Korean, Edit

Chapter 9. DNA Technology

Higher category : 【Biology】 Biology Index 


1. DNA preparation

2. Gene 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 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

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

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

Vector example 3. Ti plasmid

○ Agrobacterium tumefaciens (Rhizobium radiobacter) : Pathogens of plant myocarcinoma (benign tumor)


image

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.

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

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.

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

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.

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


image

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

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).


image

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

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

Difference 1. Extraction of samples

○ Southern blotting uses DNA extraction

○ Northern blotting effectively isolates mRNA from extracts by affinity chromatography using oligo dT.

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

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

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

Difference 1. Type of gel

○ Nucleic Acid Blotting Using Agarose Gel

○ Western blotting uses polyacrylamide gel, SDS-PAGE, etc.

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.

Difference 3. Blotting Process

○ Nucleic Acid Blotting Uses Capillary Phenomenon During Blotting

○ Western blotting uses an electric field during blotting

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

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

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


image

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


image

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:

Example 1 : Transgenic Animals - Mass production of beneficial gene products in animals (e.g., farming).

Example 2 : Edible Vaccines.

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).

GMO Debate and Principles of Safety Assessment



8. DNA-protein interaction studies

⑴ Gene footprinting assay, also known as DNA footprinting technology:


image

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


image

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


image

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.

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.


image

Figure. 9. CRISPR Processing


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.

Solutions

○ Nuclease mutation

○ PAM sequence modification

○ gRNA truncation


image

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.

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

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

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