Chapter 9. DNA Technology
Higher category : 【Biology】 Biology Index
2. Gene library
3. PCR
8. DNA-protein interaction studies
9. Protein-protein interaction studies
10. Gene therapy
1. DNA recombination : DNA synthesis
⑴ 1st. Target gene generation from mRNA (for eukaryotic DNA)
① Reverse Transcription of mRNA to Complementary DNA to mRNA (cDNA)
② RT-PCR : DNA primer added to mRNA solution and one-time reverse transcriptase treatment
○ oligo dT primer : Complete cDNA can be synthesized using poly A as a template for eukaryotic-derived mRNA
○ 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
○ Purpose to attack foreign DNA in bacteria
○ DNA that can be attacked is methylated. 4-⑽-③Protection
○ Sticky end and blunt end
○ Sticky ends : Terminals that can be cut back to restriction enzymes to form complementary hydrogen bonds
○ Smooth end : Terminal that cannot be rebound after being cut by restriction enzyme
○ Restriction enzymes that form sticky ends are used for DNA recombination
○ Palindrome awareness : If you read 5 ‘to 3’, the sense and anti-sense strands are identical.
○ TATCGTACGAAC → TATC + GTACGAAC
○ ATAGCATGCTTG → ATAGCATG CTTG
○ The above example is an example of sticky ends
○ Restriction enzyme example
○ AgeⅠ : 5’-A ▼ CCGGT-3 ‘
○ BamHⅠ : 5’-G ▼ GATCC-3 ‘
○ BglⅡ : 5’-A ▼ GATCT-3 ‘
○ EcoRⅠ : 5’-G ▼ AATTC-3 ‘
○ HindⅢ : 5’-A ▼ AGCTT-3 ‘
○ HpaⅡ : 5’-CC ▼ GG-3 ‘cleavage, 5’-CmC ▼ GG-3’ cleavage, but mC is methylated cytosine
○ MspⅠ : 5’-CC ▼ GG-3 ‘is cleaved, 5’-CmC ▼ GG-3’ is also cleaved, but mC is methylated cytosine
○ MSTⅡ : 5’-CCTN ▼ AGG-3 ‘, where N is the base sequence of 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 ‘, blunt-ended formation
○ Recombinant sequence by BamHI is also cleaved by Sau3AI, vice versa only in certain cases
○ The fragment by BglII and the fragment by BamHⅠ bind to each other and are not cut again by restriction enzymes.
○ If linear DNA is cut once with restriction enzymes, it becomes two pieces, whereas circular DNA is cut once with restriction enzymes.
○ Restriction mapping (later updated)
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. Alkiline phospatase (eg, to prevent self-ligation when only one restriction enzyme is processed) : Cal intestinal phosphatase (CIP) treatment
○ 2nd-1st-2nd. Orientation to Plasmid DNA Binding to Target DNA by Treatment with Two or More Restriction Enzymes
③ 2nd-2nd. Insert the truncated gene and plasmid into one test tube → Complementary cohesive ends bind to each other
④ 2nd-3rd. Linkage with DNA ligase to generate recombinant plasmid
⑶ 3rd. Transformation : Transformation of recombinant genes into host cells (bacteria)
① Transformation process
○ 3rd-1st. CaCl2 : Ca2+ binds to phospholipids and stabilizes cell membranes
○ 3rd-2nd. Thermal Shock 42 ℃ : Temporary perforation in the cell membrane inserts the recombinant plasmid 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, screening marker purpose (See. ↓)
③ Vector : Plasmid or bacteriophage DNA that carries and replicates recombinant DNA
④ Cloning vector : Vectors aimed at cloning genes
○ Four conditions : Origin of origin (ori), promoter, restriction enzyme recognition sequence, selectable marker (antibiotic resistance gene)
○ Additional condition : Small size, 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, cloning gene cDNA
○ Tertiary structure (example : Disulfide bond)
○ Eukaryotic Host Condition : promoter, poly A sequence
○ Example : When promoter is added after the promoter of lac operon
⑥ Vector example 1. pBR322 plasmid
○ 4361 bp, ori present
○ Restriction enzyme recognition sequences such as EcoRI, BamHI and HindIII
○ ampr : Ampicillin resistance gene in plasmid pBR322, Pst I recognition
○ tetr : Recognition of the tetracycline resistance gene, HindIII, BamHI, SalI in plasmid pBR322
○ Recombinant strains : Selecting tet non-resistant amp resistant strains
⑦ Vector example 2. pUC19 plasmid
○ 2686 bp, ori present
○ Has restriction enzyme recognition sequence such as ApaLI
○ lac Z : X-gal as a substrate to produce a blue product
○ ampr
⑧ Vector example 3. Ti plasmid
○ Agrobacterium tumefaciens (Rhizobium radiobacter) : Pathogens of plant myocarcinoma (benign tumor)
Figure 1. The mycorrhizal fungus attached to carrot cells.
○ Ti plasmid : Myocarcinoma plasmid, including T-DNA
○ T-DNA : 20 kb of DNA encodes opines and auxin and cytokinin (myocarcinoma formation) synthase
○ Opine : Nutrient Sources of Myocardium Carcinoma
○ Lapse : Myocardium carcinoma plant infection → vir (virulance DNA, 25 bp) cuts both ends of T-DNA and inserts randomly into plant nucleochromosomes
○ DNA recombination : Inserting target DNA into the T-DNA region, wounding plants and infecting recombinant myocarcinoma
⑨ Vector example 4. Gene library vector (See. 2-⑴) : BAC Library, YAC Library
⑩ Miscellaneous Vector Examples
○ Bacterial plasmid : F plasmid, R plasmid (antibiotic resistance gene insertion), approximately 1-10 kb
○ Virus vector
○ Genetic information is inserted into bacterial DNA through bacterial infection, proliferation, lysis, release and cleavage
○ λ phage is mainly used, about 25 kb
○ Sometimes use a retrovirus
○ Cosmid : Introduction of phage cos end, about 50 kb by in vitro packaging method
○ 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 following normal insertion of recombinant plasmid)
Example : Ampicillin resistance gene, tetracycline resistance gene
○ 1st. replica formation : After incubating the entire colony, the entire culture dish is buried in the filter paper
○ 2nd. Add antibiotics to some of the colonies on the filter paper
○ 3rd. Dead colonies due to antibiotics did not transform
○ 4th. Colony selection corresponding to colonies that did not die due to antibiotics on filter paper
② Antibiotic resistance genes (screened according to recombination success)
○ 1st. replica formation : After incubating the entire colony, the entire culture dish is buried in the filter paper
○ 2nd. Add antibiotics to some of the colonies on the filter paper
○ 3rd. Colonies killed by antibiotics indicate that the restriction enzyme cut the antibiotic resistance gene
○ 4th. Colony selection corresponding to dead colonies due to antibiotic on filter paper
③ X-gal (screening 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. X-gal white in some colonies containing lac Z-containing plasmids indicates transfection
④ Southern blotting (See. ↓↓)
⑸ 5th. Cloning amplification : Large quantities of product are obtained by mass fermentation and purification
① Why antibiotic-resistant plasmids and antibiotics for non-selective purposes in large scale culture
○ 1st. Plasmids in the strain randomly enter daughter cells whenever the strain divides
○ 2nd. As division progresses, some cells have no plasmids
○ 3rd. Cells with plasmids use a lot of energy to make plasmids, so growth rate is different from cells without plasmids
○ 4th. Ultimately more cells without plasmids
⑹ Success story
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
③ Agriculture : Golden rice, herbicide-tolerant crops, pest-tolerant crops
④ Ti plasmid transgenic neofunctional crop : Herbicide tolerance, anthrax crops (seed potatoes, etc.)
⑤ Recombinant Strain Development Strategy
○ feedback / antibiotics resistant mutant
○ Auxotrophic mutant
○ Overexpression mutant
2. Gene library : DNA storage
⑴ Vector for genetic library
① Bacterial Artificial Chromosome
○ Advantages : No intersection
○ Disadvantages : Match the expression system of prokaryotes
○ Used in human genome projects.
○ It is still used today
② YAC (Yeast Artificial Chromosome) (pYAC3)
○ Artificial chromosome into yeast (~ 1 Mb), ARS retention, DNA insertion between the tautomer and telomere
○ Advantages : Eukaryotes are used to make human proteins easy
○ Disadvantages : Cross from the first Meiosis electricity
③ Virus genome library
○ Disadvantages : 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 to each daughter cell.
② Telomere: A sequence of base pairs located at the end of a chromosome, serving to counteract the shortening of chromosomes during 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 Other: Promoters, gene expression regulatory mechanisms, and other related elements.
⑶ Types
① gDNA Library
○ 1st. Precipitate histones by treating with phenol.
○ 2nd. Nucleic acids exist in the upper layer of the genomic DNA (gDNA) solution.
○ 3rd. Separate the upper layer → Tri chloroacetic acid → Nucleic acid aggregates.
○ 4th. Clone all gDNA after restriction enzyme cleavage into a vector (BAC).
○ 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 separated mRNA using reverse transcriptase.
○ 3rd. Treat with low-concentration RNAase acting as primers.
○ 4th. Treat with DNA polymerase I.
○ 5th. Treat with ligase.
○ 6th. Treat with restriction enzyme.
○ 7th. Insert into a vector.
○ 8th. Clone and amplify.
○ 9th. Used in the study of gene expression.
③ Library Screening
○ Move library clones to a filter, and activate the probe.
○ Locate clones with the same sequence as the probe when screened against the library.
3. PCR (polymerase chain reaction) : DNA amplification
⑴ Defition : DNA amplification technology applying the principle of DNA replication
① Kerry Mullis, 1993 Nobel Prize in Chemistry
⑵ Sample : DNA sample, DNA primer (2 types), dNTP, Taq polymerase, Buffer (pH stabilization), MgCl2 (DNA stabilization)
① NTP RNA Polymeric Materials
② Taq polymerase
○ Taq polymerase is obtained from the archaeological bacterium Thermus aquaticus that lives in hot spring water.
○ Taq polymerase 55 ℃ optimum polymerization, 92 ~ 95 ℃ heat denaturation
○ Taq polymerase additionally adds A after polymerization to the 3 ‘end.
○ No template required, so can be applied to recombination of blunt-ended DNA
③ DNA has anion repulsion of phosphate skeleton, so unstable structure → Na + or Mg2 + can stabilize DNA
⑶ Step
① 1st. DNA denaturing : Heat to separate two strands of DNA, 94 ° C, 30-60 seconds
② 2nd. Annealing : As the mixture cools, the primers bind to the DNA template, 50-55 ° C, for 1 minute
③ 3rd. DNA extension : Polymerase starts synthesis at primer, 72 ° C, 1 minute per 1000 bp
④ 4th. ① To③ Repeating the process would theoretically duplicate an exponentially DNA sample of 2
⑤ 5th. DNA ligase (ligase) action (40 ° C) and stabilization (4 ° C)
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 Too high annealing results in poor hybridization, too low an increase in mis-hybridization
② Tm : 50% retains double strands, 50% proportionate to the released temperature, typically (A + T) × 2 + (G + C) × 4
③ Usually the annealing temperature is set as low as 5-10 ℃ at Tm
④ The presence or absence of metal ions affects the optimum annealing temperature.
⑸ Limit : Need to know the nucleotide sequence at both ends of template DNA
① Home : If there is a known sequence in the middle and both ends of the template DNa are blunt ends, the limitations can be overcome.
② Solution 1. inverse-PCR
○ 1st. Template DNA Generation through Cyclic DNA Ligase
○ 2nd. Restriction enzymes cut inside known sequences, resulting in linear DNA
○ 3rd. PCR is possible because we know sequence of both ends
③ Solution 2. anchored-PCR
○ 1st. Amplify half by connecting new primer with template DNA and DNA ligase
○ 2nd. The other half goes through the same process.
④ Solution 3. oligonucleotide-directed mutagenesis
○ Case 1. How to replicate template DNA with primers that only match a few bases
○ Case 2. Method of replicating template DNA with primer added with base sequence to form hairpin structure in the middle
○ Case 3. Method of replicating template DNA with primers without base sequence to make template DNA form hairpin structure
⑹ Kinds
① RT-PCR : Involves the creation of cDNA from RNA using reverse transcription enzymes
② Real Time PCR (QPCR, Quantitative PCR) : Check the amount of amplified DNA in real time
③ multiplex PCR : Amplification of multiple DNA samples in one PCR device
4. DNA fingerprinting : DNA discrimination, no two genetically identical twins are genetically identical
⑴ Objective : Paternity confirmation, prediction of genetic diseases, forensic perpetrator identification.
⑵ Electrophoresis : Material separation and quantity estimation by size
① Stationary phase, mobile 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
○ 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
○ SDS-PAGE : Used for more efficient separation, consisting of SDS (negative charge surfactant) and β-ME (β-mercaptoethanol)
○ SDS : Elimination of ionic bonds, hydrophobic interactions, etc., to ensure that portions of the protein have a constant negative charge density
○ β-ME : Disulfide bond removal
○ When SDS-PAGE is attached to each amino acid, all proteins become secondary structure of the same charge density due to the repulsive force between negative charges.
○ Ammonium persulfate (APS) : Role of Enzymes to Form Crosslinks of Polyacrylamide Gels and Bisacrylamide Gels
○ TEMED (N, N, N ‘, N’-tetramethylethylenediamine) : Role of stabilizing APS
○ native-PAGE : SDS-PAGE to be a modified gel and to be an unmodified gel
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
○ Molecular weight is linearly proportional to the size of special materials, which reduces the travel distance due to high resistance in the mobile phase.
○ Travel distance is proportional to time (Δt)
○ DNA ladder (size marker) : It is possible to infer the size of an unknown substance by presenting the movement distance according to the size.
○ supercoil DNA has little resistance in electrophoresis
⑥ Type 1. General electrophoresis
○ More low molecular weight nucleic acids and proteins
○ In protein electrophoresis, proteins move from the (-) pole to the (+) pole by SDS-PAGE.
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, staining with 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 : Isolation according to charge by isoelectric point electrophoresis in the presence of elements or electrophoresis under undenatured conditions
○ Element : Eliminates all R-R interactions except disulfide bonds
○ y axis : Separation Depending on Molecular Weight by Electrophoresis in the Presence of SDS
○ One can separate more than 1,000 kinds of proteins
○ Application 1. Separate the x-axis and y-axis in the same way, but add a specific substance during the y-axis separation
○ Application 2. tRNA, for small RNA molecules : 1D 10%, 2D 20% polyacrylamide gel concentration → Effective separation of various RNAs
⑶ Fingerprint method 1. RFLP (restriction fragment length polymorphism) : Paternity, genetic 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. Generating DNA fragments of different sizes with the same restriction enzyme recognition site
○ 5th. Electrophoresis : Allows you to see the pieces separated by size differences
② Cause : Single nucleotide polymorphism (SNP)
○ Defition : Difference between people due to one base being different by point mutation
○ One SNP per 1000 bp per person
○ SNPs can occur in both genes and introns
○ RFP may occur when SNP occurs at restriction enzyme recognition site
○ 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 single nucleotide polymorphisms (SNPs).
Figure. 3. Number of SNPs
③ Limit : RFLP does not vary greatly from person to person since SNPs cannot occur at the restriction sites
○ It is hard to utilize for identification of criminal
⑷ Fingerprinting 2. Repeat repeat : Targeting criminals, genetic fingerprints stronger than RFLP, and repetitive sequences
① VNTR (variable number tandem repeat) : 15-100 repetitions
○ Principle : Different sizes of repeat sequences are created between individuals
○ Cause : Uneven Crossover of Homologous Chromosome
○ 1st. DNA fragments separated and amplified along the length by electrophoresis
○ 2nd. Separated DNA fragments were transferred to filter paper
○ 3rd. Transfer to filter paper and treat chemical (basic) to break hydrogen bonds → one strand of 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 Tendom Repeat) : 2-4 repetitions
○ Not much different than VNTR
5. Hybridization : Check DNA presence
⑴ Southern blotting : Nucleic Acid Probe Hybridization to DNA
① Summary
○ Some sort of RFLP
○ Edward M 1975. Developed by Southern
② 1st. Restriction enzyme treatment → PCR amplification → electrophoresis
○ Electrophoresis : Use agarose gel
○ Master plate can be used instead of electrophoresis : List the colonies into which the recombinant plasmid was introduced in a solid medium
③ 2nd. Gel 0.Shake in 5 M HCl solution
○ Faster DNA transfer from agarose gel to membrane by HCl treatment
④ 3rd. Gel 1.5 M NaCl, 0.Shaking in 5 M NaOH solution (DNA denaturation)
○ Separate DNA into single strands by placing NaOH solution-sponge-gel
○ Sponge absorbs NaOH solution and provides it to the gel
○ Additional nucleic acid denaturant is added to prevent partial double bond formation of ssDNA
○ Nucleic acid denaturants : Form ions, such as metal ions, formamide and formaldehyde, weaken DNA binding ability
⑤ 4th. Gel 1.5 M NaCl, 1 mM EDTA, 0.5 M Tris-HCl (pH 7.2) shaking in solution
○ Trsi-HCl : Buffer
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. NaOH solution transferred to gel is absorbed by carrying single-stranded DNA and moving to the towel bundle
○ 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.
○ UV light can cause DNA to bind to the membrane
⑧ 7th. Pre-Blocking : Improved sharpness by adding blocking agent
○ 7th-1st. Identify DNA that is considered insignificant through repeated or allele-specific electrophoresis
○ 7th-2nd. Add salmon sperm DNA fragments to the DNA
○ 7th-3rd. Prevents probe DNA from binding to specific DNAs during 8th hybridization (enhances sharpness)
⑨ 8th. Hybridization
○ 8th-1st. DNA-bound nylon membranes in seal-a-meal bags
○ 8th-2nd. Add radiolabeled DNA probe to seal-a-meal bag at 62 ° C
○ Labeled probes have complementary sequences of genes of interest
○ Probe DNA : 100-500 bp, single stranded DNA
○ Probe DNA typically labels P32, a radioisotope
○ Probe DNA can be labeled with dyes such as EtBr and SYBR Green
○ 8th-3rd. Probes bind to complementary DNA
○ Other DNA that is considered insignificant through repeated experiments is covered with salmon sperm DNA by prehybridization
⑩ 9th. Eliminate unhybridized radioactive probes in seal-a-meal bags
⑪ 10th. Autoradiogram : Irradiation on nylon membrane
⑫ 11th. Identifies the location (or colony location) on the radioactive electrophoresis
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
○ 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
○ Use of Basic Solution in Northern Blots Decomposes RNA
○ Northern blotting uses a salt solution to stabilize RNA
④ Difference 4. Type of probe
○ Southern blotting uses a 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 Using 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 and 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 Using Salmon Sperm DNA Fragments
○ Western blotting uses casein, skim milk, bovine serum albumin (BSA), etc.
○ BSA can also be used for nucleic acid blotting
⑥ Difference 5. Type of probe
○ Nucleic Acid Blotting Using DNA Probes
○ Western blotting uses an antibody probe
○ Primary antibody : Target Proteins and Specific Antibodies
○ For specificity of primary antibodies, enzymes from other animals should be used
○ Secondary antibody : Combined with primary and specific antibodies, enzymes that degrade chromogenic substrates
○ For specificity of secondary antibodies, enzymes from target and primary antibody derived animals and other animals should be used
⑦ Difference 6. Imaging Method
○ Nucleic Acid Blotting Using EtBr + Self-radiation
○ Western blotting uses Coomas blue staining
⑷ DNA chip (microarray)
① Simultaneously check the expression patterns of multiple genes
○ One compartment contains one cDNA probe.
② Confirmation of tissue-specific gene expression through cDNA DNA chip
○ 1st. abrication of cDNA Chips by Dropping Human Gene cDNA Libraries on Glass Slides
○ 2nd. Treated 1% BSA Solution on cDNA Chip
○ BSA already binds to DNA, allowing only complementary DNA to bind
○ 3rd. Normal tissue, abnormal tissue (e.g., : MRNA X and Y are each prepared from cancer)
○ 4th. Add oligo-dT to X and Y, respectively
○ oligo-dT complementarily binds to poly A sequences in X and Y
○ 5th. RT-PCR : X adds dNTP and cy3 (green fluorescent substance) -dTTP, and Y adds dNTP and cy5 (red fluorescent substance) -dTTP, respectively, and performs reverse transcription reaction to generate fluorescently labeled cDNA.
○ 6th. 0 each for X and Y.1 N NaOH and reacted for 10 minutes at 70 ℃ 0.Neutralize with 1 N HCl
○ Template RNA digestion
○ 7th. The cDNA synthesized in X and Y was purified and mixed in equal amounts to hybridize with the cDNA chip.
○ 8th. Washing cDNA Chips with Buffer
○ 9th. scanning (scanning) : Measure and calibrate the fluorescence intensity of cy3, cy5
○ Black : X and Y do not express both
○ Green : X only expression
○ Red : Y only expression
○ Yellow : Expression of both X and Y
⑸ Fluorescence in situ hybridization (FISH)
① Summary
○ Experimental Techniques to Hybridize Specific DNA Sequences with Chromosome Phases
○ Used to locate RNA as well as the location of specific sequences
○ Hybridization with fluorescent probes complementary to specific sequences, followed by fluorescence microscopy
○ Diagnose chromosomal abnormalities quickly but cannot diagnose DNA abnormalities
② Process
○ 1st. Prepare epithelial tissue sections on slides
○ 2nd. Treated with RNase and incubated for 1 hour at 37 ℃
○ Probes that bind to DNA may bind to RNA, so there is a process to remove RNA
○ 3rd. Change the buffer to acidic acid, treat pepsin and react for 10 minutes at 37 ℃
○ Pepsin acts in acid, changing pH composition to acid
○ Pepson removes proteins in cells, facilitating further penetration of probes
○ 4th. Wash
○ 5th. Prepare a probe that can bind to isotope, but biotin is attached to dTTP of this probe
○ 6th. Hybridize 4th Sample and 5th Probe
○ 7th. Wash
○ 8th. Fully avidin processed and washed
○ Fluorescent material should be directly attached
○ 9th. Dye with DAPI : DAPI Nuclear Staining
○ DAPI (4’-6-diamidino-2-phenylindole)
○ Fluorescent dye that binds strongly to the AT-rich region of DNA
○ DAPI is cell membrane permeable which can be used for staining both living and fixed cells
○ UV irradiation on DNA bound to DAPI produces blue fluorescence
○ 10th. Observation of the isotope region will show a fluorescent color of avidin, DNA will show a blue fluorescent color
⑹ Dideoxy chain termination : DNA sequence determination
6. Gene deletion : DNA function check
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 plasmid vector : Contains the thymidine kinase (TK) gene, which is remote from the inactivated gene X by inserting the neor (neomycin resistant gene) gene
Figure. 5. The structure of plasmid vector
○ 1st-2nd. Insert targeting vector inside ES cell
○ 1st-3rd. Some ES cells take care of replacing existing gene X with inactivated gene X
○ Be sure to include the same gene next to gene X so that genetic recombination can occur
○ 1st-4th. Selection : Cells are cultured in medium containing G418 (geneticin, neomycin derivative), gancyclovir
○ 1st-4th-1st. Untransformed ES Cells : Killed by G418
○ 1st-4th-2nd. ES cell with site other than gene X replaced : TK gene is killed by gancyclovir (TK gene breaks down gancyclovir and causes toxicity)
○ 1st-4th-3rd. Surviving only ES cells replaced with gene X only
② 2nd. Knocked Out ES Cell Selection
③ 3rd. Injecting knocked out ES cells into the embryo
④ 4th. Germ cells of chimeric mice are produced by germ cells from normal germ cells and knocked out ES cells.
⑤ 5th. F1 mice have (+ / +) and (+/-) objects
⑥ 6th. Genetic testing for self-breeding by selecting (+/-) individuals from F1 mice
○ There is a limit to screening through mouse hair color
⑦ 7th. 25% of F2 mice are knockout mice and should be screened by genetic testing
⑵ Cre-lox
① DNA recombination by Cre occurs only between identical lox sequences
② Cre deletes DNA when the lox sequence pairs are in the same direction, and DNA inversion occurs when the lox sequences are in the opposite direction
⑶ SiRNA : RNA interference can be used to suppress all gene expression
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 gene transformation and animal cloning.
② Trait Transformation without Using a Vector
○ Can induce trait transformation using gene guns, among other methods.
○ Example 1 : Formation of transgenic plant cells by “shooting” particles containing recombinant genes into plant cells.
○ Example 2 : Transgenic plant cells → callus → organism formation (in nutrient medium).
○ Plant cells exhibit totipotency.
○ Example 3 : Maintenance and propagation of purebred, proliferation of useful plants.
○ Example 4 : Tissue culture with meristematic regions, induction of differentiation.
⑶ Examples:
① Example 1 : Transgenic Animals - Mass production of beneficial gene products in animals (e.g., farming).
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 : 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, also known as 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) : Nucleic acid-protein binding investigation
① Also called a gel shift assay
② 1st. Probe production : Labeling DNA with Radioactive Isotopes
③ 2nd. Radiophoresis after electrophoresis of protein and DNA hybrids
④ 3rd. Result analysis
Figure. 7. Example EMSA Results</center>
○ Premise : DNA is moved from top to bottom because the top is the cathode and the bottom is the anode.
○ interpretation of a : Proteins A and B bind to the probe, and A and B bind with the probe attached..)
○ interpretation of b : C protein and D protein bind to probe, C and D do not bind to each other
○ interpretation of c : E protein binds to probe, F protein does not bind to probe, but binds to E protein
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 Phage Display Assay
① 1st step: Introduce mutations or alter nucleotide sequences to create a diverse phage library.
② 2nd step: Screen for phages with desired activity and later confirm the nucleotide sequence.
⑺ Filter Binding Assay: Only transcription factors bind to the filter.
⑻ DNA Affinity Chromatography
9. Protein-protein interaction studies
⑴ Dual Hybrid System: A method for identifying proteins that interact with protein X.
① 1st. Gene of transcription factor 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: Confirming protein binding relationships using protein-protein interactions.
⑶ Protein separation and purification using GST-tagged fusion proteins.
⑷ Phage Display Method: Used to obtain monoclonal antibodies.
① Aim: Obtaining a single clone antibody that binds to the target protein.
② Method to obtain a single clone antibody in a test tube.
③ Typical process for obtaining a single clone antibody: Fusion of B lymphocytes and myeloma cells (hybridoma), becoming stem cells and proliferating to produce a large amount of antibodies.
10. Gene therapy
⑴ CRISPR-Cas9 Gene Editing Technology (also called simply CRISPR 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 unicellular organisms.
○ 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 fragments of external DNA, such as viruses, as spacers between repeats.
○ 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 gRNA (guide RNA).
○ PAM (Protospacer Adjacent Motif) site: A 5’-NGG-3’ nucleotide sequence that distinguishes self from non-self.
○ 2nd. The sgRNA-Cas9 complex cleaves both the template and non-template strands of the DNA 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) : Shows effects like the substitution of specific base pairs.
○ 6th. Research is ongoing to replace unnecessary portions of Variable Number Tandem Repeat (VNTR) genes in genetic recombination.
○ 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. 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 intake when specific genes are suppressed.
○ Gene therapy: Editing genes responsible for rare diseases.
○ Temporal sequencing (e.g., Record-seq).
⑵ siRNA, miRNA Therapeutics : Current market situation is not favorable
⑶ 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
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