ligase.

⑶ 3^rd. Transformation: Insert the recombinant gene into the host cell (bacteria) to induce transformation.

① Transformation process

○ 3^rd-1^st. CaCl₂: Ca²⁺ binds to phospholipids and stabilizes cell membranes.

○ 3^rd-2^nd. 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.

○ amp^r: The ampicillin resistance gene from plasmid pBR322, recognized by PstI.

○ tet^r: 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.

○ amp^r

○ 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. Beta-lactamidase gene creates beta-lactam ring to confer penicillin resistance.

⑷ 4^th. 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

○ 1^st. Replica formation: After incubating the entire colony, imprint the entire culture plate onto filter paper.

○ 2^nd. Add antibiotics to a portion of the colonies imprinted on the filter paper.

○ 3^rd. Colonies that die due to the antibiotic indicate that they were not transformed.

○ 4^th. Select colonies corresponding to those that survived the antibiotic on the filter paper.

② Antibiotic resistance genes (selection according to recombination success)

○ 1^st. Replica formation: After incubating the entire colony, imprint the entire culture plate onto filter paper.

○ 2^nd. Add antibiotics to a portion of the colonies imprinted on the filter paper.

○ 3^rd. Colonies that died due to the antibiotic indicate that the restriction enzyme cut the antibiotic resistance gene.

○ 4^th. 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.

○ 1^st. X-gal is originally white, but becomes blue due to lac Z.

○ 2^nd. 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 5^th. 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)

○ 1^st. Plasmids within the bacterial strain are randomly distributed to daughter cells during cell division.

○ 2^nd. As division continues, some cells end up without any plasmids.

○ 3^rd. Cells containing plasmids consume more energy to replicate them, leading to a difference in growth rate compared to plasmid-free cells.

○ 4^th. 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

○ 1^st. Precipitate histones by treating with phenol.

○ 2^nd. Nucleic acids are present in the genomic DNA supernatant.

○ 3^rd. Separate the supernatant → Trichloroacetic acid → Nucleic acid precipitate.

○ 4^th. After digesting gDNA with a restriction enzyme, clone all fragments and insert them into a BAC vector.

○ 5^th. Selectively amplify Escherichia coli containing the target DNA.

○ 6^th. 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

○ 1^st. Affinity chromatography: Attach oligo-dT (poly T) to beads.

○ 2^nd. Reverse transcribe the separated mRNA using reverse transcriptase.

○ 3^rd. Treat with a low concentration of RNase: Functions as a primer.

○ 4^th. Treat with DNA polymerase I.

○ 5^th. Treat with ligase.

○ 6^th. Treat with restriction enzyme.

○ 7^th. Insert into a vector.

○ 8^th. Cloning amplification.

○ 9^th. 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 1^st. The heating step to separate the two DNA strands. 94 ℃. 30-60 seconds.

② 2^nd. Annealing: As the mixture cools, the primers bind to the DNA template. 50-55 ℃. 1 minute.

③ 3^rd. DNA elongation: Polymerase starts synthesis at primer. 72 ℃. 1 minute per 1000 bp.

④ 4^th. Repeating steps ① to ③ theoretically results in the exponential amplification of the DNA sample by a factor of 2.

⑤ 5^th. DNA ligase action (40 ℃) and stabilization (4 ℃).

⑥ 6^th. Switch to E. coli cloning amplification after a few cycles: In reality, the amount of artificial nucleotides is insufficient, producing much less DNA.

⑷ T_m 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 T_m (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.

○ T_m value is independent of repetitive sequences.

○ The T_m 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 T_m.

○ 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 C_t (Threshold Cycle)

① Definition: The cycle at which the signal exceeds the threshold value.

② C_t 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

○ 1^st. Form circular DNA from the template DNA using DNA ligase.

○ 2^nd. Generate linear DNA by cutting within the known sequence using a restriction enzyme.

○ 3^rd. PCR is possible because we know sequence of both ends.

③ Solution 2. Anchored-PCR

○ 1^st. Attach a new primer to the template DNA using DNA ligase and amplify one half.

○ 2^nd. 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.

○ 1^st. DNA isolation from tissue.

○ 2^nd. PCR: Amplify the amount of DNA.

○ 3^rd. Cutting DNA into fragments using restriction enzymes.

○ 4^th. Generate DNA fragments of different sizes with the same restriction enzyme recognition sites.

○ 5^th. 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

○ 1^st. DNA fragments are separated and amplified based on length using electrophoresis.

○ 2^nd. Separated DNA fragments are transferred to filter paper.

○ 3^rd. After transferring to filter paper, a chemical treatment (alkaline) is applied to break hydrogen bonds → Results in single-stranded DNA.

○ 4^th. Compare the location of the VNTR fragments with a VNTR probe labeled with radioactive material.

○ 5^th. 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 1^st. 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 2^nd. 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 3^rd. 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 4^th. 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 5^th. Capillary phenomenon + blotting

○ 5^th-1^st. Place a positively charged nylon filter and a paper towel on the gel.

○ Nitrocellulose paper can be used instead of nylon filter.

○ 5^th-2^nd. The NaOH solution that moves into the gel carries single-stranded DNA and travels to the paper towel stack, where it is absorbed.

○ 5^th-3^rd. Negatively charged single-stranded DNA sticks to nylon membrane.

⑦ 6^th. 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 7^th. Blocking: A blocking agent is added to enhance clarity.

○ 7^th-1^st. Identify DNA that is considered insignificant through repeated experiments or electrophoresis for each allele.

○ 7^th-2^nd. 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 8^th. Hybridization

○ 8^th-1^st. The nylon membrane with bound DNA is placed in a Seal-a-Meal bag.

○ 8^th-2^nd. 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.

○ 8^th-3^rd. Probes bind to complementary DNA.

○ Through repeated experiments, other DNA considered insignificant is blocked and covered with salmon sperm DNA.

⑩ 9^th. Eliminate unhybridized radioactive probes in seal-a-meal bags.

⑪ 10^th. Autoradiogram: Irradiation on nylon membrane.

⑫ 11^th. 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, 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

○ 1^st. A cDNA library of human genes is spotted onto a glass slide to create a cDNA chip.

○ 2^nd. 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.

○ 3^rd. mRNA X (from normal tissue) and mRNA Y (from abnormal tissue, e.g., cancer) are extracted separately.

○ 4^{th</sup. Oligo-dT is added to X and Y, binding complementarily to poly-A sequences in mRNA.}

○ 5^th. 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.

○ 6^th. 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.

○ 7^th. Synthesized cDNA from X and Y is purified and mixed in equal amounts, then hybridized onto the cDNA chip.

○ 8^th. The cDNA chip is washed with buffer solution to remove unbound sequences.

○ 9^th. 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.

○ 1^st. Prepare epithelial tissue sections on slides.

○ 2^nd. 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.

○ 3^rd. Remove the culture medium, add a 2% formaldehyde solution, and incubate for 15 minutes.

○ This step fixes the cells.

○ 4^th. Remove the formaldehyde solution, add a 0.2% Triton X-100 solution, and incubate for 5 minutes.

○ Triton X-100 is a surfactant.

○ 5^th. 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.

○ 6^th. Wash the sample.

○ 7^th. Prepare a probe that binds to the centromere.

○ This probe contains biotin-labeled dTTP.

○ 8^th. Hybridize the sample from step 4 with the probe from step 5.

○ 9^th. Wash the sample.

○ 10^th. After sufficiently treating with fluorescently labeled avidin, wash the sample.

○ 11^th. 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.

○ 12^th. 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 1^st. Knockout induction of gene X in embryonic stem (ES) cells

○ 1^st-1^st. Construction of a plasmid vector (targeting vector): Includes gene X which is inactivated by the insertion of neo^r (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

○ 1^st-2^nd. Insert targeting vector inside ES cell.

○ 1^st-3^rd. 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.

○ 1^st-4^th. Selection: Cells are cultured in medium containing G418 (geneticin, neomycin derivative) and gancyclovir.

○ 1^st-4^th-1^st. Untransformed ES Cells: Killed by G418.

○ 1^st-4^th-2^nd. 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).

○ 1^st-4^th-3^rd. Only ES cells in which only gene X has been replaced survive.

② 2^nd. Selection of knockout ES cells.

③ 3^rd. Inject knockout ES cells into the embryo.

④ 4^th. The germ cells of the chimera mouse consist of both normal germ cells and germ cells derived from knockout ES cells.

⑤ 5^th. F1 mice include both (+/+) individuals and (+/-) individuals.

○ Reason: Because the chimera mouse can be considered a heterozygous mutant.

⑥ 6^th. Select (+/-) individuals among the F1 mice through genetic testing and perform self-crossing.

○ There are limitations in selection based on mouse fur color.

⑦ 7^th. 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 1^st. Probe production: Labels DNA with radioactive isotopes.

③ 2^nd. Electrophorese the protein-DNA hybrid, followed by radiation sensitivity analysis.

④ 3^rd. 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 1^st step: Proteins are initially subjected to Western blotting.

② 2^nd 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 1^st. Transcription factor gene is divided into two parts: DNA binding domain and transcription activation domain.

② 2^nd. Bait: Protein X + DNA binding domain.

③ 3^rd. Prey: Protein of interest for determining binding + transcription activation domain.

④ 4^th. 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 1^st step. By introducing mutagens and altering the base sequence, a vast phage library is created.

⑤ 2^nd 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.

○ 1^st. Cas5d recognizes hairpin structures and 3’ single-stranded sequences in the CRISPR repeat region, cleaving pre-crRNA into unit-length fragments.

○ 2^nd. Pre-crRNA processing: Further processing of pre-crRNA into smaller crRNAs.

○ 3^rd. Cas5d forms a complex with crRNA, Csd1, and Csd2 proteins.

○ 4^th. The crRNA portion in this complex detects and removes viral DNA.

Figure 9. CRISPR Processing

③ Step 2. Reaction to External Nucleic Acids

○ 1^st. 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

○ 2^nd. The sgRNA-Cas9 complex cleaves the template DNA, non-template DNA, and sgRNA at the same location.

○ 3^rd. Cas9 and sgRNA separate.

○ 4^th. The DNA that formed the bubble reanneals through hydrogen bonding.

○ 5^th. The cleaved template and non-template DNA undergo DNA repair mechanisms.

○ 5^th-1^st. Viruses without DNA repair mechanisms are eliminated by the CRISPR/Cas9 mechanism.

○ 5^th-2^nd. Non-homologous End Joining (NHEJ): Allows for the joining of completely different chromosomes, following the Holliday model.

○ 5^th-3^rd. Homology Directed Repair (HDR): Effects such as the substitution of specific bases or the addition of repeat sequences occur.

○ 6^th. 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.

○ Solutions

○ Nuclease mutation

○ PAM sequence modification

○ gRNA truncation

Figure 11. CRISPR/Cas9 Technology Diagram

⑤ Applications

○ For signal transduction research purposes: Multiplexing guide RNAs enable perturbation screening.

○ For imaging research purposes.

○ For drug research purposes: Studying changes in drug uptake when specific genes are suppressed.

○ Gene therapy: Editing genes responsible for rare diseases.

○ Temporal sequencing (e.g., Record-seq).

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 12. Comparison between AAV and LNP

⑹ Mega Nuclease

⑺ TALEN (Transcription Activator-Like Effector Nuclease)

⑻ ZFN (Zinc Finger Nuclease)

Input: 2015.7.03 21:57

Modify: 2019.1.25 00:18